Signalling pathways use different messenger systems acting through specific sensors and effectors to control a great variety of cell types (Module 7: Table cell inventory). Each specialized cell type has developed control mechanisms to suit their particular functions. For example, skeletal and cardiac muscle cells have very rapid signalling mechanisms capable of driving contraction within milliseconds. Smooth muscle cells tend to act more slowly, and are far more diverse in both their function and control mechanisms. Some cells contract in a conventional way in response to an external agonist, while others have more complex control mechanisms responsible for maintaining the tonic contraction of the smooth muscle cells surrounding blood vessels.
Another example is cell secretion, where there is similar diversity in function and control. Preformed secretory products packaged into vesicles are released through a process of exocytosis. In exocrine glands and the intestine, there is a process of fluid secretion that depends upon the active transport of ions to provide the driving force for a parallel flow of water. Secretion is evident in many different cell types and cellular processes, such as transmitter release during presynaptic events in neurons, enzyme and fluid secretion by pancreatic acinar cells, release of insulin by insulin-secreting β-cells and inflammatory mediators by mast cells.
Many of these mammalian cell types are controlled by more than one signalling pathway, and the object here is to understand how signalling pathways and their downstream sensors and effectors function to bring about a change in cellular activity in some typical examples of specialized cell types.
Mammalian cell types
There are a large number of mammalian cell types (Module 7: Table cell inventory). They are adapted to perform highly specific functions, which are controlled by signalling systems uniquely adapted to carry out their particular functions. These cell-specific signalsomes are a selection from the diverse array of intracellular signalling pathways (Module 2: Figure cell signalling pathways). During the process of cell differentiation, each primary cell type expresses those signalling pathways that are particularly suited to control their particular functions as illustrated by some of the main mammalian cell types shown below:
In many cases, different cell types are collected together into complex functional networks:
Module 7: Table cell inventory An inventory of mammalian primary cells that have specific cell functions.
|Tissue and cell||Comments|
|Gametes||Male and female gametes participate in the process of fertilization (Module 8: Figure mammalian fertilization)|
|Intestinal stem cells||Located in the crypt region of the intestine (Module 12: Figure colon cancer)|
|Satellite cells||Skeletal muscle stem cells that function in muscle repair and regeneration (Module 8: Figure Satellite cell function)|
|Haematopoietic stem cells (HSCs)||Located in the bone marrow and gives rise to common lymphoid progenitor (CLP) and the common myeloid progenitor (MLP) cells (Module 8: Figure haematopoiesis)|
|Epidermal stem cells||Function in the hair follicle cycle (Module 8: Figure hair follicle cycle)|
|Melanocyte stem cells||Provide melanocytes during the hair follicle cycle (Module 8: Figure hair follicle cycle)|
|Mesenchymal stem cells||The precursor of a wide range of primary cells Module 8: Figure MSC differentiation|
|Mammary gland stem cells|
|Male germ cells|
|Neural stem cells|
|Skeletal muscle||See Module 7: Figure skeletal muscle E-C coupling|
|Slow-twitch muscle||MyHC I; SERCA2a|
|Fast-twitch muscle IIa||MyHC IIa; SERCA1|
|Fast-twitch muscle IId/x||MyHC IId/x; SERCA1|
|Fast-twitch muscle IIba||MyHC IIb; SERCA1|
|Satellite cells||Skeletal muscle stem cells|
|Sinoatrial node cells||Responsible for the cardiac pacemaker (Module 7: Figure cardiac pacemaker)|
|Purkinje fibres||Specialized cells that conduct action potentials to the ventricular cells|
|Atrial cells||Responsible for atrium contraction (Module 7: Figure atrial Ca2+ domains)|
|Ventricular cells||Responsible for ventricle contraction (Module 7: Figure ventricular cell E-C coupling)|
|Smooth muscle||Responsible for contraction of smooth muscle (Module 7: Figure smooth muscle cell E-C coupling)|
|Atypical smooth muscle cell||Smooth muscle pacemaker cell (Module 7: pelviureteric organization)|
|Interstitial cells of Cajal||Smooth muscle pacemaker cell (Module 7: Figure ICC pacemaker)|
|Airway smooth muscle cells||See Module 7: Figure bronchiole-arteriole contraction|
|Corpus cavernosum smooth muscle cell||See Module 7: Figure corpus cavernosum|
|Detrusor smooth muscle cell||See Module 7: Figure bladder SMC activation|
|Gastrointestinal smooth muscle cells||See Module 7: Figure ICC pacemaker|
|Urethral smooth muscle cells||See Module 7: Figure ICC pacemaker|
|Uterine smooth muscle cells||See Module 7: Figure uterus activation|
|Vascular smooth muscle cells||See Module 7: Figure SMC cytosolic oscillator|
|Vas deferens||See Module 7: Figure vas deferens activation|
|Dorsolateral prefrontal cortex (DLPFC)||See Module 10: Figure dorsolateral prefrontal cortex|
|Cortical pyramidal neuron|
|Cortical inhibitory interneurons|
|Double bouquet neurons|
|Wide arbor (basket) interneurons|
|Hippocampus||See trisynaptic hippocampal circuit (Module 10: Figure hippocampus)|
|Hippocampal CA1 neurons|
|Hippocampal CA3 neurons||See Module 10: Figure CA3 hippocampal neurons|
|Hippocampal interneurons||See Module 10: Figure hippocampal interneurons|
|Axo-axonic cell||See Module 10: Figure CA3 hippocampal neurons|
|Basket cell (PV+ CCK−)|
|Basket cell (PV− CCK+)|
|Basket cell (PV− CCK+VIP+)|
|Oriens-lacunosum molecular (O-LM) cell|
|Schaffer-collateral-associated cell||See Module 10: Figure CA3 hippocampal neurons|
|Lacunosum-moleculare-perforant path (LM-PP)|
|Lacunosum-moleculare-radiatum-perforant path (LM-R-PP)|
|Hippocampal septal cell|
|Hypothalamus||See Module 10: Figure hypothalamic pituitary regulation|
|Magnocellular (MCN) neurons||See Module 10: Figure magnocellular neurons|
|Oxytocin neurons||See Module 10: Figure oxytocin neuron|
|Vasopressin neurons||See Module 10: Figure vasopressin neuron|
|Cerebellum||See Module 10: Figure cerebellar neural circuit|
|Cerebellar Purkinje neurons||See Module 10: Figure Purkinje cell input-specific Ca2+ signals|
|Cerebellar climbing fibre||See Module 10: Figure cerebellar climbing fibres|
|Basket cells||See Module 10: Figure basket cell Ca2+ transients|
|Starburst amacrine cells||Directionally selective retinal ganglion cells|
|Thalamic interneurons||See Module 10: Figure dendritic GABA release|
|Suprachiasmatic nuclei||Location of the circadian clock neurons (Module 6: Figure circadian clock location)|
|Medium spiny neurons||See Module 10: Figure medium spiny neuron signalling|
|Hypothalamic neurons||See Module 10: Figure hypothalamic Ca2+ syntilla|
|Pinealocytes||Synthesize and secrete melatonin|
|Outer hair cells|
|Inner hair cells||See Module 10: Figure hair cell|
|Hypoxia-sensing||Functions in O2 sensing (Module 10: Figure carotid body chemoreception)|
|Glomus cells||Associates with the glomus cell|
|Nociception||See Module 10: Figure inflammatory soup|
|Aα sensory fibres||Function in proprioception|
|Aβ sensory fibres||Function in proprioception|
|Aδ sensory fibres||Function in nociception|
|C fibres||Function in nociception|
|Olfactory receptor cells||See Module 10: Figure olfaction|
|Organum vasculosum of the lamina terminalis (OVLT) neuron|
|Subfornical organ (SFO) neuron|
|Photoreception||See Module 10: Figure rod and cone structure|
|Taste||See Module 10: Figure taste receptor cells|
|Type I glial-like cells|
|Type II receptor cells|
|Type III presynaptic cells|
|Merkel cells||See Module 10: Figure Merkel cell|
|Astrocytes||See Module 7: Figure astrocyte structure|
|Bergmann glia||See Module 7: Figure Ca2+ microdomains in Bergmann glia|
|Microglia||See Module 7: Figure microglia interactions|
|Pituicytes||See Module 10: Figure magnocellular neurons|
|Satellite glial cells|
|Anterior pituitary||See Module 10: Figure hypothalamic pituitary regulation|
|Corticotrophs||See Module 10: Figure corticotroph regulation|
|Gonadotrophs||See Module 10: Figure gonadotroph regulation|
|Lactotrophs||See Module10: Figure lactotroph regulation|
|Somatotrophs||See Module 10: Figure somatotroph regulation|
|Thyrotrophs||See Module 10: Figure thyrotroph regulation|
|Folliculostellate (FS) cells|
|Adrenal gland||See Module 7: Figure adrenal gland|
|Zona glomerulosa||See Module 7: Figure glomerulosa cell signalling|
|Zona fasciculata/reticularis cells||See Module 7: Figure adrenal gland|
|Chromaffin cell||See Module 7: Figure chromaffin cell secretion|
|Chief cell||See Module 7: Figure parathyroid gland|
|Stomach||See Module 7: Figure stomach structure|
|Parietal cell||See Module 7: Figure parietal cell|
|D cell||See Module 7: Figure stomach structure|
|G cell||See Module 7: Figure stomach structure|
|Enterochromaffin-like cell||See Module 7: Figure stomach structure|
|X/A-like cells||See Module 7: Figure stomach structure|
|Intestine||See Module 7: Figure small intestine|
|Small intestine||See Module 7: Figure intestinal secretion|
|Enterochromaffin cell||See Module 7: Figure small intestine|
|L cell||See Module 7: Figure L cell|
|Colon||See Module 7: Figure colon function|
|Islets of Langerhans||See Module 7: Figure pancreas|
|α-Cells||See Module 7: Figure α-cell signalling|
|Insulin-secreting β-cells||See Module 7: Figure β-cell signalling|
|Exocrine pancreas||See Module 7: Figure exocrine pancreas|
|Exocrine pancreatic acinar cells||See Module 7: Figure control of pancreatic secretion|
|Exocrine pancreatic centroacinar cells|
|Salivary gland||See Module 7: Figure salivary gland secretion|
|Liver||See Module 7: Figure liver cell structure|
|Liver cells||See Module 7: Figure liver cell signalling|
|Hepatic stellate cell||See Module 7: Figure hepatic stellate cell|
|White fat cells||See Module 7: Figure lipolysis and lipogenesis|
|Brown fat cells||See Module 7: Figure brown fat cell|
|Endothelial cells||See Module 7: Figure endothelial cell|
|Haematopoietic cells||Many of these cells participate in inflammation (Module 11: Figure inflammation)|
|Blood platelets||See Module 11: Figure platelet activation|
|B cells||See Module 9: Figure B cell activation|
|T cells||See Module 9: Figure TCR signalling|
|Neutrophils||See Module 11: Figure neutrophil chemotactic signalling|
|Macrophages||See Module 11: Figure macrophage signalling|
|Tingible body macrophages||See Module 8: Figure germinal centre|
|Mast cells||See Module 11: Figure mast cell signalling|
|Kidney||See Module 7: Figure kidney tubule|
|Proximal convoluted tubule||See Module 7: Figure kidney tubule|
|Loop of Henle||See Module 7: Figure kidney tubule|
|Distal convoluted tubule||See Module 7: Figure kidney tubule|
|Collecting duct cells||See Module 7: Figure collecting duct function|
|Juxtaglomerular apparatus||See Module 7: Figure juxtaglomerular apparatus|
|Mesangial cell||See Module 7: Figure mesangial cell|
|Macula densa||See Module 7: Figure macula densa|
|Renin-producing glomerular cell||See Module 7: Figure renin secretion|
|Osteoblasts||See Module 7: Figure osteoblast function|
|Osteoclasts||See Module 7: Figure bone cells|
|Chondrocytes||See Module 7: Figure bone cells|
|Skin||See Module 7: Figure skin cells|
|Arrector pilli muscle||See Module 7: Figure skin cells|
|Keratinocytes||See Module 7: Figure melanogenesis|
|Melanocytes||See Module 7: Figure melanogenesis|
|Merkel cells||See Module 10: Figure Merkel cell|
|Sebaceous gland||See Module 7: Figure skin cells|
|Sweat gland secretory coil cells||See Module 7: Figure sweat gland function|
|Sweat gland reabsorptive duct cells||See Module 7: Figure sweat gland function|
Each cell type has a unique signalling system that is put in place during development. There are a large number of intracellular signalling pathways (Module 2: Figure cell signalling pathways) and each cell type selects out those pathways to establish a cell-specific signalsome that enables it to precisely regulate its activity.
Each cell type has a unique signalling system that is put in place during development. There are a large number of intracellular signalling pathways (Module 2: Figure cell signalling pathways) and each cell type selects out those pathways to establish a cell-specific signalsome that enables it to precisely regulate its activity.
Metabolic energy network
The primary function of the metabolic energy network is to manage the energy status of the cell (Module 7: Figure metabolic energy network). Energy is derived from the intake of food, which is processed in the intestine to form the high-energy metabolites such as amino acids, free fatty acids and glucose that then circulate in the plasma to be dealt with by cells that participate in the metabolic network. In addition, there are other cells that monitor various parameters of the energy supply and then release appropriate hormonal signals to make adjustments to different components of the network. The energy supply is dealt with in two ways: most of the energy is consumed by a variety of cellular processes, while any excess is then stored either as triacylglycerols in the white fat cells or as glycogen in the liver cells. Metabolic homoeostasis depends on energy consumption closely matching energy supply. If consumption exceeds supply, then energy is mobilized from the energy stores to maintain equilibrium. Since the energy stores are finite, such an imbalance in energy supply begins to cause severe wasting as the process of autophagy kicks in to begin to mobilize energy from the structural components of cells. Conversely, if the supply of energy exceeds energy consumption, the excess is diverted into the energy stores, resulting in obesity. This balance between energy uptake and expenditure is determined by the control of food intake and body weight.
The numbers on Module 7: Figure metabolic energy network refer to some of the major components of the network that determines energy homoeostasis:
All cells have to consume energy to survive and to carry out their different functions. Three examples of energy-consuming cells are illustrated in Module 7: Figure metabolic energy network:
The two major energy storage cells are the white fat cells and the liver cells. They are specialized to store energy when it is in excess and then deliver it to the energy consumers during periods of fasting (Module 7: Figure metabolic energy network):
Control of food intake and body weight
Energy homoeostasis is achieved by balancing energy supply and energy consumption (Module 7: Figure metabolic energy network). The latter depends on a variety of processes such as basal metabolisim, physical activity and heat production through adaptive thermogenesis. Energy supply comes from food intake, which is usually balanced against energy consumption to protect against weight gain or loss. Control of food intake and hence body weight depends upon endocrine and neural signalling systems interacting with each other to control the feeding and satiety centres in the brain. Meals are usually terminated by satiation signals released from the gastrointestinal tract. The stomach responds mainly to mechanical distension to provide some of the earliest hormones whereas the chemical composition of the food provides the main signals for the enteroendocrine cells in the intestine. The release and function of these gut hormones is outlined in the following sequence of events (Module 7: Figure control of food intake):
Mutations in MC4R, which carry out the anorexigenic action of the POMC/CART neurons (step 7 in Module 7: Figure control of food intake), account for approximately 2% of adult obesity. Subjects carrying this mutation eat excessively and display increases in fat and lean mass as well as increases in bone mineral density.
The neurotransmitter 5-hydroxytryptamine (5-HT) is a potent suppressor of appetite. Some of its action occurs in the arcuate nucleus (ARC) where it simultaneously can inhibit the orexigenic NPY/AgRP neurons while activating the POMC/CART neurons. Analogues of 5-HT have been used to treat obesity.
The brain is composed of two principal cell types: neurons and glial cells. The neurons form the neural circuits that are responsible for carrying out the computational functions of the brain. The glial cells, which are present in the brain in approximately equal numbers as the neurons, have a variety of functions. The first point to recognize is that there are a number of glial cell types (Module 7 Table cell inventory) with different functions. There are oligodendrocytes and Schwann cells that are responsible for myelination in the central nervous system and the peripheral nervous system respectively. The microglia are phagocytic cells that function in host defence. Finally, there are the astrocytes, which are the major glial cells of the brain. There are many different astrocytes and some of these have been given special names, such as the Bergmann glia found in the cerebellum, the Müller cell found in the retina and the satellite glial cells. It is the astrocytes that will be described in more detail below.
For a long time, astrocytes were thought to play a secondary role in the brain by supporting neuronal activity by carrying out a number of essential housekeeping duties. They are known to provide neurons with nutrients and the redox buffer glutathione (GSH) (Module 12: Figure astrocyte-induced neuronal cell death). They provide a mechanisms for the spatial buffering of K+ and they remove neurotransmitters and other waste products. Recently, however, it has become apparent that glial cells play a much more active role in regulating neural function because they not only respond to information coming from the neurons, but also can transmit information back to the neurons. This two-way dialogue, which depends upon the neuronal–astrocyte communication and the reciprocal astrocyte–neuronal communication systems, may play a critical role in modulating neuronal activity and information processing in the brain. It is becoming evident that this active participation in brain function depends upon a process of astrocyte excitability. This excitability has two important functional consequences. First, it results in the formation of intra- and inter-cellular Ca2+ waves that spread information throughout individual cells or between groups of cells respectively. For example, this excitability is critical for the astrocyte regulation of cerebral blood flow, where astrocytes integrate the degree of neural activity and use this information to regulate contractile tone in arteriole smooth muscle cells. Secondly, astrocyte excitability can lead to the formation of spontaneous astrocyte Ca2+ oscillations that may have a critical role to play in the astrocyte–neuronal communication system. The complex astrocyte structure reveals the intimate associations with both neurons and blood vessels and shows how these cells can perform these different functions.
The problem with trying to deal with astrocyte function is that astrocytes come in many different shapes and sizes (Module 7: Figure astrocyte diversity). Some of these astrocytes have precise locations, such as the Bergmann glia (cell II in Module 7: Figure astrocyte diversity) and the Müller cells found in the retina. These astrocytes are also defined by the kinds of connections that they make within the brain. For example, the tanycytes have their cell bodies facing the ventricles, and then have long extensions connecting to either the pia or the blood vessels. A characteristic feature of many of the astrocytes is a long extension with an endfoot that is attached to a blood vessel (e.g. cells Ib and IV–VII in Module 7: Figure astrocyte diversity). It is this cell type that co-ordinates neural activity with cerebral blood flow.
The most important structural feature of these astrocytes is their extraordinary degree of branching. Each of the major arms that radiate out from the cell body divides repeatedly to produce a highly complex tree-like arrangement (Module 7: Figure astrocyte organization). The long extensions of each astrocyte are often connected to those of other astrocytes through gap junctions made up from connexion 43 (Cx43) (Module 7: Figure astrocyte structure). Astrocytes are often mistaken for neurons because the branching pattern can closely resemble the dendritic tree found on neurons (Module 10: Figure Purkinje cell input-specific Ca2+ signals). This analogy is apt because it is this highly branched structure that makes contact with the neuronal synaptic endings. The reason for this high degree of branching is that each astrocyte makes contact with an enormous number of synaptic endings. It is estimated that each astrocyte makes contact with approximately 150 000 synaptic connections. Since each neuron has roughly the same number of synaptic connections, there has to be a similar number of astrocytes, and this seems to be the case because it is estimated that there are approximately 1.4 astrocytes for every neuron in the human brain. It is quite remarkable to find that all the synaptic endings in the brain are closely associated with astrocytic processes to form tripartite junctions (Module 10: Figure synaptic organization).
The structural organization of the astrocyte is dominated by this close relationship with the synaptic connections between neurons (Module 7: Figure astrocyte structure). The presynaptic neuron (shown in green) sends its axons into the astrocyte domain, where they make connections with the synaptic endings of another set of neurons, the postsynaptic neurons (shown in red). Each synaptic ending comes into close contact with one of the myriad astrocytic processes to form the tripartite junction. Only two such junctions are shown, but one has to imagine that within each astrocyte domain there are approximately 150 000 similar junctions. In addition to this intimate contact with the neuronal synaptic connections, many of the astrocytes also make intimate contact with the cerebral blood vessels. Some of the branches extend out towards the blood vessels, where they end in an endfoot that is planted on the surface to make a junctional contact with the smooth muscle cells.
These astrocytes with their connections to neuronal synapses and blood vessels are packed together to form pseudo-crystalline arrays, which is an important organizing principle within the nervous system (see panel b in Module 7: Figure astrocyte organization). This structural organization forms the basis for the spatial buffering of K+, neuronal–astrocyte communication, astrocyte–neuronal communication and the astrocyte regulation of cerebral blood flow.
Spatial buffering of K+ by astrocytes
The astrocytes have a key role in maintaining a constant concentration of K+ in the narrow extracellular spaces that surround each neuron. The K+ released during periods of neuronal activity is buffered by a ’K+ siphoning’ process whereby the K+ is taken up by astrocytic process near the neurons and is then transferred throughout the astrocytic syncytial system finally to be released from the end foot processes on the blood vessels (see red arrows on Module 7: Figure astrocyte structure). The flow of K+ in and out of the astrocytes is regulated by a number of different types of K+ channels. The K+ that is taken up at the astrocytic processes near the synapses enters the astrocytes through the glial-specific Kir4.1 (Module 7: Figure astrocyte tripartite synapse). Kir4.1 is one of the inward rectifier K+ (Kir) channel family that form either homo- or heteromeric channels. In the case of the uptake of K+ by the astrocytes, this seems to depend on the strongly rectifying heteromeric channels formed by Kir4.1 combining with either Kir2.1 or Kir5.1(Module 3: Table inward rectifier K+ (Kir) channel). At the other end of the siphon, the K+ leaves the end foot processes that line up on the blood vessels through two types of channel. It can leave through either the weakly rectifying homomeric Kir4.1 channels or through the large-conductance (BK) channels (Module 7: Figure astrocyte endfoot signalling). The role of the latter is described in more detail in the section on the astrocyte regulation of cerebral blood flow.
Astrocytes are sensitive to a large number of stimuli such as glutamate, γ-aminobutyric acid (GABA), noradrenaline (also known as norepinephrine), ATP and dopamine. Many of these stimuli are released from the synaptic endings of the tripartite synapse (Module 7: Figure astrocyte tripartite synapse). The transmitter that escapes from the synaptic cleft diffuses out to make contact with the astrocyte. The amount of transmitter spilling out of the synapse will depend on the level of synaptic activity. At low levels of neural activity, the astrocyte receives little stimulation, but this increases markedly when there is rapid neural communication. In addition to this indirect supply of transmitter, there may be specialized ectopic release sites on the presynaptic ending that release transmitter directly on to the astrocytes. The response of the astrocytes to these transmitters depends on the receptors that they express, and this can vary between the different astrocytes. In most cases, however, there is an increase in the intracellular level of Ca2+ achieved by activating different Ca2+ signalling modules. For many of the astrocytes, there are metabotropic receptors that respond to glutamate by activating the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette (i.e. Ca2+ module 6 in Module 2: Figure Ca2+ modules). In addition, astrocytes such as the Bergmann glia have ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) composed of the glutamate receptor 1 (GluR1) and 4 (GluR4) subunits that enable them to gate Ca2+ (i.e. Ca2+ module 1 in Module 2: Figure Ca2+ modules).
The increase in Ca2+ within the astrocyte that results from this neuronal–astrocyte communication system can have at least two functions. Firstly, it is responsible for triggering the reciprocal astrocyte–neuronal communication system by releasing glutamate (Module 7: Figure astrocyte tripartite synapse). The mechanism of glutamate release is still not absolutely certain: both vesicular- and channel-mediated release mechanisms have been proposed. With regard to release by exocytosis, typical vesicles have been observed in astrocytes. Since there are no large membrane depolarizations, astrocytes may use a mechanism of exocytosis triggered by Ca2+ release from internal stores (Module 4: Figure Ca2+-dependent exocytosis). Alternatively, the release of Ca2+ may activate hemichannels formed from pannexin 1 (Panx1) to release ATP and glutamate (See Module 3: Figure hemichannels and gap junctions). The glutamate then triggers astrocyte–neuronal communication.
The second function of the astrocytic Ca2+ signal is to set up a regenerative intracellular Ca2+ wave that spreads the local signal generated near the synaptic sites to more distant parts of the astrocyte. This signalling mechanism is described in more detail in the section on astrocyte excitability.
Astrocytes have the ability to initiate neural activity. The stimulus, which is an increase in intracellular Ca2+, is induced either as a consequence of the neuronal–astrocyte communication pathway (Module 7: Figure astrocyte tripartite synapse) or it can occur spontaneously through the generation of astrocyte Ca2+ oscillations. In both cases, the increase in the intracellular level of Ca2+ within the astrocytic processes, which are a part of the tripartite synapse, release stimuli such as glutamate that then act on receptors on the neuronal endings to either excite or inhibit neuronal activity. An example of how an astrocytic Ca2+ can lead to the activation of neural activity has been described in the hippocampus (Module 7: Figure astrocytic synchronization of hippocampal neurons). A spontaneous Ca2+ transient in the astrocyte was able to activate increases in Ca2+ in three neurons. The implication of this observation is that the astrocyte administers the synaptic connections of more than one output neuron. This organization is depicted in Module 7: Figure astrocyte structure, which illustrates how a single astrocyte can make contact with the synapses that go to two neurons. This arrangement can account for the observation that astrocytes are able to synchronize the activity of groups of neurons. This synchronization is not due to coupling between the neurons because current injection into one of the synchronized neurons fails to elicit a response in the other neuron. Also, blocking the ability of neurons to generate action potentials with tetrodotoxin failed to inhibit this synchronicity. The astrocyte-evoked responses in the neuron tend to have a very slow time course, which is consistent with the fact that they are driven by the relatively slow spontaneous transients in the astrocytes and they also occur with a frequency that matches those observed in the astrocytes. The next question to consider is how the astrocyte Ca2+ transient can trigger these neuronal responses.
The increase in Ca2+ within the astrocytic process is thought to trigger the release of glutamate from the astrocyte (Module 7: Figure astrocyte tripartite synapse). The release of glutamate then acts on receptors on the neuronal synapse to activate neuronal activity. In the case of the synchronized Ca2+ transients just described in hippocampal neurons, the glutamate acts on extrasynaptic ionotropic N-methyl-D-aspartate (NMDA) receptors that gate Na+ and Ca2+ to provide sufficient inward current to depolarize the neurons to give the Ca2+ signals recorded in the experiment shown in Module 7: Figure astrocytic synchronization of hippocampal neurons.
The astrocyte–neuronal communication system indicates that the excitability of astrocytes can extend to regulation of neural activity. The astrocyte regulation of cerebral blood flow is another example of how astrocytes can regulate another cell type, in this case the smooth muscle cells.
Astrocytes are not excitable in the classical sense of being able to conduct action potentials as occurs in neurons and muscle. However, they have an element of excitability in that they are able to generate and conduct Ca2+ signals. This depends upon the role of the endoplasmic reticulum (ER) being an excitable system that can generate propagating Ca2+ waves. This property is similar to that described in neurons, where it is the basis of the neuron-within-a-neuron hypothesis. The ER is a continuous network that has release channels that have the Ca2+-induced Ca2+-release (CICR) mechanism to enable them to set up regenerative Ca2+ waves (Module 2: Figure Ca2+-induced Ca2+ release). Astrocytes appear to have a similar excitable system in that they can generate both intracellular and intercellular Ca2+ waves (Module 7: Figure astrocyte Ca2+ signalling). These waves appear to initiate at the tripartite synapses, from where they spread through the astrocyte and down to the endfoot that sits on the muscle. Since the astrocytes are linked together through gap junctions, such waves can also spread from one cell to the next to create a long-range communication system. Such long-range communication depends on the neighbouring cells being sufficiently excitable to detect and propagate the wave.
Astrocytes have different modes of Ca2+ signalling that depend on the intensity of the signals they receive from the neuronal–astrocyte communication pathway at the tripartite synapse. When neuronal signalling at the synapses is at a low level, there is little stimulation, and the resulting Ca2+ signal is restricted to local microdomains in the astrocytic processes in immediate contact with the active synapses. Such local Ca2+ microdomains have been observed in Bergmann glial cells following stimulation of the parallel fibres (Module 7: Figure Ca2+ microdomains in Bergmann glia). Since these astrocytic processes are at the end of fine branches, the increase in Ca2+ is effectively isolated within a small compartment. This is exactly analogous to the isolation of Ca2+ signals in the individual spines on the dendritic tree of neurons (see Module 10: Figure hippocampal input-specific Ca2+ signals). This is an example of how the astrocyte endings appear to be matched to the neighbouring synaptic region.
When the neuronal synapse is stimulated at higher intensities, the Ca2+ in the microdomains spreads out to the rest of the astrocyte and this usually travels as an intracellular Ca2+ wave (Module 7: Figure astrocyte Ca2+ signalling). It is this propagating wave that determines astrocyte excitability. The initiation and wave propagating mechanism is shown in Module 7: Figure astrocyte tripartite synapse. The initiation is triggered by the formation of inositol 1,4,5-trisphosphate (InsP3) during the operation of the neuronal–astrocyte communication pathway. The InsP3 then releases Ca2+, which can then excite neighbouring InsP3 receptors to set up the regenerative wave that spreads through the process of Ca2+-induced Ca2+ release (CICR). In order for the InsP3 receptors to participate in CICR, they must be excitable, and there are a number of factors that determine this excitability. One of these is the level of InsP3 and the other is the luminal level of Ca2+. The lumen must be charged up with Ca2+, and the level of this loading can regulate the excitability of the InsP3 receptors. This loading is very dependent on the entry of external Ca2+, and astrocytes have a number of entry channels, such as L-type Ca2+ channels and members of the transient receptor potential (TRP) ion channel family, that may function to load the intracellular store.
The intracellular waves are often restricted to single astrocytes, but there are numerous examples of intercellular Ca2+ waves (lower panel in Module 7: Figure astrocyte tripartite synapse). The Ca2+ spreads from one cell to the next to co-ordinate the activity of a large population of cells. The function of such intercellular waves is still not clear. However, they are most often observed during extremes of cell stimulation and have been linked with pathological states, such as spreading depression.
Another aspect of astrocyte excitability is the generation of astrocyte Ca2+ oscillations.
Astrocyte Ca2+ oscillations
One of the remarkable aspects of astrocytes is that they can generate spontaneous Ca2+ oscillations apparently independently of external stimulation (Module 7: Figure astrocyte Ca2+ oscillations). While there may be no obvious stimulation, it is important to stress that such oscillations usually depend upon the intracellular release channels such as the inositol 1,4,5-trisphosphate (InsP3) receptors being in an excitable state. Despite this reservation, what is evident is that these oscillations can occur in the absence of neural activity at the tripartite synapse because they can occur under conditions where neuronal action potentials are suppressed with tetrodotoxin. When groups of astrocytes are analysed simultaneously, it is evident that the individual cells oscillate independently of each other. When these oscillations were analysed in finer detail, it was discovered that they could be restricted to individual processes, thus providing further evidence that the individual astrocytic processes can function independently of each other.
The spontaneous oscillations generated by the astrocytes are of particular significance with regard to astrocyte–neuronal communication because they indicate that the astrocytes are able to initiate a signalling process to control neural activity.
Astrocyte regulation of cerebral blood flow
The vasculature tone of the cerebral arterioles, like those found elsewhere in the body, depends on the contractile state of smooth muscle cells (SMCs). SMC tone depends upon the cell being in a dynamic condition, poised between states of contraction and relaxation. There are indications that this dynamic state may be driven by an endogenous smooth muscle cell cytosolic oscillator that delivers the regular pulses of Ca2+ to maintain SMC tone (Module 7: Figure smooth muscle cell Ca2+ oscillations). This Ca2+ oscillator generates pulses of Ca2+ with a frequency of about 0.2 Hz. Such oscillations can be generated by either a membrane or a cytosolic oscillator (Module 6: Figure membrane and cytosolic oscillators). It has not been established which one of these oscillatory mechanisms is operating in these arteriole SMCs, but it is likely to be a cytosolic oscillator similar to that found in other vascular smooth muscle cells (Module 7: Figure SMC cytosolic oscillator). However, what is clear is that this Ca2+ oscillator is rapidly switched off when the Ca2+ wave invades the endfoot. It has been suggested that this Ca2+ activates the large-conductance (BK) channels to release K+, which then acts to depolarize the SMC, resulting in inhibition of the oscillator, a decrease in contraction and dilation of the vessel (Module 7: Figure astrocyte endfoot signalling). Some of the other mechanisms that have been proposed to explain how a Ca2+ signal in the endfoot causes the SMC to relax are based on the formation of lipid messengers. The idea is that the Ca2+ activates phospholipase A2 (PLA2) to hydrolyse phospholipids to release arachidonic acid (AA), which is then converted into agents such as 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2) (Module 1: Figure eicosanoids) that are known to influence muscle tone.
Although the signalling mechanisms operating between the endfoot and the SMCs remain to be fully established, what seems certain is that an astrocytic Ca2+ signal initiated by neural activity at the tripartite synapse spreads down to the endfoot to bring about a change in blood flow through the cerebral arterioles.
Satellite glial cells
The satellite glial cells, which surround the primary sensory neurons located in the sensory ganglia, have a very similar function to the astrocytes in the brain. For example they have a mechanism that resembles the spatial buffering of K+ in astrocytes. The inward rectifier K+ (Kir) channel family, particularly the Kir4.1 channel, contribute to both the entry and release of K+ that is a feature of this buffering of the extracellular concentration of K+ that controls neuronal excitability. A decrease in the expression of this Kir4.1 channel may contribute to the onset of neuropathic pain.
The microglia, which resemble macrophages, are phagocytic cells located specifically in the central and peripheral nervous systems. The microglia express many of the markers normally associated with macrophages such as colony-stimulating factor 1 receptor (CSF1R), CD11b, CD14 and EGF-like module-containing mucin-like hormone receptor-like-1 (EMR1). Their main function within the nervous system is to maintain the status quo by responding to adverse changes such as the death of neurons as occurs following ischaemia or due to localized breaches of the blood–brain barrier that allows entery of plasma components and pathogens (Module 7: Figure microglia interactions). They have two functional states. For most of their life they exist as star-shaped cells with numerous long processes that are moving constantly as they survey their local environment. This surveillance mode switches into an activation mode during injury to the nervous system or in response to stimuli received from the surrounding neurons. The activated microglia transform into amoeboid cells that can proliferate and migrate thereby producing a cohort of cells capable of releasing neurotrophic factors and pro-inflammatory mediators [chemokines, cytokines, reactive oxygen species (ROS) and nitric oxide (NO)]. Like macrophages, the microglia removes debris through phagocytosis. This phagocytotic response appears to be induced by the G protein-coupled receptor P2Y6 (Module 1: Table G protein-coupled receptors) that respond to the UDP produced by damaged and dying cells.
In addition to this sentinel role, microglia are tightly integrated into neuronal activity through a two-way communication network of neuronal–microglia interactions largely convened by chemokine signalling pathways.
A characteristic feature of microglia is their very large receptor repertoire and their capacity to express and release an enormous number of stimuli (Module 7: Figure microglia interactions):
The signalling mechanisms used by microglial cells have not been established in the sort of detail described for macrophages. Since both cell types have many receptors in common and seem to display similar inflammatory responses, it is likely that the microglia will have signalling pathways very similar to those used by macrophages (Module 11: Figure macrophage signalling). However, there is a major difference between these two cell types and that concerns the special relationship that exists between the microglia and neurons and this is explored further in the section on neuronal–microglia interactions.
A number of neurodegenerative diseases, such as Alzheimer's disease and Nasu-Hakola disease (NHD), have been linked to the inflammatory conditions created by excessive and prolonged activation of microglial cells.
There is a two-way communication operating between the neurons and the microglia (Steps 4–6 in Module 7: Figure microglia interactions). Much of the interaction between these two cells seems to be carried out through a chemokine signalling network (Module 7: Figure neuronal chemokine function). The basis of this network is that both neurons and microglia can release and respond to chemokines. It has been proposed that this network might be responsible for a long-term up-regulation of neuronal activity within the pain transmission pathway leading to the onset of neuropathic pain. One of the current ideas is that the excessive stimulation during the acute phase of injury or disease results in a heightened sensitization of the participating neurons that can persist for a long time. In response to peripheral nerve injury, for example, this sensitization reaches a peak after about a week and can then persist for many weeks before declining. The following hypothesis, based on the idea of phenotypic signalsome remodelling (Module 12: Figure signalsome remodelling), attempts to describe how this switch in neuronal sensitization may develop. The basis of the hypothesis is that excessive neuronal activation due to peripheral injury brings about a phenotypic remodelling of the neurons that become sufficiently sensitized that they begin to conduct painful stimuli long after the original painful stimuli have gone. This remodelling may occur through the following reciprocal interactions operating between the neurons and microglia (Module 7: Figure neuronal chemokine function):
Up-regulation of the chemokine signalling network as outlined above provides a plausible mechanism to describe both the onset and persistence of neuropathic pain.
Endothelial cells are flattened cells that line the inside of blood vessels and provide a variable barrier between the circulating blood and the interstitial space. In larger vessels, they are surrounded by smooth muscle cells, but in the finer capillaries, they often are the only cell type. Endothelial cells have a number of signalling pathways that regulate their numerous functions in vascular biology:
Endothelial regulation of paracellular permeability
The endothelial cells that line the inside of blood vessels provide a semi-permeable barrier that separates the plasma from the interstitial space (Module 7: Figure endothelial cell). This barrier is made up of two components. Firstly, there is an extracellular matrix (ECM) composed of a meshwork of different proteins (collagen IV, fibronectin, entactin, laminin, chondroitin sulphate, heparin sulphates, perlecan and syndecan). Endothelial cells are attached to this ECM through focal adhesion complexes based on integrin receptors. The second component of the barrier is the interendothelial junction (IEJ), which contains different junctional complexes. The tight junction is particularly important in restricting the movement of substances with molecular radii above about 3.0 nm. Ions and small molecules can pass through this paracellular pathway. However, anything above this radius, such as plasma proteins including albumin, are restricted and cross the endothelium through a transcellular pathway that is based on caveolae. Endothelial cells have a large number of caveolae, which pinch off at the luminal surface to form vesicles containing a cargo of proteins (endocytosis) that are then carried across to the basal surface. The vesicles fuse with the basal surface through an exocytotic process to release proteins to the interstitial space. Both the transcellular and paracellular pathways can be regulated. Considerable attention has focused on this paracellular pathway.
The paracellular pathway can be opened up by a contractile mechanism that displays some remarkable similarities to events in smooth muscle cells. This increase in endothelial permeability is particularly evident during inflammation, when inflammatory mediators such as thrombin stimulate the endothelial cells to contract and pull apart the cell junctions (Step 4 in Module 11: Figure inflammation). The contractile event is carried out by a cytoskeletal network of actin and non-muscle type II myosin filaments that are attached to cell junctions and to the cell adhesion complexes that face the ECM (Module 7: Figure endothelial cell). The myristoylated alanine-rich C-kinase substrate (MARCKS) protein may play a role in attaching the actin filaments to the membrane. Vascular endothelial growth factor (VEGF), which plays a critical role in inducing angiogenesis, can also increase vascular permeability by opening up the junctions between the endothelial cells. The signalling pathways that control permeability resemble those used to induce endothelial cell proliferation (Module 9: Figure VEGF-induced proliferation). The proteins that function to hold endothelial cells together are the targets for these signalling pathways and once phosphorylated the junctions weaken and the cells can be pulled apart.
At the tight junctions, occludin and claudin molecules function to fuse the two membranes at small patches to form the barrier. In addition, the C-terminal regions of occludin and claudin are attached to the zona occludens protein (ZO-1), which is linked to actin fibres through α-catenin and spectrin. Similarly, one of the classical cadherins (Module 6: Figure classical cadherin signalling) called vascular endothelial cadherin (VE-cadherin), which forms the adherens junction, is also linked to actin through β-catenin and p120 catenin. It seems likely that the actin filaments coming out from the tight junction and the adherens junction form an interconnected meshwork by interacting with the actin filaments coming from the integrin receptors. The VEGF receptors appear to be associated with the VE-cadherins and are thus positioned within the junction where they can control adhesive properties through phosphorylation of components such as β-catenin, p120 catenin, occluding and ZO-1.
It has been suggested that there is a dynamic tension within this network, with the adhesive properties of the junctional complexes resisting the forces within the cytoskeleton trying to pull the junctions apart. Under normal conditions, the resting tension is relatively small and the junctions can resist the separation forces within the cytoskeleton to maintain the semi-permeable barrier (Module 7: Figure regulation of paracellular permeability). However, in response to mediators such as thrombin, the cytoskeletal network contracts to pull the junctions apart, thus opening up the barrier. The ability of thrombin to stimulate contraction depends upon the combined action of both the Ca2+ and RhoA signalling mechanisms (Module 7: Figure endothelial cell contraction).
The control of contraction in endothelial cells is remarkably similar to that found in smooth muscle cells (Module 7: Figure smooth muscle cell E-C coupling). In both cases, contraction is activated by two separate signalling pathways, one controlled by Ca2+ and the other by the RhoA signalling mechanism (Module 2: Figure Rho signalling).
The endothelial cells that line blood vessels are constantly subjected to fluctuating haemodynamic forces resulting from the pulsatile flow of blood. Mechanosenstive channels, which remain to be properly characterized, respond to these shear stresses by opening to allow entry of external Ca2+. Ca2+ signals can also be generated by thrombin receptors that are coupled through the heterotrimeric protein Gq to phospholipase C (PLC) to generate inositol 1,4,5-trisphosphate (InsP3), which then releases Ca2+ from the endoplasmic reticulum (Module 7: Figure endothelial cell contraction). Endothelial cells also express a number of transient receptor potential (TRP) ion channel family members. The canonical TRP 1 (TRPC1) channel may function as a store-operated channel (SOC) in endothelial cells to provide the prolonged Ca2+ signal that has been observed during the action of thrombin. This increase in Ca2+ acts through calmodulin (CaM) to stimulate the CaM-dependent myosin light chain kinase (MLCK) to phosphorylate the myosin light chain (MLC) that then enables the non-muscle myosin II to interact with actin to induce contraction.
Endothelial contraction can also be activated by the RhoA signalling mechanism. Thrombin is also known to act through G12/13, which is one of the G protein subunits capable of activating RhoA (Module 2: Figure Rho signalling). The active RhoA/GTP complex has been implicated in a number of endothelial processes (Module 7: Figure endothelial cell contraction). It can stimulate the Rho kinase (ROCK) to phosphorylate the myosin phosphatase targeting subunit (MYPT1), which results in inhibition of protein phosphatase 1 (PP1). RhoA thus facilitates contraction by reducing the dephosphorylation of MLC. In addition, RhoA can increase actin polymerization and appears to act by converting cortical actin into the stress fibres responsible for contraction. Finally, there also are indications that the ability of RhoA to induce actin polymerization seems to enhance the entry of Ca2+ through the TRPC1 channels, perhaps by facilitating the mechanism of store-operated channel (SOC) activation (Module 3: Figure STIM-induced Ca2+ entry).
Skeletal muscle is responsible for locomotion and for moving various parts of the body, such as the diaphragm. Skeletal muscle structure is characterized by the high degree of organization of the contractile proteins actin and myosin that are arranged into striated myofibrils. The co-ordinated activation of these myofibrils is mostly under voluntary nervous control, as opposed to the involuntary control of smooth muscle cells. Control of contraction is regulated by motor neurons that release acetylcholine to initiate the process of excitation–contraction (E-C) coupling in skeletal muscle. In addition to this control over contraction, there also is a tight regulation of energy metabolism in skeletal muscle. A process of excitation–metabolism coupling in skeletal muscle ensures that the levels of ATP remain constant despite variations in demand. There are sophisticated feedback loops, such as the Ca2+-dependent breakdown of glycogen, that ensure that ATP levels remain relatively constant during normal contraction. During intense activity usually associated with stress, energy metabolism can be enhanced further through a process of adrenaline (epinephrine)-induced glycogenolysis. Storage of glycogen in skeletal muscle is regulated by insulin-induced glycogen synthesis.
Skeletal muscle structure
The basic contractile unit in skeletal muscle is the muscle fibre (Module 7: Figure skeletal muscle structure). This fibre is not a conventional cell, but is a syncytium formed by the fusion of myoblasts during the differentiation of skeletal muscle (Module 8: Figure skeletal muscle myogenesis). The individual muscle fibres are held together by connective tissue to form the compact muscles that connect to different parts of the skeleton. The individual fibres are often very long, and can stretch from one end of the muscle to the other, but usually the fibres are shorter than the muscle as a whole and are bound together by connective tissue septa. The contractile machinery, which makes up the bulk of the fibre, is arranged in the form of myofibrils that have characteristic striations due to the precise organization of the contractile proteins actin and myosin (Module 7: Figure skeletal muscle structure). The process of excitation–contraction (E-C) coupling in skeletal muscle functions to deliver the global Ca2+ signal that drives the interaction between these two contractile proteins (Module 7: Figure skeletal muscle E-C coupling).
Excitation–contraction (E-C) coupling in skeletal muscle
The process of excitation–contraction (E-C) coupling in skeletal muscle cells concerns the way in which an electrical signal in the sarcolemma is transduced into the Ca2+ signal that activates contraction. In addition to the control of contraction, there are signalling mechanisms to regulate energy metabolism. The way in which all these signalling events are co-ordinated to regulate skeletal muscle function are outlined in the following steps, as shown in Module 7: Figure skeletal muscle E-C coupling:
Excitation–metabolism coupling in skeletal muscle
The energy for muscle contraction comes from glucose, which is stored within the muscle in the form of glycogen. An important control of energy supply depends upon the rate at which glycogen is hydrolysed to glucose by the enzyme phosphorylase. Inactive phosphorylase b is converted into active phosphorylase a by the enzyme phosphorylase kinase, which is the focal point for two signalling systems. One operates through cyclic AMP, the other through Ca2+, as illustrated in Steps 6–8 in Module 7: Figure skeletal muscle E-C coupling:
Insulin control of skeletal muscle glycogen synthesis
Skeletal muscle is an important component of the metabolic energy network (Module 7: Figure metabolic energy network). It is able to take up glucose and synthesize glycogen through processes that are regulated by insulin through Steps 9–13, as shown in Module 7: Figure skeletal muscle E-C coupling:
The heart is an organ superbly designed to pump blood around the body with a remarkable degree of regularity and adaptability. The regularity is controlled by a pacemaker system located in the sinoatrial node pacemaker cell that generates the repetitive action potentials that travel through gap junctions to excite all the contractile cells to drive each heartbeat (Module 7: Figure heart summary). This communication through electrical signals is used to spread the action potentials from the sinoatrial node throughout the atrium, where it triggers atrial cell contraction. The action potential then invades the atrial–ventricular node that is coupled to the Purkinje fibres responsible for transmitting action potentials to the ventricles to stimulate ventricular cell contraction.
One of the remarkable properties of the heart is its ability to adapt to changing demands. In the short term, the modulation of ventricular Ca2+ signals in response to different hormones can account for both positive and negative inotropy. In the longer term, ventricular cells can adapt to persistent modulator signals by undergoing a process of phenotypic remodelling of the signalsome. For example, an increased load on the heart results in cardiac hypertrophy that can lead to congestive heart failure (CHF).
Sinoatrial node pacemaker cells
The rhythmical contraction of the heart is driven by the cardiac pacemaker located in the sinoatrial (SA) node. This SA node consists of a small group of specialized muscle cells that has an oscillatory mechanism, which consists of a membrane oscillator that is coupled to a cytosolic Ca2+ oscillator, that spontaneously generates repetitive action potentials (Module 7: Figure cardiac pacemaker). The electrical activity of these cells is characterized by a slow pacemaker depolarization of the membrane potential (Vm), which is responsible for triggering each cardiac action potential. The steepness of this pacemaker potential determines the frequency of the rhythm. The main pacemaker channel currents operating during the pacemaker depolarization can be divided into two groups depending on whether they are driven by the membrane oscillator or the cytosolic Ca2+ oscillator.
Pacemaker channel currents
The slow depolarization that occurs during the pacemaker phase (the green shaded area in Module 7: Figure cardiac pacemaker) depends upon a number of currents that are generated during the operation of two interacting oscillators: a membrane oscillator that is driven by various voltage-sensitive channels and a cytosolic Ca2+ oscillator that depends on Ca2+ release from the RYRs. The fact that these two oscillators act together, means that the pacemaker mechanism has some degree of redundancy.
In the case of the membrane oscillator, the primary pacemaker channel is If (f stands for funny) that is carried by Na+ that provides the inward current that slowly depolarizes the membrane. This If current is carried by the HCN4 member of the hyperpolarizing-activated cyclic nucleotide (HCN)-gated channels (Module 3: Figure HCN channels). The key properties of this channel is that it is activated by hyperpolarization, which means that If switches on when the action potential hyperpolarizes and then slowly decays throughout the rest of the pacemaker period (the green shaded area in Module 7: Figure cardiac pacemaker). The channels are somewhat non-selective, and since they tend to carry Na+ rather than K+, If generates an inward current that slowly depolarizes the membrane potential (Vm) towards the threshold for activating the next action potential.
In the case of the cytosolic Ca2+ oscillator, the INa/Ca current comes into play towards the end of the final stages of the pacemaker phase to speed up the onset of the action potential (Module 7: Figure cardiac pacemaker). Release of Ca2+ from the SR close to the sarcolemma plays a primary role in this pacemaker mechanism by controlling the onset of this INa/Ca current by activating the Na+/Ca2+ exchanger (NCX). Opening of the RYRs during the action potential contributes to the global elevation in Ca2+. During the interspike interval, the SERCA pumps replenish the store and there is a slow build-up of Ca2+ within the SR lumen and this may contribute to the pacemaker mechanism by sensitizing the RYRs to generate the periodic Ca2+ sparks. When pacemaker cells are imaged, a series of Ca2+ sparks is evident in the run up to the action potential (Module 7: Figure pacemaker Ca2+ sparks). These sparks occur immediately below the membrane, where there is close apposition between the sarcoplasmic reticulum (SR) and the sarcolemma (Module 7: Figure SA node junctional zone). The appearance of these sparks appears to be facilitated by the activity of the CaV3 family of T-type Ca2+ channels. As If depolarizes the membrane, the T-type channels begin to open and the Ca2+ that flows into the cell will help to load the store and may also activate the ryanodine receptor 2 (RYR2) channels in the SR to create localized Ca2+ sparks (Module 7: Figure pacemaker Ca2+ sparks). Some of the Ca2+ from these sparks is extruded from the cell via the Na+/Ca2+ exchanger (NCX). As three Na+ ions enter for each Ca2+ that is extruded, the INa/Ca is a net inward current that acts to further depolarize the membrane and to activate more T-type channels. The T-type channel, the RYR2 and the NCX thus function as a feedback amplifier that comes in to play towards the end of the pacemaker phase to initiate the onset of the action potential (Module 7: Figure cardiac pacemaker).
In summary, the activation of INa/Ca amplifies the unitary T-type currents, thereby prolonging the inward current flow. It is estimated that only a few sparks are necessary to enhance the pacemaker towards the threshold for the onset of the action potential. This is a feedback amplifier based on the release of internal Ca2+ that is coupled to the plasma membrane pacemaker mechanism.
The frequency of the oscillator is regulated by a signalling system that can be increased by adrenaline or reduced by acetylcholine both of which act through the cyclic AMP signalling pathway. Adrenaline acts through β-adrenoceptors and GS to stimulate type 1 adenylyl cyclase (AC1) (Module 2: Table adenylyl cyclases). AC1 is one of the isoforms that is activated by Ca2+. The increase in cyclic AMP accelerates the oscillator in two ways. Firstly, cyclic AMP can increase membrane depolarization by binding to the cyclic nucleotide-binding domain (CNBD) on the C-terminal tail to stimulate the hyperpolarizing-activated cyclic nucleotide (HCN)-gated channels (Module 3: Figure HCN channels) responsible for If. Secondly, cyclic AMP stimulates the phosphorylation of phospholamban (PLB) to accelerate the SERCA pump to enhance the loading of the SR with Ca2+ to initiate the Ca2+ sparks.
Acetylcholine acting through Gi reduces the frequency through two mechanisms. First, Gαi inhibits AC1 to reduce the level of cyclic AMP thereby reducing the activation of HCN4 channels responsible for If. Secondly, the βγ subunit activates the GIRK channel to hyperpolarize the membrane.
Action potential currents
The sinoatrial (SA) node action potential is driven by two main currents, the ICa,L that flows through the CaV1.2 L-type channel and IK that flows through K+ channels (blue traces in Module 7: Figure cardiac pacemaker). When the pacemaker depolarization reaches the activation threshold for the L-type channel, the latter opens and the inward ICa,L rapidly depolarizes the membrane to give the rapid upstroke of the action potential. The rapid depolarization then switches on the K+ channel, and this strong outward current (IK) rapidly curtails the action potential by hyperpolarizing the membrane. It is this strong hyperpolarization that then activates If to begin the next pacemaker phase, as described earlier.
The large striated muscle cells that make up the ventricles are collectively responsible for generating the force that pumps blood out of the heart. This pumping action depends on the co-ordinated activation of all the ventricular cells. This co-ordination is achieved by the action potential that sweeps through all the cells very quickly to ensure that they all contract in unison. In addition to co-ordinating the activity of cells throughout the ventricle, the action potential also serves to synchronize the large number of contractile units within each cell. This internal synchronization is achieved through the highly ordered ventricular and atrial cell structure that ensures that the 20000 individual dyadic junctions (the signalling units that generate Ca2+ signals) are located in register with the contractile units. The dyadic junctions are responsible for the process of excitation–contraction (E-C) coupling during which the electrical signal in the plasma membrane triggers ventricular cell Ca2+ release from the sarcoplasmic reticulum. One of the remarkable properties of the ventricular signalling system is its ability to adapt to changing demands. In the short term, the modulation of ventricular Ca2+ signals in response to different hormones can account for both positive and negative inotropy. In the longer term, ventricular cells can adapt to persistent modulator signals by undergoing a process of phenotypic remodelling of the signalsome. For example, an increased persistent load on the heart results in cardiac hypertrophy that can lead to congestive heart failure (CHF).
Ventricular and atrial cell structure
The two major contractile cells in the heart (ventricular and atrial cells) differ markedly both in their structure and in the way that electrical stimulation induces the release of Ca2+ by the type 2 ryanodine receptors (RYR2s) on the sarcoplasmic reticulum (SR). This difference results from the way the SR is organized relative to the plasma membrane (Module 7: Figure ventricle and atrial cell organization). In ventricular cells, the plasma membrane has tubular invaginations (T-tubules) that make regular contacts with specialized regions of the SR at the junctional zones (Module 7: Figure ventricle cell). Atrial cells lack T-tubules, and the junctional SR makes contact with the sarcolemma at the cell surface, and the rest forms the non-junctional SR that projects into the cell interior (Module 7: Figure ventricle and atrial cell organization). The junctional SR and the non-junctional regions are separated by a gap that is populated by mitochondria, which may function as a firewall to restrict Ca2+ released by the junctional SR from diffusing in to excite the RYR2s on the internal non-junctional SR. This mitochondrial firewall may feature significantly in the modulation of atrial Ca2+ signals. The difference in the structural organization between these two cell types is clearly apparent in sections that have been stained with an antibody against RYR2 (Module 7: Figure RYR2 location in cardiac cells).
The critical region of the ventricular cell with regard to excitation–contraction (E-C) coupling is the junctional zone where the ryanodine receptors 2 (RYR2s) on the junctional SR face the T-tubule (Module 7: Figure ventricular junctional zone). Note how the junctional zone makes close contact with the T-tubule. A critical component of the E-C coupling mechanism is the relationship between the Ca2+ channels on the two membranes (Module 7: Figure junctional zone Ca2+ channels). There are approximately 10000 junctional zones in each ventricular cell. The T-tubule region that faces each junctional zone contains about ten CaV1.2 L-type channels, which provide the trigger Ca2+ responsible for activating the RYR2s. There are about 100 RYRs located in each junctional zone arranged in a pseudo-crystalline array. These L-type Ca2+ channels and RYRs are critical components of ventricular cell Ca2+ release. This release of Ca2+ is activated by the ventricular cell action potential, which is generated by the sinoatrial node pacemaker cells.
Ventricular cell action potential
The ventricular cell action potential depends on the sequential activation and inactivation of a number of inward and outward currents (Module 7: Figure ventricular action potential). The membrane potential passes through a series of distinct phases during the course of the action potential: 1. resting potential; 2. rapid depolarization; 3. brief notch; 4. prolonged plateau; 5. repolarization back to the resting potential. The inward and outward currents responsible for these different phases are generated by a number of ion channels:
Ventricular cell Ca2+ release
The action potential generated by the sinoatrial node pacemaker cells sweeps through the heart to initiate the process of excitation–contraction (E-C) coupling in the ventricular cells (Module 7: Figure heart summary). The function of the heart is dominated by Ca2+, which not only drives contraction during each heartbeat, but also plays a role in stimulating gene transcription. The latter is particularly important in cardiac hypertrophy, where alterations in Ca2+ signals can lead to congestive heart failure. The following overview of ventricular Ca2+ signalling will include information on how Ca2+ signalling changes in the diseased heart. Ventricular cells have a complex signalsome, and the control of ventricular Ca2+ signalling is summarized in the following sequence of steps as outlined in Module 7: Figure ventricular Ca2+ signalling:
The sequences of events outlined above provide an overview of Ca2+ signalling in the ventricular cell. To better understand how Ca2+ controls contraction it is necessary to consider in more detail the ventricular cell excitation–contraction (E-C) coupling process at a single junctional complex.
Ventricular cell excitation–contraction (E-C) coupling
The key event in ventricular cell excitation–contraction (E-C) coupling is the link between Ca2+ entry across the sarcolemma and the release of Ca2+ from the sarcoplasmic reticulum (SR) (Steps 1 and 2 in Module 7: Figure ventricular Ca2+ signalling). This process is critically dependent on the structural organization of the individual diadic junctions (Module 7: Figure junctional zone Ca2+ channels). Each junctional zone is an autonomous signalling unit that depends upon a close association of approximately ten CaV1.2 L-type channels in the T-tubule and about 100 type 2 ryanodine receptors (RYR2s) in the junctional SR membrane. The integrity of this diadic junction seems to be maintained by junctophilin 2 (JP2). The way in which these two channels communicate with each other during E-C coupling is shown in Module 7: Figure ventricular cell E-C-coupling.
As the action potential invades the T-tubule, it depolarizes the sarcolemma, which then activates the L-type channels to allow Ca2+ to enter the cell to form a sparklet. Rapid imaging studies have been able to visualize a sparklet at one of these junctional zones (Module 3: Figure Ca2+ sparklet). At each of the 10 000 junctional zones, the individual sparklets contribute a flow of charge that sums to produce a measurable electrical current (ICa) (Module 7: Figure Ca2+ cardiac kinetics). Notice how this inward Ca2+ current precedes the Ca2+ transient because it is this entry of Ca2+ that is responsible for activating the RYR2s to release Ca2+ from the junctional SR, which is the basis of E-C coupling. It is estimated that each sparklet will activate about ten of the 100 available RYR2s in each junctional zone to produce a spark, which then diffuses out to activate myofibrils in the immediate vicinity to complete the process of E-C coupling.
The next problem to consider is how these individual sparks are recruited to provide the global Ca2+ signal that activates contraction. There are approximately 10 000 junctional zones in each ventricular cell. In order to get a fast contraction, it is necessary to activate sparks in all of them as synchronously as possible, and this is achieved through a process of electrical recruitment. The action potential provides the rapid synchronization signal to trigger all these autonomous units simultaneously (Module 7: Figure ventricular and atrial cell kinetics). A critical aspect of this E-C mechanism is that each of the junctional zones is an autonomous unit that produces its spark independently of its neighbours. Evidence for this autonomy can be seen in line scans of ventricular cells activated by increasing levels of depolarization (Module 7: Figure autonomous Ca2+ sparks). At low membrane depolarizations, when relatively few junctional complexes are being recruited, the individual sparks are clearly evident. This is an example of how the SR can be divided into modular Ca2+ signalling units that produce local Ca2+ signals that can then be recruited to provide the rapid global Ca2+ signal necessary to contract a large muscle cell.
A critical question in cardiac physiology concerns the inactivation of ventricular RYR2s. What causes the channels to suddenly close once they have delivered sufficient Ca2+ to activate a contraction? A related question arises when one considers the sequence of events that occur in a junctional zone during the formation of a spark. An obvious question is why only about 10% of the available RYR2s contribute to a spark. Why do the majority of RYRs remain quiescent even though they must be experiencing a high level of Ca2+ as the spark spreads out of the junctional zone? It seems that rapid inactivation of ventricular type 2 ryanodine receptors (RYR2s) may be caused by depletion of Ca2+ within the lumen of the SR.
Inactivation of ventricular type 2 ryanodine receptors (RYR2s)
During the course of a ventricular Ca2+ transient, the type 2 ryanodine receptors (RYR2s) open for a brief period, then they inactivate, and the recovery process begins. The nature of this inactivation mechanism is also relevant to the question of why only a small proportion of the available RYR2s are activated during each spark. It has been estimated that each junctional zone contains about 100 RYR2s packed together in the sarcoplasmic reticulum (SR) membrane (Module 7: Figure junctional zone Ca2+ channels). Only about 10% of these channels seem to be employed to produce the individual Ca2+ sparks at each junctional zone. It is not clear why the majority of the RYR2s remain quiescent. The luminal regulation of Ca2+ release channels may provide an explanation for how the RYR2s might be rapidly inactivated. The idea is that the Ca2+ spark inactivates the RYR2s by causing a rapid depletion of Ca2+ within the lumen of the junctional zone (Module 7: Figure ventricular cell Ca2+ blink). When the Ca2+ sparklet activates the RYR2s to produce a spark, the amount of Ca2+ leaving the lumen of SR is so large that it causes a temporary depletion, and this has been called a blink. The effect of this blink is to inactivate the RYR2s because the sensitivity of these channels is very dependent on the level of Ca2+ within the internal store. If indicators are used to simultaneously monitor Ca2+ in the cytoplasm and within the lumen of the SR, it is possible to visualize the appearance of a spark and the simultaneous appearance of a blink (Module 7: Figure sparks and blinks).
Modulation of ventricular Ca2+ signals
A remarkable feature of cardiac function is the way that contractility can be modulated rapidly to change the force of contraction. The sympathetic nervous system controls the positive inotropic response, which is an increase in the force of contraction that is activated through the β-adrenergic receptors acting through the cyclic AMP signalling pathway (Step 7 in Module 7: Figure ventricular Ca2+ signalling). The β-adrenergic receptors are located on the T-tubules, where they appear to produce two cyclic AMP microdomains (Module 6: Figure ventricular cyclic AMP microdomains). These microdomains are responsible for phosphorylating some of the key components responsible for regulating both the generation and action of the Ca2+ transients that control each heartbeat (Module 7: Figure ventricular cell E-C coupling):
These three effects of cyclic AMP on the L-type entry channels, the SERCA pump and the RYR2s all contribute to the positive inotropic effect by increasing the amount of Ca2+ that is released during each beat.
The striated atrial cells responsible for contracting the atrial chambers of the heart have a superficial resemblance to the ventricular cell, but there are a number of marked differences. The first obvious difference concerns atrial cell structure in that there are no T-tubules. In ventricular cells, the T-tubules are used to carry the electrical signal deep into the cell to trigger a rapid synchronous release of Ca2+. In atrial cells, the electrical signal activates a Ca2+ signal that is normally confined to the cell surface, but under some circumstances it can be amplified by a process of Ca2+-induced Ca2+ release (CICR), which is much slower because it spreads through a Ca2+ wave. The mechanism of atrial cell Ca2+ release is thus very different from that in ventricular cells. The modulation of atrial Ca2+ signals depends on whether or not the sparks at the cell surface can activate the global Ca2+ wave.
Atrial cell structure
Atrial cells have a long elongated structure and are somewhat smaller than the ventricular cells (Module 7: Figure RYR2 location in cardiac cells). The type 2 ryanodine receptors (RYR2s) have two distinct locations: they lie along the surface sarcolemma and they also form parallel strands at right angles to the surface. This arrangement is shown diagrammatically in Module 7: Figure ventricle and atrial cell organization. As there are no T-tubules, like those in the ventricular cells, the junctional zones are restricted to the sarcolemma at the cell surface, and it is here where the process of atrial cell Ca2+ release begins. There also are non-junctional sarcoplasmic reticulum (SR) sheets that spread perpendicularly into the cell, and these are lined with RYR2s that can amplify the initial signal at the cell surface. Whether or not these non-junctional RYR2s are brought into play seems to depend upon a mitochondrial firewall located between the junctional and non-junctional SR.
Atrial cells also express type 2 inositol 1,4,5-trisphosphate receptors (InsP3R2), which have very specific locations. One group is located together with the RYR2s in the junctional zone, and the other group is clustered around the nucleus. The former have an important role in mediating the positive inotropic response to stimuli such as endothelin-1 during the modulation of atrial Ca2+ signals.
Atrial cell Ca2+ release
Atrial cells use two distinct mechanisms of Ca2+ release. The junctional zones on the sarcolemma generate Ca2+ sparks using a mechanism similar to that described for ventricular cells (Module 7: Figure ventricular and atrial cell kinetics). L-type Ca2+ channels in the sarcolemma produce a sparklet that then triggers a spark in the junctional zone at the cell surface. These sparks at the surface then activate the second mechanism of Ca2+-induced Ca2+ release (CICR) that spreads the Ca2+ signal into the cell. The gradual development of this atrial Ca2+ signal can be visualized using fluorescent imaging, which clearly reveals how the signal originates at the periphery and then spreads into the cells (Module 7: Figure atrial cell Ca2+ signalling). Globalization is achieved by the regenerative process of CICR that forms a wave to activate contraction as it spreads into the cell from the plasma membrane (Module 7: Figure ventricular and atrial cell kinetics). This mode of diffusional recruitment in atrial cells is much slower than the electrical recruitment mechanism found in ventricular cells. It also is the basis for the modulation of atrial Ca2+ signals that depend upon stimuli such as β-adrenergic agents and endothelin-1 being able to couple the sparks at the cell surface with the release of Ca2+ from the non-junctional sarcoplasmic reticulum (SR).
Modulation of atrial Ca2+ signals
The modulation of Ca2+ signals in atrial cells seems to depend upon the coupling of cell-surface Ca2+ sparks to the release of Ca2+ from the non-junctional sarcoplasmic reticulum (SR) (Module 7: Figure atrial Ca2+ domains). In response to membrane depolarization, the L-type channels in the junctional zone introduce a small pulse of Ca2+ that then triggers Ca2+ release from the type 2 ryanodine receptors (RYR2s) to create a Ca2+ spark. Under normal conditions, this spark fails to breach the mitochondrial firewall because the Ca2+ is rapidly removed by uptake into the mitochondria and by uptake into the SR by the sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA) pumps. Not enough Ca2+ gets across the firewall to activate the internal RYR2s. This small signal spatially localized to the cell periphery accounts for why the normal atrial contractions are very weak. However, stronger contractions are induced following stimulation of the atrial cells by either β-adrenergic agonist or by endothelin (Module 7: Figure atrial arrhythmias). These two stimuli induce a large positive inotropic response in two ways. Firstly, they enable the cell-surface sparks to breach the mitochondrial firewall so as to stimulate the non-junctional RYR2s to ignite the Ca2+ wave that produces the global signal responsible for the stronger contractions. Secondly, they enhance the sensitivity of the RYR2s so that they can respond to the signal coming through the firewall.
The action of β-adrenergic agonists such as isoproterenol (isoprenaline) seems to depend on the same mechanisms that have been described for the modulation of ventricular Ca2+ signals. By activating the L-type channels and the SERCA pumps, cyclic AMP increases the load of Ca2+ within the SR, and this is responsible for creating the larger Ca2+ sparks to breach the mitochondrial firewall and for sensitizing the RYR2s to respond to this signal.
The ability of endothelin-1, which acts through the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette, is still not fully understood. InsP3 receptors in the junctional zone (Module 7: Figure ventricle and atrial cell organization) are appropriately positioned to contribute to the initial Ca2+ spike. Since the InsP3 receptors are sensitive to both InsP3 and Ca2+, they may make little contribution to the spark under normal conditions. However, when endothelin-1 increases the level of InsP3, the InsP3 receptors will be brought into play and may augment the Ca2+ spark sufficiently to break through the firewall to recruit the RYR2s on the non-junctional SR. This enhancement of Ca2+ signalling by endothelin often leads to the development of atrial arrhythmias (Module 7: Figure InsP3-induced arrhythmias).
Atrial arrhythmias are the most common form of long-lasting arrhythmias that carry a high risk of stroke. However, little is known about the cause of these arrhythmias. Recently, it has been shown that the type 2 inositol 1,4,5-trisphosphate receptors (InsP3R2s), which are strongly expressed in atrial cells, may play a role in initiating this arrhythmic activity. If atrial myocytes are stimulated with endothelin-1, which acts to increase the level of InsP3, there is a gradual increase in extra Ca2+ transients in the intervals between the depolarization-induced transients (see traces c in Module 7: Figure atrial arrhythmias). These arrhythmias seem to be caused by InsP3, because they can be triggered simply by adding a membrane-permeant InsP3 ester (Module 7: Figure InsP3-induced arrhythmias). What is remarkable about this action of InsP3 is that some of the transients it induces are as large as those produced by the membrane depolarization that was applied at each of the arrowheads in this figure. Just how InsP3 acts to trigger these extra transients is still unclear.
The fact that the InsP3 receptors are located in the atrial cell junctional zone intermingling with the type 2 ryanodine receptors (RYR2s) means that they are in the right place to have a role in excitation–contraction (E-C) coupling (Module 7: Figure ventricle and atrial cell organization). A likely scenario therefore is that high levels of InsP3 in the region of the junctional zone can induce sufficient Ca2+ release to trigger the RYR2s to produce a Ca2+ spark. This spark will then act to open non-specific cation channels to depolarize the sarcolemma sufficiently to initiate an action potential to produce the large Ca2+ transients. This action of InsP3 thus constitutes an alternative mechanism that comes into play during pathological conditions to produce atrial arrhythmias.
Smooth muscle cells
Smooth muscle cells are a heterogeneous collection of cell types that function in many different organs, where they regulate a variety of functions such as vascular smooth muscle control of blood vessel tone, emptying of the bladder, tonic contractions of sphincters such as the urethra and the rhythmical contraction of the gastrointestinal tract, uterus and ureter. In order to carry out these multiple functions, a number of different signalling mechanisms have evolved to control smooth muscle contractility. There is a certain degree of uniformity with regard to smooth muscle cell structure and there also are some general mechanisms that are responsible for smooth muscle cell excitation–contraction coupling. In addition to this excitatory mechanism, there also are signalling pathways responsible for smooth muscle cell relaxation. However, when it comes to the smooth muscle activation mechanisms, each muscle has to be considered separately especially with regard to the way in which excitatory stimuli bring about the increase in Ca2+ necessary to trigger contraction. Most smooth muscle cells are excited by membrane depolarization that then activates VOCs to provide the Ca2+ signal to induce contraction. For many of these muscles, this depolarization is induced by the interstitial cells of Cajal that have a pacemaker function resembling that of the sino-atrial node in the heart. Other smooth muscles have an endogenous pacemaker mechanism while others are activated directly by neurotransmitters released by nerves.
Smooth muscle cell structure
Smooth muscle cells have a spindle shape, and are usually lined up alongside each other and are connected through gap junctions (Module 7: Figure smooth muscle cell structure). They are about 100 μm long and 5–10 μm wide. The cell surface has numerous caveolae and many of these make close contact with the sarcoplasmic reticulum (Module 6: Figure smooth muscle caveolae). By comparison with skeletal and cardiac muscle, the contractile system composed of actin and myosin is somewhat disorganized, with actin filaments radiating away from localized densities dotted around the plasma membrane. Smooth muscle cell contraction also differs from that found in skeletal and cardiac muscle in that there are two different mechanisms for regulating the contractile processes (Module 7: Figure smooth muscle cell E-C coupling):
Smooth muscle cell excitation–contraction coupling
Excitation–contraction coupling refers to the mechanism whereby the smooth muscle cell contracts in response to excitatory stimuli. Most smooth muscle activation mechanisms result in depolarization of the membrane that then initiates the smooth muscle Ca2+ signalling cascades that lead to contraction. In addition, there is a smooth muscle Rho/Rho kinase signalling pathway that bring about contraction.
Smooth muscle Ca2+ signalling
Like other muscle cells, smooth muscle cells use actomyosin to generate force. However, the way in which the interaction between actin and myosin is controlled in smooth muscle is very different to that in other muscles. The sensor is not troponin C (TnC), as it is in skeletal and cardiac muscle, but is calmodulin (CaM) that responds to Ca2+ by stimulating myosin light chain kinase (MLCK) to phosphorylate the myosin light chains (MLCs) enabling myosin and actin to interact and drive contraction (Module 7: Figure smooth muscle cell E-C coupling).
The way in which smooth muscle cells produce their Ca2+ signals varies between the different cell types (Module 7: Figure smooth muscle cell E-C coupling):
A feature of the Ca2+ signalling system in smooth muscle cells is that there is considerable variation with regard to the spatial and temporal properties of their Ca2+ signals. In some cells, there are global signals where Ca2+ rises uniformly throughout the cell. In other cell types, the signal appears as waves that sweep regularly through the cell to drive a corresponding wave of contraction. Finally, there are highly localized elementary events that resemble the Ca2+ sparks found in cardiac cells. These smooth muscle cell Ca2+ sparks are one of the mechanisms used to control smooth muscle relaxation, since they can act on cell-surface large-conductance (BK) channels to hyperpolarize the membrane, thereby reducing the entry of Ca2+ through the VOCs.
Smooth muscle cell relaxation occurs when these Ca2+ signalling mechanisms are inhibited either by the cyclic GMP signalling pathway, as outlined in the section on nitric oxide (NO)/cyclic GMP and smooth muscle relaxation, or through the action of smooth muscle cell Ca2+ sparks.
Smooth muscle Rho/Rho kinase signalling
In addition to the direct Ca2+-dependent control of contraction, smooth muscle cells can also regulate contraction through a Rho signalling mechanism, as illustrated in Module 2: Figure Rho signalling. In the case of smooth muscle cells, activation of various receptors can contribute to the contraction through Steps 8 and 9 in Module 7: Figure smooth muscle cell E-C coupling. Like the Ca2+ signalling pathway, this Rho/Rho kinase signalling pathway is also directed towards the phosphorylation status of the myosin light chain (MLC). However, instead of acting on the myosin light chain kinase (MLCK), it acts to inhibit the protein phosphatase 1 (PP1) that dephosphorylates MLC (Module 7: Figure smooth muscle cell E-C coupling).
Smooth muscle cell relaxation
Many smooth muscle cells maintain a tone whereby they are poised between states of contraction and relaxation. To understand this tone, it is necessary to understand how the contractile mechanisms described above are balanced by processes that cause relaxation. There are two main mechanisms for controlling smooth muscle relaxation:
Nitric oxide (NO)/cyclic GMP and smooth muscle relaxation
Smooth muscle tone depends upon a balance between the contractile mechanisms and the mechanisms that promote relaxation. The nitric oxide (NO)/cyclic GMP signalling pathway plays an important role in controlling relaxation by acting at a number of sites, and particularly those concerned with Ca2+ signalling (Module 7: Figure smooth muscle cell cyclic GMP signalling):
The ability of cyclic GMP to relax smooth muscle has been exploited to develop the drug Viagra, which is used in the treatment of male erectile dysfunction.
A decrease in the operation of this cyclic GMP-dependent relaxation of smooth muscle might be one of the causes of hypertension.
Viagra (sildenafil) is the drug used in the treatment of male erectile dysfunction. It acts to enhance the level of cyclic GMP by inhibiting the cyclic GMP-specific phosphodiesterase PDE5, which hydrolyses cyclic GMP in smooth muscle cells (Module 7: Figure smooth muscle cell cyclic GMP signalling). By stimulating an increase in the level of cyclic GMP, Viagra will enhance the nitric oxide (NO)/cyclic GMP and smooth muscle relaxation mechanisms.
Rho-mediated Ca2+-sensitization contributes to the flaccid state of the corpus cavenosum, and this can be reversed by a Rho kinase inhibitor (Y-27632), which might be suitable for therapy of erectile dysfunction.
Smooth muscle cell Ca2+ sparks
Smooth muscle cell relaxation can also be activated by Ca2+, which is somewhat counterintuitive because an increase in Ca2+ is usually associated with contraction. An explanation of how Ca2+ can control both contraction and relaxation can be found in the spatial organization of smooth muscle Ca2+ signalling. When the cell is activated to give global Ca2+ signals through the entry and release of internal Ca2+, then contraction occurs (Module 7: Figure smooth muscle cell spark). Smooth muscle cells also produce Ca2+ sparks that act locally to switch on either Cl− or K+ channels. The relationship between sparks and the activation of a K+ current carried by large-conductance (BK) channels is shown in Module 3: Figure smooth muscle cell Ca2+ sparks. If sparks occur in the vicinity of Cl− channels, they will depolarize the membrane, resulting in global Ca2+ signals and contraction. However, if the sparks activate large-conductance (BK) channels, the resulting hyperpolarization prevents Ca2+ entry through the L-type channels and the muscle relaxes.
Smooth muscle activation mechanisms
The activation mechanisms responsible for generating the Ca2+ signal that controls contraction varies considerably between different smooth muscle cell (SMC) types. Smooth muscle Ca2+ signalling plays a major role in the process of smooth muscle cell excitation–contraction coupling (Module 7: Figure smooth muscle cell EC coupling). The different cell types put in place a Ca2+ activation mechanism that suits their particular function. Those muscles such as the bladder and vas deferens, which have to produce rapid phasic contractions at a particular point in time, have activation mechanisms that respond quickly to nerve stimulation. In other smooth muscles, which have to maintain a particular tone over an extended period of time, are more dependent on pacemaker mechanisms that generate regular pulses of Ca2+.
Some muscles increase Ca2+ by opening of VOCs whereas others have a cytosolic oscillator that generates the Ca2+ pulses to drive the contractions that maintain smooth muscle tone. There are three main smooth muscle activation mechanisms (Module 7: Figure SMC activation mechanisms):
These different smooth muscle activation mechanisms are illustrated by describing some specific examples from the many different smooth muscle and pacemaker cell types listed below:
The vas deferens functions to propel spermatozoa into the ejaculate. The peristaltic contractions of the smooth muscle cells that surround the vas deferens are activated by sympathetic neurons (Module 7: Figure urinogenital tract). These neurons have numerous varicosities that release ATP and noradrenaline (NA) that activate contraction. These transmitters act through mechanism A in Module 7: Figure SMC activation mechanisms. Unlike some of the other smooth muscle cells that use this mechanism, such as the bladder and uterus, the vas deferens shows little evidence of the pacemaker activity characteristic of muscles that have to maintain contractile activity over a protracted period. In the case of the vas deferens, contractile activity has to be switched on rapidly to induce sperm transfer during the brief period of ejaculation. This rapid activation is achieved by the transmitters released from the sympathetic neurons that form an extensive network throughout the vas deferens with numerous varicosities that make close contact with each muscle cell. The signalling pathways activated by ATP and NA are illustrated in Module 7: Figure vas deferens activation:
Alterations in the signalling pathways that control vas deferens contraction and particularly the α1-adrenoreceptor signalling pathway may be one of the causes of ejaculatory dysfunction.
Uterine smooth muscle cells
The uterus is made up of three parts: the body that makes up the major part, a domed-shape fundus at the top and the cervix at the bottom (Module 7: Figure urinogenital tract). Most of the uterus is composed of the myometrium, which is a thick layer of longitudinal and circular smooth muscle cells. Within each layer, the smooth muscle cells are arranged in individual bundles (fasciculus) that lie parallel to each other and are connected through gap junctions.
During early pregnancy, the uterus is relatively quiescent. There are a few weak twitches resulting from unsynchronized smooth muscle cell contractions. With the onset of labour, the uterus begins to develop stronger and more frequent contractions that appear first in the fundus and then spread rapidly through the body of the uterus. This remarkable switch from quiescence to a cycle of strong contractions is an example of the phenotypic remodelling of the signalsome, which in this case is the endogenous uterine smooth muscle activation mechanism. For example, there is an increase in gap junctional coupling just before labour. There is an increase in the expression of PDE4B, which decreases the effect of tocolytic agents such as β-adrenergic agents that use cyclic AMP to inhibit uterine contractions. There is an increase in the expression of the oxytocin receptors that act by inducing and accelerating contractions. The expression of the ryanodine receptor 2 (RYR2) is up-regulated during pregnancy and this may facilitate the process of excitation–contraction coupling. A decline in the activity of the Na+-K+-ATPase pump during labour may accelerate the uterus smooth muscle cell membrane oscillator and thus contribute to the increase in the frequency of contractions. There may also be a decrease in the activity of potassium channels such as TREK-1 and the large-conductance (BK) channels that contributes to the quiescence of the uterus during pregnancy. The next aspect to consider is how this remodelled signalsome switches the uterus out of its quiescent state such that it begins to generate the repetitive electrical impulses responsible for driving contractions during labour.
The uterine contractions that occur during labour have a periodicity of approximately 3–5 min (see Module 7: human uterine contractions and Module 7: Figure Ni2+ slows uterine oscillations). They have a characteristic shape; there is a slow rising phase (15 s), a plateau that lasts for about 30 s that is followed by a gradual relaxation phase that is roughly symmetrical with the rising phase. Each of these protracted uterine contractions depends upon the co-ordinated activation of most of the smooth muscle cells and this synchronicity is achieved by an action potential spreading through the smooth muscle cell layer with a conduction velocity of approximately 4 cm/s. The wave of excitation appears to begin at the fundus and then spreads through the body of the uterus towards the cervix. Even though all smooth muscle cells in the uterus are capable of generating a rhythm, those in the fundus are inherently more rhythmical and thus initiate each contraction wave.
Since there appear to be no interstitial cells of Cajal and there is little neural innervation, the uterine myometrium is driven by an endogenous pacemaker mechanism (see A in Module 7: Figure SMC activation mechanisms). Some of the features of this pacemaker mechanism are illustrated in Module 7: human uterine contractions. The regular rhythm is accelerated reversibly by oxytocin. Perhaps the most interesting feature of the rhythm is that it continues and is accelerated by the drug cyclopiazonic acid (CPA) that blocks the sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA) pump and thus empties the internal store of Ca2+. This observation rules out the presence of a cytosolic oscillator and suggests that the pacemaker is driven by a membrane oscillator. The activation mechanism thus has two main components (Module 7: Figure uterus activation). A membrane oscillator (horizontal box), which generates a pacemaker depolarization (ΔV), and L-type VOCs that respond to depolarization with a fairly typical smooth muscle cell excitation–contraction coupling mechanism (vertical green box). The primary signal for triggering contraction is the Ca2+ that enters the cell through the L-type VOC. There appears to be little role for release of Ca2+ from the internal stores since contractions continue when these stores have been depleted. However, there is an increased expression of RYR2s during the end of pregnancy and these may open in response to the entry of external Ca2+ to provide an additional boost of Ca2+ to increase the force of contraction (Module 7: Figure uterus activation). However, the major activation mechanism for contraction is the opening of the L-type channels by the membrane depolarization provided by the action potential that sweeps through the uterine smooth muscle cell syncytium.
While the nature of the excitation–contraction coupling mechanism is fairly well established, there is less information on the nature of the uterus smooth muscle cell membrane oscillator that is responsible for generating the periodic action potentials.
Uterus smooth muscle cell membrane oscillator
The nature of the pacemaker mechanism responsible for generating the action potentials that drive the smooth muscle contraction cycles during labour is still a mystery. The following membrane oscillator model attempts to explain the mechanism responsible for the slow pacemaker depolarization that triggers each contraction and how the frequency of this oscillator is modulated by stimuli such as oxytocin (Module 7: Figure uterus activation). The shape of the membrane potential fluctuation that drives each contractile cycle has two main components: there is a slow pacemaker depolarization that brings the membrane potential to the threshold (dashed line in Module 7: Figure uterus activation) for the action potential that is driven by the opening of the L-type VOCs. At the end of the action potential, the membrane potential switches back to a hyperpolarized state before beginning the next pacemaker depolarization phase. The critical question with regard to uterine contractility is the nature of the membrane oscillator that drives this slow pacemaker depolarization.
At any moment in time, membrane potential depends upon the co-ordinated activity of ion channels, pumps and exchangers that can either depolarize or hyperpolarize the membrane. At the end of the action potential, the hyperpolarizing components are dominant but as their influence wanes the depolarizing components begin to take over to produce the pacemaker depolarization. There are a number of candidates that could contribute to this hyperpolarizing/depolarizing switch (see the membrane oscillator box in Module 7: Figure uterus activation where the components that contribute to depolarization or hyperpolarization are coloured green and red respectively). At present, it is not clear how or when these possible contenders might contribute to the membrane oscillator during the course of the pacemaker depolarization. These different candidates have been included in the model based on either physiological evidence or the fact that they are expressed in human uterine or other smooth muscle cells as outlined below (Module 7: Figure uterus activation).
A number of K+ channels regulate uterine contraction. Both the large-conductance (BK) channels and the small-conductance (SK) channels are expressed in uterine smooth muscle and may contribute to the membrane oscillator. Activation of the BK and SK3 channels by Ca2+ contribute to the repolarizing phase of the action potential and may thus determine the maximal hyperpolarizing potential at the start of the pacemaker phase. As Ca2+ is pumped out of the cell, their hyperpolarizing influence will wane and this will contribute to the gradual pacemaker depolarization. Inhibition of the SK3 channel with apamin results in an acceleration of the oscillator. Conversely, if SK3 is overexpressed in rat uterus, there is a drastic reduction in oscillator frequency.
In addition to contributing to the membrane oscillator during labour, the BK channel may also play a role in maintaining the quiescent state of the uterus during pregnancy. These BK channels appear to be concentrated in the numerous caveolae that are a characteristic feature of smooth muscle cells (Module 7: Figure smooth muscle cell structure). These infoldings of the surface membrane, which often come into close contact with the endoplasmic reticulum (ER) (Module 6: Figure smooth muscle caveolae), have numerous signalling components and are rich in lipids such as cholesterol and sphingomyelin (SM) (Module 6: Figure caveolae organization). The cholesterol appears to function by enhancing the activity of the BK channels to reduce membrane excitability and thus contribute to the quiescent state. The increases in cholesterol that accompany obesity may increase the risk of complications in pregnancy by reducing the uterine contractility during labour and may account for the increased incidence of Caesarean sections in obese women.
The normal uterus contracts when stretched, but this excitability is reduced during pregnancy through the expression of stretch-sensitive TREK-1 channels and this enanles the uterus to distend. At the end of pregnancy, a decrease in TREK-1 expression enhances excitability and thus contributes to the onset of labour.
During the course of the action potential, opening of the L-type VOC results in Na+ entering the cell together with Ca2+. A hyperpolarization is generated when the Na+ is removed by the Na+ pump, which is electrogenic because it extrudes 2Na+ for 3K+. The pump will be activated immediately after an action potential to extrude Na+ and this will contribute to the early hyperpolarizing phase. But as the Na+ concentration returns to resting levels, this hyperpolarizing effect will decline and this may contribute to the depolarizing pacemaker.
The Na+/Ca2+-exchanger (NCX) has a somewhat ambiguous role because it can operate in both a forward and reverse mode (Module 5: Figure Na+/Ca2+-exchangers) and thus can contribute to either depolarization or hyperpolarization. The resting membrane potential of the uterus as it begins to oscillate is approximately −50 mV, which is probably more positive than the equilibrium potential for Na+, so it is likely that the exchanger will be operating in its reverse mode as shown in Module 7: Figure uterus activation. When operating in its reverse mode, 1Ca2+ is exchanged for 3Na+ so this stoichiometry may also favour hyperpolarization.
Store-operated Ca2+ channel (SOC)
Smooth muscle cells are known to express store-operated channels (SOCs) and these have been included as one of the possible channels for providing the inward current that is necessary to provide the depolarizing drive during the pacemaker depolarization. In addition, such a SOC mechanism may help to describe how oxytocin can accelerate the membrane oscillator. The suggestion is that oxytocin stimulates the formation of InsP3 that then acts to empty part of the endoplasmic reticulum (ER) store that lies near the membrane to promote the opening of the SOC and this increase in inward current will steepen the pacemaker depolarization to accelerate the oscillator. Another possibility is that the Ca2+ that enters the cell may act to switch on channels such as the Ca2+-sensitive chloride channels (CLCA). Ca2+ may also activate TRPM4 or TRPM5 that gate an inward monovalent current and have been implicated in the operation of other membrane oscillators.
CaV3 T-type channels
Uterine smooth muscle cells express CaV3.1, which is one of the isoforms of the CaV3.1 family of T-type channels (Module 3: Table VOC classification), which have been implicated in the pacemaker activity of other excitable cells. At the low membrane potential of activated uterine smooth muscle cells, these T-type channels are probably inactivated. However, at these inactivation potentials, a small population may still contribute to a pacemaker depolarization by gating an inward flux of Ca2+. Such a role for the T-type channel is consistent with the observation that low concentrations of Ni2+ slow the oscillator (Module 7: Figure Ni2+ slows uterine oscillations). The entry of Ca2+ through these T-type channels may also contribute to the depolarization by activating other inward currents such as those gated by TRPM4 or TRPM5 as mentioned above.
Ca2+-sensitive chloride channels
A proportion of human uterine cells express Ca2+-sensitive chloride channels (CLCA), which could play an important role in providing the inward current to drive pacemaker depolarization (Module 7: Figure uterus activation).
TRPM4 and TRPM5
TRPM4 and TRPM5, which are members of the melastatin-related transient receptor potential (TRPM) family of ion channels, respond to an increase in Ca2+ by gating an inward current carried by monovalent cations such as Na+. Although there is no information yet on their expression in uterine smooth muscle, they are known to occur in other smooth muscle cell types. These channels are not shown in Module 7: Figure uterus activation, but they should also be considered in the model because they could play a critical role in the pacemaker mechanism by providing an inward current to drive the pacemaker depolarization.
Hyperpolarizing-activated cyclic nucleotide-gated (HCN) channels
A number of cells that display pacemaker activity express members of the hyperpolarizing-activated cyclic nucleotide-gated (HCN) channels, which have been described in rat uterine smooth muscle. They have been included in the model (Module 7: Figure uterus activation) because they are one of the major pacemaker channel currents in the sinoatrial (SA) node pacemaker cells that has a similar membrane oscillator (Module 7: Figure cardiac pacemaker).
In conclusion, uterine contraction is driven by a membrane oscillator that consists of multiple components that can either depolarize or hyperpolarize the membrane. At the end of the action potential, the hyperpolarizing elements predominate but as these wane and the depolarizing components begin to take over the membrane gradually depolarizes to the point where the next action potential and contraction are triggered. Many of the hormones that modulate contractility are likely to influence some of these oscillatory components. For example, it is argued that oxytocin may act to switch on a SOC to provide an inward Ca2+ current that not only contributes a depolarizing signal but it also may act to switch on some of the TRPM channels. Oxytocin may have additional actions because it can increase the force of contraction in muscle cells where the internal stores have been depleted of Ca2+ following treatment with CPA (Module 7: human uterine contractions). In this case, it seems likely that oxytocin may switch on the smooth muscle Rho/Rho kinase signalling pathway to enhance the sensitivity of the contractile mechanism.
It is difficult to speculate further on how contractility is modulated until the nature of the membrane oscillator has been established. This is an important priority because problems remain concerning an effective treatment of controlling premature labour, which is one of the major causes of neonatal morbidity.
Detrusor smooth muscle cell
The bladder, which functions to store and expel urine, is lined with an uroepithelium that is surrounded by a lamina propia and layers of detrusor smooth muscle cells. These detrusor cells line up in bundles called fascicles that are arranged in a circular layer sandwiched between two longitudinal layers. The concerted contraction of these detrusor smooth muscle cells during micturition, is regulated by an extensive neural innervation (Module 7: Figure urinogenital tract) that orchestrates both the slow refilling of the bladder and the periodic and co-ordinated contraction of the detrusor muscles during micturition. Sensory neurons (shown in blue), which respond to stretch as the bladder fills, sends information into the spinal cord as part of the micturition reflex. Sympathetic nerves originating from the hypogastric plexus have two functions: they inhibit the detrusor muscles to enable the bladder to fill and they excite the urethral smooth muscle cells that keep the internal urethral sphincter closed to prevent urine escaping from the bladder. Parasympathetic pelvic nerves from the pelvic plexus provide the major excitatory input to trigger detrusor smooth muscle cell contraction during micturition.
The detrusor smooth muscle cells thus have two operational modes. During bladder filling the muscle cells remain relaxed for an extended period to enable the bladder wall to expand. During micturition, they respond to parasympathetic stimulation by contracting vigorously to increase the intraluminal pressure to expel urine. This bladder contraction phase must be co-ordinated with relaxation of the urethral smooth muscle cells that control the internal urethral sphincter and the opening of the external urethral sphincter by contraction of the striated muscles that make up the urogenital diaphragm. To understand detrusor smooth muscle cell activation, therefore, it is necessary to consider both the control of the relaxation phase and how this is switched into the active contraction phase by the neurotransmitters released from the parasympathetic neural innervation.
Detrusor smooth muscle cell activation
Detrusor muscle seems to be activated by mechanism A in Module 7: Figure SMC activation mechanisms. They have an endogenous membrane oscillator capable of producing repetitive trains of action potentials (Module 7: Figure detrusor action potentials). The pattern of action potentials varies between species. In pig and human, the action potentials are arranged in bursts whereas in guinea-pig and some other mammals they appear as a continuous train. This electrical activity is present during both the resting state when the bladder is filling and during the active contraction phase. The major difference is that this activity and associated contractions are restricted to small areas of the bladder during the resting phase. During the active phase, neurotransmitters released from the parasympathetic neurons not only induces such pacemaker activity in most of the detrusor muscles but it also results in an increase in frequency (Module 7: Figure bladder pacemaker acceleration). With regard to detrusor smooth muscle cell activation, therefore, there are two main questions: how is this pacemaker generated and how is it induced or accelerated during the contractile phase of bladder emptying?
The pacemaker mechanism that generates the repetitive action potentials is driven by a membrane oscillator, which has not been fully characterized. However, it contains many of the ionic components found in the oscillator in the uterus (Module 7: Figure uterus activation). An L-type VOC is responsible for the upstroke of the action potential, which is terminated by both Ca2+-induced desensitization of the VOC and by the activation of the Ca2+-sensitive large-conductance (BK) channels and the small-conductance (SK) channels. Activation of BK and SK3 set the potential at its maximum hyperpolarizing potential at the start of the pacemaker phase following each action potential. As Ca2+ is pumped out of the cell, their hyperpolarizing influence will wane and this will contribute to the gradual pacemaker depolarization. The frequency of the oscillator is acutely sensitive to changes in membrane potential being accelerated by depolarization and slowed or stopped when the membrane is hyperpolarized. The role of inward depolarizing currents in this pacemaker depolarization has not been fully determined. The main point is that the detrusor has an endogenous membrane oscillator that generates the spontaneous action potential that occurs in the resting muscle.
Each of these spontaneous action potentials is capable of inducing an increase in intracellular Ca2+ and contraction, which develop with much slower time courses (Module 7: Figure bladder E-C coupling). Each of the action potentials (trace a) is associated with an increase in Ca2+ (trace b) and contraction (trace c). The temporal relationship between these three events is shown in trace d. The Ca2+ signal lags behind the action potential because it spreads into the cell in the form of a Ca2+ wave (see panel Aa in Module 7: Figure bladder Ca2+ wave). About 10 ms after the depolarization, Ca2+ appears in the form of a spark at two sites (α and β) near the membrane. These sparks then develop into waves that spread into the cell to produce a global signal after about 200 ms. If the ryanodine receptors are blocked by 100 μM ryanodine, the Ca2+ signal is restricted to a smaller elevation immediately below the membrane, which might represent Ca2+ sparklets. The signalling components responsible for these spatiotemporal patterns are illustrated in Module 7: Figure bladder SMC activation. During this process of excitation–contraction coupling, membrane depolarization (ΔV) activates the L-type VOCs to produce sparklets, which provide the Ca2+ signal to activate ryanodine receptors (RYRs) located near the membrane to produce sparks. These sparks then recruit RYRs deeper in the cell to initiate the regenerative Ca2+ wave responsible for the global Ca2+ signal that triggers contraction. Ryanodine inhibits both the sparks and the wave leaving behind the sparklets that produce the small Ca2+ signal that appears as a ring immediately below the membrane (see panel Ba in Module 7: Figure bladder Ca2+ wave).
It is likely that the excitation–contraction (E-C) coupling process associated with the spontaneous action potentials are similar to those that occur when the whole bladder contracts during the emptying process. In the latter case, neural stimulation induces action potentials throughout the bladder thus recruiting the contractile activity of most of the bladder cells. This neural activation process is complicated by the fact that the parasympathetic neurons that innervate the bladder (Module 7: Figure urinogenital tract) release both acetylcholine (ACh) and ATP. These two transmitters have different effects on action potential frequency and contractile behaviour (Module 7: Figure neural evoked contractions). If the nerves are activated by a brief train of stimuli (solid bar), there is rapid acceleration of action potential frequency both during the burst and after the burst following a brief delay (see trace A). A strong contraction began during the neural stimulus. In the presence of α,β-methylene ATP, which inhibits ATP to reveal the action of ACh (trace B), there were no action potentials during the 1 s stimulus period, but thereafter the membrane depolarized and there was a marked increase in the frequency of action potentials similar to those seen in trace A. During the action of ACh, the onset of an oscillatory period of contraction was delayed. A similar delayed action potential acceleration and enhanced contraction is observed following stimulation of the cholinergic pathway with carbachol (Module 7: Figure bladder pacemaker acceleration). The acceleration of the membrane oscillator resulted in a tonic contraction that probably resembles what happens in vivo during bladder emptying. The ATP and ACh released during neural stimulation thus have similar effects but with different time courses: ATP acts quickly whereas ACh acts more slowly.
The next aspect to consider is how ACh and ATP act to induce/accelerate the membrane oscillator to stimulate contraction (Module 7: Figure bladder SMC activation):
Ureter smooth muscle cell
Transfer of urine from the kidney to the bladder is carried out by the pelviureteric system that has two components, the renal pelvis and the ureter (Module 7: Figure pelviureteric organization). Urine emerging from the kidney tubules is passed via the pelvicalyceal junction (PCJ) to the proximal renal pelvis and then into the distal renal pelvis and finally into the ureter. Urine is propelled through the pelvis and ureter by peristaltic smooth muscle contractions. This peristalsis is initiated in the proximal region and then spreads through the distal renal pelvis and ureter by an action potential that triggers smooth muscle contraction. Within the renal pelvis, a wave of Ca2+ sweeps through the typical smooth muscle cells at a rate of approximately 1mm/s (Module 7: Figure renal pelvis SMC Ca2+ wave), which is very much faster than classical intercellular Ca2+ waves that have an upper propagation rate of about 100 μm/s. In the case of the pelvis, the faster rate depends on the fact that the Ca2+ signal in each cell is driven by an action potential that travels through the smooth muscle cell syncitium. With regard to cell signalling in ureter smooth muscle cells, therefore, there are two main questions. First, what is the nature of the pelviureteric pacemaker mechanism that initiates the propagating action potential? Secondly, how does the action potential drive the process of ureter smooth muscle contraction?
Pelviureteric pacemaker mechanism
Activation of smooth muscle cells in the renal pelvis and ureter resembles most closely mechanism C in Module 7: Figure SMC activation mechanisms, but with some minor variations. In this mechanism, smooth muscle cells (SMCs) are activated by depolarization produced by current flow from other cells, such as the interstitial cells of Cajal (ICC). In the case of the renal pelvis, where pacemaker activity is located, there are two cell types that could function as pacemakers to drive contraction (Module 7: Figure pelviureteric organization). One is the interstitial cells of Cajal-like cells that resemble the classical interstitial cells of Cajal (ICC) both in their structure and their expression of c-KIT. They are spontaneously active and generate rhythmical action potentials that have very long plateau phases. At present, most evidence indicates that they may not be the primary pacemakers. Rather, attention has focused on the atypical smooth muscle cells, which form a loose network lying on top of the smooth muscle layer. These atypical muscle cells produce regular spontaneous transient depolarizations (STDs) that are thought to provide the depolarizing drive that triggers the action potentials to drive the Ca2+ waves that spread through the layer of typical smooth muscle cells (Module 7: Figure renal pelvis SMC Ca2+ wave).
Atypical smooth muscle cells
The atypical smooth muscle cells, which are restricted to the proximal renal pelvis (Prox. RP), are long thin cells that lie on the surface of the typical smooth muscle cells (/1Module 7: Figure pelviureteric organization). The contractile filaments are rather sparse and separated by various organelles such as the Golgi, endoplasmic reticulum and many mitochondria. With regard to their pacemaker role, the atypical cells form numerous gap junctions with the typical smooth muscle cells, which presumably enables them to pass depolarizing current to the smooth muscle cells (see mechanism C in /1Module 7: Figure SMC activation mechanisms). The nature of the pacemaker mechanism in these atypical smooth muscle cells, which depends upon a tonic production of prostaglandins and the release of tachykinins from sensory neurons, is still being worked out but seems to resemble the /1ICC cytosolic Ca2+ oscillator (/1Module 7: Figure ICC pacemaker). One important difference is that the oscillations in cytosolic Ca2+ do not activate Ca2+-sensitive Cl− channels in these atypical cells. The spontaneous transient depolarizations (STDs), which create the current flow to excite /1ureter smooth muscle contraction, depend upon the activation of an inward Na+ current carried by channels that remain to be determined. In addition, there appears to be a role for L-type Ca2+ channels that function to synchronize STDs into large enough pacemaker events capable of triggering the action potentials in the typical smooth muscle cells to drive /1ureter smooth muscle contraction.
Ureter smooth muscle contraction
The current flow created by the spontaneous transient depolarizations (STDs) in the atypical smooth muscle cells provides the depolarization drive responsible for activating the periodic action potentials that travel through the typical smooth muscle cells (Module 7: Figure pelviureteric organization). Despite the fact that the atypical smooth muscle cells generate STDs at a high frequency (10–40/min), the action potentials that they initiate in the typical smooth muscle cells occur at a much lower frequency (6–15/min) and this is caused by a prolonged refractory period induced by the Ca2+ that enters the cell during the action potential. During the recovery period, much of this Ca2+ is sucked up by the endoplasmic reticulum and this loading of the store sensitizes the ryanodine receptors 2 (RYR2s), which immediately begin to generate smooth muscle cell Ca2+ sparks. These sparks then activate large-conductance (BK) channels; the resulting hyperpolarization prevents the activation of action potentials thus resulting in a refractory period. Once the Ca2+ level in the stores declines back to normal, the sparks decline and the membrane can now respond to the depolarizing drive coming from the atypical smooth muscle cells to initiate another action potential.
This action potential then drives the process of smooth muscle cell excitation–contraction coupling (Module 7: Figure smooth muscle cell E-C coupling). To what extent contraction depends upon the entry or the release of internal Ca2+ remains to be determined, but it has to be fast to account for the rapid rates of Ca2+ wave propagation (Module 7: Figure renal pelvis SMC Ca2+ wave) that establish the peristaltic waves necessary to propel urine to the bladder.
Airway smooth muscle cells
Maximal gas exchange in the lung depends upon a precise matching of ventilation to blood perfusion. Smooth muscle cells (SMCs), which have been coloured green, surround the airways and arterioles that contribute to this gas exchange (Module 7: Figure lung morphology). Contraction of these SMCs can be activated by a number of stimuli (Module 7: Figure bronchiole-arteriole contraction). The lung arterioles, which contracted strongly in response to 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1) and an increase in KCl but not to acetylcholine (ACh), are described in the section on vascular smooth muscle cells. The airway bronchioles responded to ACh and 5-HT. The action of ACh on the muscarinic receptors is modulated by GRK3 and GRK5, which are members of the family of G protein receptor kinases (GRKs). At the single-cell level, these airway smooth muscle cell contractions are driven by oscillations in the intracellular level of Ca2+, which is driven by a smooth muscle cell cytosolic oscillator (Module 7: Figure SMC cytosolic oscillator). Airway smooth muscle cell activation thus conforms to mechanism B in Module 7: Figure SMC activation mechanisms.
Hydrogen sulfide (H2S) is turning out to be an important bronchodilator of airway smooth muscle cells. The relaxing action of H2S might depend on it being an inositol 1,4,5-trisphosphate receptor (InsP3R) antagonist (Module 3: Figure InsP3R regulation). By inhibiting the InsP3-induced release of Ca2+, H2S reduces the Ca2+ oscillations that trigger contraction.
Alterations in airway smooth muscle function are important for a number of respiratory disorders such as asthma.
Vascular smooth muscle cells
The blood vessels that make up the vasculature are surrounded by a layer of smooth muscle cells (SMCs). Vascular SMCs have been studied in a number of different vessels such as mesenteric arteries, cerebral arteries, coronary arteries and arterioles located in the lung. In the case of the lung, the arterioles are located close to the bronchioles (Module 7: Figure lung morphology). The SMCs, which have been coloured green, surround both vessels. Contraction of these SMCs can be activated by a number of stimuli (Module 7: Figure bronchiole-arteriole contraction). The lung arterioles contracted strongly to 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1) and an increase in KCl but not to acetylcholine (ACh). On the other hand, the airway bronchioles responded to both ACh and 5-HT but not KCl. These experiments illustrate that the SMCs express different receptors and are likely to have slightly different signalling pathways. Despite these differences, most vascular SMCs are responsible for maintaining the myogenic tone of blood vessels. By constricting or dilating the resistance arteries, these SMCs regulate the flow of blood and thus blood pressure. Many of these vascular SMCs have a myogenic vasoconstriction response that enables them to contract when the intraluminal pressure increases and then relax when the pressure falls. Blood pressure is determined by this myogenic tone. In addition, neurotransmitters released from the nerves that innervate the blood vessels can also control these vascular SMCs. Before considering how these various transmitters act to generate muscle contraction, it is appropriate to consider the neural mechanisms responsible for delivering these transmitters on to the surface of the vascular smooth muscle cells.
In the case of mesenteric arteries, the perivascular nerves that ramify over the smooth muscle surface (Module 7: Figure arterial nerves) release both ATP and noradrenaline. When these nerves are stimulated, small Ca2+ signals appeared in the underlying muscle cells at discrete points close to the nerve tracts (white boxes in panel B Module 7: Figure arterial nerves). These small signals in the muscle cells are called junctional Ca2+ transients (jCaTs) and are best seen in spatiotemporal plots (Module 7: Figure junctional Ca2+ transient). An image of one of these small jCaTs was captured at 4.8 s in the line-scan image in panel B. The fine line running along the length of the plot is the Ca2+ signal in the fine nerve indicated by the white arrow in panel A. In these experiments, the effect of noradrenaline was inhibited by prazosin so the appearance of the jCaTs was the result of ATP acting on the smooth muscle cells to open P2X receptor channels to introduce a local pulse of Ca2+ (Module 7: Figure SMC cytosolic oscillator). There is little contraction in response to these small ATP-dependent jCaTs. Larger contractions are seen when prazosin is removed so that the muscle can respond to both ATP and noradrenaline (Module 7: Figure neurogenic contractions). The ATP acts first to produce a small initial contraction that is then followed by a much larger contraction when noradrenaline initiates a series of Ca2+ transients, which are a characteristic feature of vascular SMC contractility. In the case of the astrocyte regulation of cerebral blood flow, the activity of cerebral arterioles is controlled by factors coming from the astrocytes (Module 7: Figure astrocyte endfoot signalling).
Contractile activity of many vascular SMCs depends on oscillatory Ca2+ signals. In response to stimuli such as noradrenaline (NA), 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), individual cells are found to generate repetitive Ca2+ transients (Module 7: Figure neurogenic contractions). Another example is the rhythmical contractions of cerebral arterioles (Module 7: Figure smooth muscle cell Ca2+ oscillations). In some blood vessels, a specific tone is maintained by the spatial averaging of asynchronous oscillations. However, there are some vessels where the oscillations in individual cells are synchronized resulting in pulsatile contractions known as vasomotion. Another important feature of this oscillatory activity is that variations in transmitter concentration are translated into a change in contractile tone through a frequency modulation mechanism (Module 7: Figure frequency modulation of SMCs). Frequency modulation is one of the mechanisms used for the encoding and decoding of Ca2+ oscillations. The signalling mechanisms that generate these oscillations conform to mechanism B in Module 7: Figure SMC activation mechanisms. The primary activation mechanism for vascular smooth muscle is a cytoplasmic Ca2+ oscillator that generates periodic pulses of Ca2+ that then drives contraction. Transmitters operate by controlling the frequency of the oscillator. Entry of Ca2+ also plays an important role in modulating the oscillator. In some cells, the oscillator can alter membrane potential (ΔV) and this can act both as a feed-forward signal and as a way of entraining the oscillators in neighbouring cells to create the synchronicity responsible for vasomotion. The central feature of vascular SMC activation is a smooth muscle cell cytosolic oscillator.
Vascular SMCs thus have sophisticated signalling mechanisms that maintain precise control over blood pressure. Since an alteration in the delicate balance between contraction and relaxation is one of the defects leading to hypertension, there is a clear imperative to understand how these cells are activated.
Smooth muscle cell cytosolic oscillator
A smooth muscle cytosolic Ca2+ oscillator drives the contractile activity of both vascular smooth muscle cells, airway smooth muscle cells and perhaps also in the corpus cavernosum smooth muscle cells. The Ca2+ oscillation mechanism depends upon a sequential series of events: store loading, spike initiation, spike development and spike recovery (Module 6: Figure Ca2+ oscillation model). The way in which these components function in the smooth muscle cell oscillator is illustrated in Module 7: Figure SMC cytosolic oscillator:
Myogenic vasoconstriction, which is also known as the Bayliss effect, is a dynamic mechanism that enables small-resistance arterial blood vessels to contract in response to an increase in intraluminal pressure. This response is important for maintaining vascular tone and peripheral vascular resistance that ensures that organs such as the brain and kidney retain a normal perfusion. This myogenic response seems to be carried out by the smooth muscle cells that have a mechanotransduction mechanism that enables them to detect mechanical deformation and to generate the internal signals necessary to activate contraction. It has been proposed that the mechanosensor might be a G protein-coupled receptor (GPCR) such as the angiotensin II AT1 receptor. In response to mechanical deformation, the GPCR activates Gq/11, which then stimulates phospholipase β (PLCβ) to generate InsP3 and diacylglycerol (DAG). The DAG then activates the TRPC6 to provide the inward current necessary to depolarize the membrane such that the L-type Ca2+ channel can open to bring about contraction.
Corpus cavernosum smooth muscle cells
The corpus cavernosum, which are two elongated sponge-like bodies that lie on either side of the penis, fill up with blood during an erection (Module 7: Figure urinogenital tract). The interconnected cavernous spaces, which give the corpora cavernosum a sponge-like appearance, are connected to the penile arteries. Both the arteries and the spaces are lined with smooth muscle cells that regulate the flow of blood into the corpora cavernosum. The penis is flaccid when these muscles are contracted, but becomes rigid when these muscles relax to allow blood to flow into the cavernous spaces. The tunica albuginea that surrounds this erectile tissue is relatively inextensible and resists the increased entry of blood resulting in an increase in pressure within the corpora cavernosa. As the tissue distension continues, the emissary veins that carry blood out of the penis are compressed resulting in further engorgement and penile rigidity. In summary, penile erection is critically dependent on the contractile state of the corpora cavernosum smooth muscle cells.
The corpora cavernosum smooth muscle cells are regulated by a large number of stimuli that determine whether the muscle is relaxed or contracted (Module 7: Figure corpus cavernosum). Like other vascular smooth muscle cells, contraction seems to depend on mechanism B in Module 7: Figure SMC activation mechanisms. The central feature of this mechanism is a smooth muscle cytosolic Ca2+ oscillator that resembles that found in both vascular and airway smooth muscle cells (Module 7: Figure SMC cytosolic oscillator). There is some debate as to where this oscillator is located. Interstitial Cells of Cajal (ICC), which have a pacemaker role in other tissues such as the gastrointestinal tract, have been described in the corpus cavernosum but whether they play a direct role in regulating contraction is still an open question. However, the ICC may play an indirect role by providing stimuli such as the eicosanoid prostaglandin F2α (PGF2α). In the model depicted in Module 7: Figure corpus cavernosum, the oscillator is located in the corpus cavernosum.
This oscillator generates the pulses of Ca2+ that maintains the contractile state (Module 7: Figure corpus cavernosum oscillations). The pulses of Ca2+ spread through the muscle cells as fast waves traveling at about 800μm/s. This wave may spread to neighbouring cells by current flow through the gap junctions to synchronize the oscillators of neighbouring cells. The membrane depolarization will activate voltage-operated channels (VOCs) in neighbouring cells and thus provide a Ca2+ pulse that will initiate Ca2+ waves in phase with the cell with the fastest rhythm. The following description provides further details of how this central oscillator is controlled during both contraction and relaxation (Module 7: Figure corpus cavernosum):
Alterations in the signalling systems that control the contractile activity of corpus cavernosum smooth muscle cells are responsible for erectile dysfunction.
Gastrointestinal smooth muscle cells
The intestine and stomach that make up the gastrointestinal tract maintain a peristaltic rhythm. The waves of contraction are driven by a pacemaker mechanism located within the Interstitial cells of Cajal (ICC) (Module 7: Figure ICC morphology). These ICC cells have characteristic positions within the gastrointestinal tract (Module 7: Figure ICC subtypes). The stomach and intestinal smooth muscle ICCs have smooth muscle activation mechanisms that depends on the ICC generating slow waves of membrane depolarization that spread through the gap junctions to drive the periodic contractions (Module 7: Figure SMC activation mechanisms). There are two main components of the pacemaker mechanism: an ICC cytosolic Ca2+ oscillator, which generates the slow waves, and the modulation of ICC pacemaker activity by hormonal and neural stimuli (Module 7: Figure ICC pacemaker).
A remarkable feature of the ICC network is its ability to co-ordinate the contractile activity of large groups of smooth muscle cells by synchronizing their individual oscillators (Module 7: Figure ICC network synchrony). This synchronicity, which has been referred to as a phase wave, might be achieved by coupling oscillators together through a depolarizing signal spreading through the gap junctions (Module 7: Figure ICC coupling). The nature of this synchronicity and how the separate oscillators are coupled together is described in the section on phase waves.
Urethral smooth muscle cells
Urethral smooth muscle cells control the internal urethral sphincter located at the base of the bladder (Module 7: Figure urinogenital tract). There is a reciprocal relationship between the operation of the bladder and the urethra. During urine storage, the detrusor smooth muscle cells of the bladder are relatively relaxed whereas the urethral smooth muscle cells are contracted to close off the sphincter to prevent urine leaving the bladder. These roles are reversed when urine is voided, the urethra relaxes and the bladder contracts. These different functions are under neural control. Like the bladder, the urethra receives a parasympathetic (cholinergic) and a sympathetic (adrenergic) innervation. The latter releases noradrenaline (NA) responsible for maintaining the contractile tone whereas acetylcholine (ACh) released from the parasympathetic nerves provides the nitric oxide (NO) that mediates relaxation.
The neurogenic-dependent contractile tone of the urethral smooth muscle is a dynamic process driven by regular oscillations in the level of intracellular Ca2+. These oscillations depend on a pacemaker mechanism located within a population of interstitial cells of Cajal (ICC) that are connected to the smooth muscle cells by gap junctions. The ICC come in two shapes, bipolar and multipolar (Module 7: Figure ICC morphology). The urethral smooth muscle thus have smooth muscle activation mechanisms that depend on the ICCs generating slow waves of membrane depolarization that spread through the gap junctions to drive the periodic contractions (mechanism C in Module 7: Figure SMC activation mechanisms). When Ca2+ is recorded simultaneously in these two cells, each ICC slow wave is associated with a Ca2+ transient in the urethral smooth muscle cell (Module 7: Figure ICC–SMC synchronization). There are two main components of the pacemaker mechanism that drives these slow waves: an ICC cytosolic Ca2+ oscillator, which generates the slow waves and the modulation of ICC pacemaker activity by hormonal and neural stimuli (Module 7: Figure ICC pacemaker). Activation of this pacemaker by noradrenaline induces the urethral cells to contract to close off the sphincter to prevent urine from escaping from the bladder. Stress urinary incontinence may result from a defect in the control of this closure mechanism.
Relaxation of contraction during bladder emptying depends both on the sympathetic nerves being switched off to reduce the release of noradrenaline and the activation of the parasympathetic nerves that increase the release of NO. The latter then acts through the nitric oxide (NO)/cyclic GMP and smooth muscle relaxation mechanisms (Module 7: Figure smooth muscle cell cyclic GMP signalling).
Interstitial cells of Cajal
The Spanish histologist Ramon y Cajal first described the elongated cells that are located in many organs and have been well-characterized in various smooth muscles. These Interstitial Cells of Cajal (ICC) are particularly important in the gastrointestinal tract and urethra where they function as pacemaker cells to provide one of the major smooth muscle activation mechanisms (see C in Module 7: Figure SMC activation mechanisms). The ICC has two distinct morphologies, bipolar and multipolar (Module 7: Figure ICC morphology). Since the bipolar ICC closely resemble the spindle-shaped smooth muscle cells, it has proved difficult to distinguish the two cell types in situ. However, the finding that the ICC can be picked out by using an antibody against the Kit receptor has greatly facilitated their identification in the gastrointestinal tract, urethra, lymphatic system, prostate and fallopian tube.
The ICC have characteristic distributions within smooth muscle. Much of the work on these cells has been done on the gastrointestinal tract where the terminology of the different cell types is based on their characteristic regional locations (Module 7: Figure ICC subtypes):
It remains to be determined whether these ICC subtypes, as defined by their tissue locations, are functionally different. There are indications that ICC can carry out a variety of functions. They are pacemaker cells responsible for activating many smooth muscle cells. As part of this pacemaker activity they also function to propagate electrical activity to the smooth muscle cells. The close association between ICC and neurons supports physiological evidence that the ICC can also function as neuroeffector cells to receive and to relay excitatory and inhibitory signals to the smooth muscle cells (see C in Module 7: Figure SMC activation mechanisms).
The ICC pacemaker activation mechanism generates the stimuli that maintain repetitive smooth muscle cell contractility. This activation process depends upon an ICC cytosolic Ca2+ oscillator that generates periodic pulses of Ca2+, which are known as slow waves, that then activates membrane channels that carry inward currents. The resulting current flow and depolarization spreads through the gap junctions to the smooth muscle cells. Since the ICC form an interconnected network that extends through the smooth muscle cell layer, they can co-ordinate the activity of large populations of smooth muscle cells to function in near synchrony. This co-ordinated activity is evident in the form of the peristaltic waves of contraction that spread through the gastrointestinal tract. It is important to emphasize, however, that this excitation is not propagated through an action potential because the ICC are not excitable. Rather, the near synchronous contraction of large populations of smooth muscle cells, which has been referred to as a phase wave, depends on the synchronization of the ICC cytosolic Ca2+ oscillators located in each of the ICC that make up the excitatory network.
ICC pacemaker activation mechanism
Studies on ICC in both the gastrointestinal tract and urethra have indicated that pacemaker activity depends upon a typical cytosolic oscillator (Module 6: Figure membrane and cytosolic oscillator). The oscillator in the ICC provides the depolarizing drive responsible for triggering smooth muscle cell contraction (see C in Module 7: Figure SMC activation mechanisms). There are two main components of this pacemaker mechanism: the ICC cytosolic Ca2+ oscillator, which provides the depolarization to activate smooth muscle contraction and the modulation of ICC pacemaker activity by hormonal and neural stimuli (Module 7: Figure ICC pacemaker).
ICC cytosolic Ca2+ oscillator
The following hypothesis, which attempts to integrate the main features of this ICC oscillator, is based on the basic mechanism of Ca2+ oscillations with a few additional features. This ICC cytosolic Ca2+ oscillator resembles that of the smooth muscle cell cytosolic oscillator but there are enough differences for them to be described separately. These differences may well relate to how they function in the activation process. The muscle oscillator provides the internal Ca2+ signal that triggers contraction (Module 7: Figure SMC cytosolic oscillator), whereas the ICC oscillator is designed to generate a depolarizing signal to activate a local population of muscle cells (Module 7: Figure ICC pacemaker).
The basis of this ICC oscillator is that Ca2+ is periodically released from InsP3-sensitive stores (Module 6: Figure Ca2+ oscillation model). The timing of each release depends upon the entry of external Ca2+, which loads up the stores resulting in the gradual sensitization of the internal release channels such as the ryanodine receptors (RYR) and possible the InsP3 receptors to a point where they trigger the next phase of Ca2+ release. The enhanced sensitivity of the channels prior to each release is often accompanied by the appearance of Ca2+ puffs, which then coalesce to form a global Ca2+ signal that activates the repetitive membrane depolarizations that constitute the slow wave.
This basic oscillatory model has a central role in driving ICC pacemaker activity as outlined in the following sequence of events (Module 7: Figure ICC pacemaker):
Modulation of ICC pacemaker activity
Pacemaker activity can be modulated by the neural input that forms synaptic junctions with the ICC cells (Module 7: Figure SMC activation mechanisms). Many of the neurotransmitters released on to the ICC, such as acetylcholine (ACh) and noraderenaline (NA), accelerate oscillator frequency (Module 7: Figure ICC modulation). In the absence of ACh, there are very few transients but after the addition of 100 nM ACh there is a reversible acceleration of frequency. These transmitters act through the phosphoinositide signalling pathway to increase the formation of InsP3 that then increases the sensitivity of the InsP3 receptors to the stimulatory action of Ca2+ (Step 11 in Module 7: Figure ICC pacemaker). Conversely, inhibitory nerves that release nitric oxide (NO) had the opposite effect of reducing both the amplitude and the frequency of slow waves. This overall reduction in the slow wave reduces the pacemaking drive responsible for smooth muscle contraction. This action of the nitric oxide (NO)/cyclic GMP signalling pathway also seems to be directed towards the InsP3 receptors. In this case, the InsP3R modulation by protein phosphorylation depends upon the action of cyclic GMP-dependent protein kinase Iβ (cGKIβ), which reduces the Ca2+ sensitivity of the receptor.
A remarkable feature of the network of interstitial cells of Cajal (ICC), which co-ordinate the contractile activity of large groups of smooth muscle, is that their oscillatory activity can be closely synchronized (Module 7: Figure ICC modulation). Slow waves in cells that are 4.5 mm apart occur at the same time. The basis of this experiment is shown in Module 7: Figure ICC coupling. If the slow wave is recorded from two cells widely separated from each other (e.g. electrodes 1 and 2), the regular waves of membrane depolarization occur in phase with each other. This synchronicity has been referred to as a phase wave to indicate that the repetitive Ca2+ transients responsible for the slow wave occur closely in phase with each other within a large population of cells. In reality, there are small phase shifts that result from the fact that excitation spreads quickly down the network at 5–40 mm/s to account for the waves of peristalsis. Phase waves can also be observed when the Ca2+ transients responsible for these slow waves are simultaneously recorded from a number of ICC cells that form a network (Module 7: Figure ICC network synchrony). Trace E shows that all the cells highlighted by the coloured circles in panel A produced Ca2+ transients in close synchrony with each other. However, when the gap junctions are blocked, this synchrony is lost and the individual cells produce Ca2+ transients at random (Panel F). It is clear, therefore that the ICC cytosolic Ca2+ oscillator in each cell is tightly coupled to that of its neighbours and this phase locking seems to depend on a signal being passed through the gap junctions.
Just how the oscillatory activity of ICCs are coupled together to form a phase wave is still somewhat of a mystery. The phase wave spreads too fast (5–40 mm/s) to be explained by an intercellular Ca2+ wave (Module 6: Figure intercellular Ca2+ wave), which has an upper propagation rate of about 100 μm/s. Most attention is now focused on the idea that coupling depends upon membrane depolarization spreading through the gap junctions and is thus an example of communication through electrical signals (Module 1: Figure cell communication). This depolarization is initiated by the Ca2+ oscillator in each ICC (Module 7: Figure ICC pacemaker). The upstroke of each slow wave is initiated by the release of Ca2+ that then opens the chloride and Ca2+ channels to produce each depolarization. The idea is that this depolarization spreads quickly to entrain the oscillatory activity of neighbouring cells. Since this oscillatory activity depends upon the spontaneous activation of InsP3 receptors, depolarization may entrain oscillations by enhancing the supply of either the Ca2+ or InsP3 necessary to trigger release. It seems unlikely that a depolarization-induced synthesis of InsP3 would be fast enough to be the coupling mechanism. In the following hypothesis, therefore, it is suggested that a depolarization-induced influx of Ca2+ provides a mechanism for coupling together the oscillators in separate cells (Module 7: Figure ICC coupling):
The critical feature of this mechanism is that all the oscillators in the network are coupled together through a voltage-dependent mechanism. At any moment in time, an individual cell is either functioning as a pacemaker to initiate a Ca2+ transient (e.g. cell B in Module 7: Figure ICC coupling) or it is responding to the depolarization emanating from a neighbouring pacemaker cell (e.g. cells A and C). It seems that these two functions are not fixed and cells can switch roles on a beat by beat basis. This switching is clearly evident when three cells are monitored during successive slow waves (Module 7: Figure ICC phase switching). During wave 1, the cell recorded by electrode 1 (el1, red trace) fired first and this was followed by el2 (green trace) and then el3 (blue trace). This sequence was reversed during wave 3. The main point to make, therefore, is that this system of coupled oscillators is highly dynamic in that each cell is not only capable of initiating each wave but it is also finely tuned to respond to the entrainment signal coming from any of its neighbours and this ensures that each beat is synchronous within a large population of cells.
The concentration of Ca2+ in plasma is maintained within a narrow physiological range by a number of interacting hormonal and organ systems (Module 7: Figure Ca2+ homoeostasis). This constancy of the level of serum Ca2+ is crucial not only to maintain the skeleton but also because the Ca2+ signalling system that controls so many cellular processes is very sensitive to variations in extracellular Ca2+ concentration. A number of cell types located in different tissues contribute to Ca2+ homoeostasis.
The average human skeleton contains about 1000 g of Ca2+ and is the major reservoir from which Ca2+ can be added or removed. When the dietary supply of Ca2+ is adequate, the rate of bone formation and bone resorption are approximately equal at about 400 mg/day. Bone is not a static organ, and bone remodelling occurs continuously in response to changing skeletal requirements. Control over this ebb and flow of Ca2+ between bone and plasma is a major component of the control mechanisms that regulate Ca2+ homoeostasis. The kidney, which filters almost the total plasma content of Ca2+ each day, is another important site of hormone regulation. Almost all of this Ca2+ is reabsorbed, with a small amount (200 mg/day) being lost in the urine. Control over this Ca2+ reabsorption by the kidney is a major component of Ca2+ homoeostasis. There also are large fluxes of Ca2+ associated with the intestine. There is an input of Ca2+ from food and the secretions coming from the salivary glands and pancreas. A little less than half of the Ca2+ entering the intestine is reabsorbed and slightly more is lost in the faeces, resulting in a net uptake of approximately 200 mg/day, which thus balances the urinary losses (Module 7: Figure Ca2+ homoeostasis). Control over this Ca2+ reabsorption by the intestine is another major component of Ca2+ homoeostasis.
All the systems that participate in Ca2+ homoeostasis are carefully regulated by three main hormones:
PTH is released from chief cells in the parathyroid glands, whereas calcitonin is released from C cells in the thyroid (Module 7: Figure parathyroid gland). Circulating PTH plays a major role in regulating 1,25(OH)2D3 formation, which is synthesized by a series of steps located in the skin, liver and kidney (Module 7: Figure vitamin D metabolism). The hormonal regulation of Ca2+ homoeostasis is based on the ability of these three hormones to regulate Ca2+ fluxes in tissues, such as bone intestine and kidney.
Hormonal regulation of Ca2+ homoeostasis
The nicely balanced system of Ca2+ homoeostasis is regulated by three main hormones: parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and calcitonin. PTH and 1,25(OH)2D3 act primarily to increase the level of Ca2+ in the plasma, whereas calcitonin has the opposite effect. PTH is formed and released by the parathyroid gland, which is located alongside the thyroid gland (Module 7: Figure parathyroid gland). C cells dispersed throughout the thyroid are responsible for forming and releasing calcitonin. 1,25(OH)2D3 is formed through a series of steps located in different cells (Module 7: Figure vitamin D metabolism). There are three major processes that are regulated by these hormones:
The parathyroid gland, which synthesizes and releases parathyroid hormone (PTH), has a somewhat varied location in mammals. In humans, it has four separate parts, each about the size of an apple seed, that are located in close association with the thyroid gland (Module 7: Figure parathyroid gland). A thick capsule of connective tissue surrounds the sheets of chief cells that synthesize and release PTH. Chief cells are typical protein secretory cells with the usual stacks of rough endoplasmic reticulum and Golgi bodies that function to package PTH into storage vesicles. An unusual feature of these chief cells is that they have relatively few storage granules, which means that the store of hormone is rather limited in relation to the amount being released. PTH thus turns over quickly and the level of Ca2+ in the plasma is one of the main determinants of parathyroid hormone (PTH) synthesis and release.
The calcitonin-producing parafollicular cells (C cells) are located amongst the thyroid follicular cells (Module 7: Figure parathyroid gland). These C cells express the Ca2+-sensing receptor (CaR) that respond to elevations in the level of serum Ca2+ by releasing calcitonin.
Parathyroid hormone (PTH)
Parathyroid hormone (PTH), which is the most important hormonal regulator of Ca2+ homoeostasis, is synthesized and released from the parathyroid gland (Module 7: Figure parathyroid gland). PTH is released as a linear protein of 84 amino acids. It shares considerable homology with parathyroid hormone (PTH)-related peptide (PTHrP), which also has a function in regulating bone, especially during development.
One of the main actions of PTH is to elevate blood Ca2+ levels by affecting bone remodelling by increasing bone resorption by the osteoclasts, by stimulating Ca2+ reabsorption by the kidney and by stimulating the kidney synthesis of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (Module 7: Figure vitamin D metabolism). With regard to bone resorption, one of the actions of PTH is to stimulate the osteoblasts to increase the expression of receptor activator of nuclear factor κB (NF-κB) ligand and to decrease the release of osteoprotegerin (OPG), which combine to increase the differentiation of the osteoclasts (Module 8: Figure osteoclast differentiation).
Parathyroid hormone (PTH)-related peptide (PTHrP)
Parathyroid hormone (PTH)-related peptide (PTHrP) has some homology with parathyroid hormone (PTH), especially with regard to the first 13 amino acids. The similarity between these two molecules extends to the fact that they both act on the same PTH receptor 1 (PTHR1). Like PTH, PTHrP plays a role in bone remodelling, especially during embryogenesis. In adult life, PTHrP has also been implicated in the regulation of a number of cellular processes, such as control of cell proliferation, smooth muscle cell relaxation and the transplacental flux of Ca2+. With regard to pathology, PTHrP appears to be one of the main components of humoral hypercalcaemia of malignancy (HHM).
Parathyroid hormone (PTH) synthesis and release
The synthesis and release of parathyroid hormone (PTH) is regulated by a number of factors. The primary control is through the plasma level of Ca2+, which is monitored by a Ca2+-sensing receptor (CaR) located on the surface of the chief cells (Module 7: Figure parathyroid gland). The parathyroid gland is richly supplied with blood vessels, thus enabling the CaR on the chief cells to constantly monitor the level of Ca2+ in the plasma. The Ca2+ in the plasma thus functions as an agonist by acting on the CaR to regulate the release of PTH. There is an inverse relationship between the plasma level of Ca2+ and the release of PTH (Module 7: Figure PTH secretion). PTH secretion is maximal at low levels of Ca2+ and declines steeply over the narrow range of Ca2+ concentrations that occur normally in the plasma. This relationship is somewhat unusual in that it is opposite to what might be expected. Since the CaR is coupled to the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette, it might be expected that an increase in the stimulation of CaR should increase the level of Ca2+ signalling and hence the level of PTH secretion, but the opposite is the case. Since the CaR acts to increase the level of intracellular Ca2+, it seems reasonable to assume that Ca2+ somehow acts to inhibit exocytosis contrary to its normal stimulatory effect. This paradoxical situation is currently unresolved.
The CaR is also known to be coupled to the cyclic AMP signalling pathway, where it acts through Gαi (Module 2: Table heterotrimeric G proteins) to reduce the level of cyclic AMP. Cyclic AMP does appear to play a role in modulating the parathyroid gland, which is richly innervated by a sympathetic neural input. Like Ca2+, cyclic AMP may also exert a negative-feedback effect on PTH secretion because inhibition of the β-receptor that acts by increasing cyclic AMP enhances PTH secretion.
The secretory activity of the parathyroid gland is also regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is the hormonally active form of vitamin D (Module 7: Figure PTH secretion). The 1,25(OH)2D3 acts on a longer time scale in that it regulates the synthesis of PTH by controlling the expression of the PTH gene. 1,25(OH)2D3 enters the nucleus, where it binds to the vitamin D receptor (VDR), which binds to the VDR element (VDRE) to inhibit transcription of preproPTH and hence the synthesis of PTH.
Many of the disorders of Ca2+ homoeostasis arise from an increase in the release of PTH. There are two main types of hyperparathyroidism:
Changes in PTH secretion are also a feature of the inherited disorders of Ca2+ that arise from mutations in CaR:
The hormone calcitonin, which is a polypeptide containing 32 amino acids, is released from the parafollicular cells, which are also known as C cells, located in the thyroid gland (Module 7: Figure parathyroid gland). Work on some of the lower vertebrates such as fish seemed to indicate that calcitonin might function by counteracting the effects of parathyroid hormone (PTH) by reducing the level of Ca2+ in the plasma. For example, it can reduce Ca2+ absorption by the intestine and kidney tubules. It can also act by decreasing the activity of the osteoclasts (Step 7 in Module 7: Figure osteoclast function). In humans, there is less evidence for a role of calcitonin in regulating Ca2+ homoeostasis. For example, when calcitonin levels are increased (i.e. in medullary thyroid cancer) or decreased following thyroidectomy, there is little change in the level of serum Ca2+. However, there are indications that calcitonin may have a role in maintaining Ca2+ homoeostasis during the physiological mineral stress associated with the reproductive cycle. This is especially the case during lactation, when there is a large requirement for Ca2+ during milk production. It seems that calcitonin may help to protect the skeleton by reducing mineral loss during pregnancy and lactation.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3]
The hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is the active component of vitamin D3, has an important role in the feedback mechanisms that regulate Ca2+ homoeostasis. 1,25(OH)2D3 formation and metabolism depends upon a series of reactions that take place in a number of different tissues (Module 7:Figure vitamin D metabolism). The role of 1,25(OH)2D3 in the hormonal regulation of Ca2+ homoeostasis occurs in a number of tissues. One of its main actions is to promote Ca2+ reabsorption by the kidney (Module 7: Figure kidney Ca2+ reabsorption) and Ca2+ reabsorption by the intestine (Module 7: Figure intestinal Ca2+ reabsorption). It also plays an import role in reducing the expression of PTH synthesis by the parathyroid gland (Module 7: Figure PTH secretion). Vitamin D deficiencies, which result in a decline of 1,25(OH)2D3, removes this inhibitory effect on PTH synthesis resulting in secondary hyperparathyroidism. The relationship between vitamin D and Ca2+ regulation in neurodegeneration suggest that deficiencies in vitamin D may contribute to a number of the major neural diseases in humans such as Alzheimer's disease, Parkinson's disease and multiple sclerosis.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] formation and metabolism
Formation of 1,25-dihydroxyvitamin D3 [1,25-dihydroxycholecalciferol or 1,25(OH)2D3], which is the hormonally active form of vitamin D3, depends upon a series of reactions, most of which are carried out by the cytochrome P-450-containing (CYP) enzymes, and take place in different tissues (Module 7: Figure vitamin D metabolism):
These metabolic pathways responsible for forming this hormone are carefully regulated depending on the level of Ca2+ in the plasma. One of the actions of parathyroid hormone (PTH), which is released when Ca2+ levels are low, is to stimulate the activity of the 1α-hydroxylase to increase the formation of 1,25(OH)2D3 (Module 7: Figure Ca2+ homoeostasis). On the other hand, 1,25(OH)2D3 operates a negative-feedback loop whereby it can activate the 24-hydroxylase to reduce its level should it become too high.
The formation of 1,25(OH)2D3, often acting together with PTH, plays a major role in the hormonal regulation of Ca2+ homoeostasis.
Vitamin D receptor (VDR)
The vitamin D receptor (VDR) is a member of the nuclear receptor family (Module 4: Table nuclear receptor toolkit) and is widely distributed in many different cell types. The VDR functions as a heterodimer together with the retinoid X receptor (RXR), which is another nuclear receptor (Module 7: Figure vitamin D receptor activation). The VDR has a number of functional domains: a DNA-binding (DNB) domain, which has two Zn fingers, a nuclear localization signal (NLS), which directs the receptor into the nucleus, a ligand-binding domain (LBD), and a transactivation domain that binds to activation factor-2 (AF-2).
When VDR binds 1,25(OH)2D3, it is phosphorylated on serine-51 in the DNB domain by protein kinase C (PKC) and on serine-208 by casein kinase II and this alters its conformation resulting in the removal of co-repressors such as nuclear receptor co-repressor (N-CoR) and the closely related silencing mediator for retinoid and thyroid hormone receptor (SMRT). The liganded VDR then interacts with Retinoid X receptor (RXR) and the heterodimer binds to the vitamin D response element (VDRE). A number of coactivators such as CBP/p300, steroid receptor coactivator 1 (SRC-1) and the vitamin D receptor interacting protein 205 (DRIP205) contribute to this gene transcription. The DRIP205 is part of the mediator complex that links transcription factors to the initiation factor II (TFII) that are associated with RNA polymerase II (pol II) at the core promoter where the target genes are transcribed. There are a large number of vitamin D-sensitive target genes most of which are activated by 1,25(OH)2D3, but there are some that are repressed. The transcription of these genes contributes to the control of many different cellular processes:
There are multiple polymorphisms of the VDR gene and some of these have been associated with various disorders including autoimmune diseases and cancer. Expression of the VDR is reduced in a large proportion of the population in the Mediterranean island of Sardinia that suffer from Multiple sclerosis (MS). These MS patients have reduced expression of the Ifng gene that encodes Interferon-γ (IFN-γ), which plays a role in regulating the expression of the VDR.
The cancer drug bexarotene acts by stimulating RXR and may thus increase the activity of vitamin D. Such an action might explain the reported beneficial effects on both Parkinson's disease and Alzheimer's disease, but these findings are somewhat controversial.
Vitamin D control of bone function
One of the important actions of vitamin D is to regulate bone remodelling by regulating the expression of many of the genes that control the function of the osteoblasts and osteoclasts and thus regulates both bone formation and bone resorption (Module 7: Figure bone cells).
The vitamin D hormone 1,25(OH)2D3 (Module 7: Figure vitamin D metabolism) acting through the vitamin D receptor (VDR) activates a number of genes that regulate bone formation (Module 7: Figure vitamin D receptor activation):
Vitamin D control of cell proliferation
Vitamin D (calcitriol) can have a profound effect on cellular proliferation. Deficiencies of vitamin D have been identified in many cancers and supplementation with this vitamin D has been used as a preventive measure to reduce cancer risk and prevalence.
Vitamin D control of Ca2+ homoeostasis
One of the important functions of vitamin D is to contribute to the mechanisms responsible for whole body Ca2+ homoeostasis (Module 7: Figure Ca2+ homoeostasis). The vitamin D hormone 1,25(OH)2D3 (Module 7: Figure vitamin D metabolism) acting through the vitamin D receptor (VDR) activates a number of genes that regulate different aspects of the mechanisms that regulate the serum level of Ca2+ (Module 7: Figure vitamin D receptor activation):
Expression of this gene is increased in intestinal cells to provide the calbindin D-9k, which acts as a buffer to facilitate the diffusion of Ca2+ from the apical membrane across the cell to the basolateral surface where it is pumped out of the cell by the plasma membrane Ca2+-ATPase 1b (PMCA1b) pumps that are also regulated by 1,25(OH)2D3 (Module 7: Figure intestinal Ca2+ reabsorption).
Expression of this gene is increased in kidney cells to provide the calbindin D-28k, which acts as a buffer to facilitate the diffusion of Ca2+ from the apical membrane across the cell to the basolateral surface where it is pumped out of the cell by the plasma membrane Ca2+-ATPase 1b (PMCA1b) pumps that are also regulated by 1,25(OH)2D3 (Module 7: Figure kidney Ca2+ reabsorption).
This gene regulates the expression of the plasma membrane Ca2+-ATPase 1b (PMCA1b) that extrudes Ca2+ from cells.
This gene regulates the expression of the plasma membrane Ca2+-ATPase 2c (PMCA2c) that extrudes Ca2+ from cells.
Vitamin D control of inflammation
Vitamin D can exert a beneficial effect by dampening down inflammatory responses to reduce the formation of inflammatory cytokines such as Tumour necrosis factor (TNF). The primary action of Vitamin D seems to depend on the suppression of T lymphocyte-mediated inflammation as is the case in Multiple sclerosis (MS). Vitamin D may also act on microglia in the brain to reduce inflammation in Alzheimer's disease (AD) (Module 12: Figure Inflammation and Alzheimer's disease).
Ca2+ reabsorption by the intestine
Absorption of Ca2+ by the intestine is a key component of Ca2+ homoeostasis (Module 7: Figure Ca2+ homoeostasis). Most of this absorption takes place in the small intestine. Movement across the intestinal epithelium occurs through either a passive paracellular pathway or an active transcellular pathway (Module 7: Figure intestinal Ca2+ reabsorption). The paracellular pathway seems to occur down the length of the intestine, whereas the transcellular mechanism is found mainly in duodenum and upper jejunum.
The active transcellular pathway consists of three main steps. Firstly, there is the passive entry of Ca2+ from the lumen through vanilloid transient receptor potential 6 (TRPV6), which is a member of the transient receptor potential (TRP) ion channel family (Module 3: Figure TRP channel phylogeny). TRPV6 functions in much the same way as TRPV5 does in Ca2+ reabsorption by the kidney (Module 7: Figure kidney Ca2+ reabsorption). An important property of TRPV5 and TRPV6 is that they inactivate rapidly when the intracellular level of Ca2+ increases. As occurs in the kidney, this inactivation of TRPV6 may be alleviated by a Ca2+ buffer, in this case calbindin D-9k. As it charges up with Ca2+, the calbindin D-9k is thought to leave the channel and then diffuse across the cell to the basolateral surface, where it gives up its cargo to the plasma membrane Ca2+-ATPase 1b (PMCA1b) pumps that extrude Ca2+, thus completing the transcellular flux of Ca2+(Module 7: Figure kidney Ca2+ reabsorption).
The transcellular reabsorption of Ca2+ by the intestinal cells is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which acts by increasing the expression of all of the main components responsible for transporting Ca2+ across the cell. The 1,25(OH)2D3 binds to the vitamin D receptor (VDR), which then acts on the VDR response element (VDRE) to increase the expression of TRPV6, calbindin D-9k and PMCA1b.
Bone is a living organ that serves both mechanical and physiological functions. The mechanical function is obvious; bone is the main structural component of the skeleton. The physiological function is mainly concerned with mineral metabolism and particularly the maintenance of Ca2+ homoeostasis (Module 7: Figure Ca2+ homoeostasis). The average human skeleton contains about 1000 g of Ca2+ and is the major internal reservoir from which Ca2+ can be added or removed. When the dietary supply of Ca2+ is adequate, the rate of bone formation and bone resorption are approximately equal at about 400 mg/day. Bone is not a static organ, but is constantly being remodelled in response to changing skeletal requirements. During bone remodelling, there is an ebb and flow of Ca2+ between bone and plasma that is carried out by the bone cells.
A cross-section through a piece of compact bone reveals that much of the matrix is organized into long circular rods called osteons (Module 7: Figure bone structure). Blood vessels ramify through the central Haversian canals, which are connected to the Volkmann's canals. The matrix is composed of proteins (e.g. collagen, fibronectin and osteopontin) that provide a framework for the deposition of Ca2+ and phosphate. The main matrix protein is collagen, which is cross-linked in such a way that the individual fibrils retain their ordered structure despite the massive impregnation with minerals.
During growth, bones increase in diameter by resorbing matrix material from the endosteal surface while depositing new material at the periosteal surface. Bone growth is thus an example of how the skeleton is constantly been remodelled by the addition and removal of the matrix. This constant turnover of bone is regulated by three main bone cell types (Module 7: Figure bone cells):
An important aspect of bone remodelling is the way the processes of bone formation and resorption are co-ordinated through a process of bone cell coupling. During development, growth of the skeleton occurs through an increase in the rate of bone formation over that of bone resorption. In adulthood, these processes come into balance, and this accounts for the rate of Ca2+ flux in and out of the bone being equal at 400 mg/day. Osteoporosis occurs when this balance is perturbed so that the rate of bone resorption exceeds that of bone formation, whereas the opposite occurs in osteopetrosis.
Osteoblasts are derived from mesenchymal stem cells (MSCs) and their maturation into mature bone forming cells (a process of osteoblastogenesis) is driven by the canonical Wnt signalling pathway (Module 8: bone cell differentiation). Osteoblasts cover the bone-forming surfaces (Module 7: Figure bone structure), where they are responsible for synthesizing and secreting the collagen fibrils and osteocalcin, which constitute the organic phase of the bone matrix (Module 7: Figure osteoblast function). They are typical protein-secreting cells with extensive arrays of rough endoplasmic reticulum (Module 7: Figure bone cells). As these secrete bone, they become trapped in the forming bone and gradually transform into the osteocytes.
A large number of signalling pathways have been implicated in the control of bone formation by the osteoblasts. There appear to be two main mechanisms. First, there are the signalling pathways that drive osteoblastogenesis to promote bone formation by increasing the number of cells participating in this activity. Secondly, there are mechanisms that modulate the activity of fully differentiated osteoblasts. With regard to the former, the canonical Wnt signalling pathway acting through β-catenin has a primary role in driving osteoblast proliferation and differentiation (Module 7: Figure osteoblast function). In addition, the insulin-like growth factor 1 (IGF-1), which acts through the PtdIns 3-kinase signalling pathway, promotes osteoblast survival.
A number of pathways have been invoked with regard to the regulation of the osteoblasts once they have differentiated. The cyclic AMP signalling pathway, which is activated or inhibited by different agonists, controls the expression of the extracellular matrix components, such as collagen I and osteocalcin responsible for bone formation. They also control the expression of RANKL that is part of a feedback interaction with the osteoclasts (Module 8: bone cell differentiation). Positive signals for bone formation are provided by β2 adrenoreceptors that activate adenylyl cyclase (AC) and parathyroid hormone (PTH) that also stimulates AC, but may also be coupled to phospholipase C (PLC). The cyclic AMP signalling pathway, which results from AC activation, stimulates protein kinase A (PKA) that then phosphorylates various transcription factors. Phosphorylation of CREB increases expression of activating transcription factor (ATF), which then increases expression of RANKL, collagen I and osteocalcin. The collagen I and osteocalcin are released to form the bone matrix. The RANKL is inserted into the membrane where it functions as an agonist to stimulate RANK in the osteoclasts. In addition, the PKA inhibits the formation and release of osteoprotegerin (OPG), which inhibits this interaction between RANKL and RANK and thus enhances the activation of osteoclasts. This RANKL/RANK pathway, which enables osteoblasts to excite osteoclasts, enhances bone resorption and opposes the bone forming function of the osteoblasts. This interaction between the osteoblasts and osteclasts is thus an example of the dynamic nature of the bone cell coupling mechanisms that ensure that bone formation and bone resorption are precisely balanced.
Osteoblasts also express 5-HT1B receptors that respond to 5-hydroxytryptamine (5-HT, also known as serotonin) (Module 1: Figure G protein-coupled receptors) by inhibiting the cyclic AMP signalling pathway thus resulting in a decrease in bone resorption (Module 7: Figure osteoblast function). The circulating 5-HT is released by the enterochromaffin cells located in the gut (Module 7: Figure small intestine).
Mutations in LRP5 which codes for the LRP5 co-receptor that operates in the Wnt signalling pathway, can cause either extremely high or low bone mass traits. A high bone mass syndrome (HBM) is caused by gain-of-function mutations of LRP5. On the other hand, loss-of-function LRP5 mutations are linked to osteoporosis pseudoglioma (OPPG). The effect of the LRP5 mutations may either influence the operation of the osteoblasts directly or they act indirectly by altering the way that the enterochromaffin cells release 5-HT.
Osteoblasts can also be considered as an endocrine cell in that the osteocalcin released during bone formation (Module 7: Figure osteoblast function) can function as a circulating hormone. Osteocalcin is released from the osteoblast in a carboylated form in that three of the Gla residues are carboxylated and this enhances its affinity for the bone matrix during extracellular matrix formation. A portion of this carboxylated osteocalcin is decarboxylated to form undercarboxylated osteocalcin that enters the circulation to function as a hormone. This circulating osteocalcin can enhance the release of insulin by the insulin-secreting β-cells, it can enhance insulin sensitivity by increasing the release of adiponectin by white fat cells and it has been reported to support the survival of germ cells by boosting testosterone production. The ability of osteocalcin to stimulate insulin release by the insulin-secreting β-cells sets up a positive-feedback loop in that the osteoblasts provide a signal that then enhances the release of insulin which then acts to increase bone formation.
Osteocytes, which are by far the most numerous of the bone cells, are derived from osteoblasts that are buried by the growing bone (Module 8: bone cell differentiation). As the osteoblasts form bone, they transform into the osteocytes that are embedded within the bone matrix. As they transform, the volume of the cell body declines and they develop long radiating extensions that lie within an interconnected canalicular system (Module 7: Figure bone cells). A tracer molecule such as horseradish peroxidase, when added to the blood, can permeate throughout this system of canals, implying that plasma can penetrate throughout the matrix. The finger-like extensions of individual osteocytes lying within this fluid-filled space communicate with the other bone cells on the surface through gap junctions. It is this interaction with the other bone cells that appears to be crucial for the function of these osteocytes and may contribute to bone cell coupling.
The osteocytes are not able to form or resorb bone, but they do appear to play a critical role in bone remodelling by modulating the activity of the osteoblasts and osteoclasts. The osteocytes appear to function as strain sensors to provide information about the degree of mechanical loading. When the loading stress on bone is increased, more bone is formed, but when loading declines, bone is removed. Osteocytes may thus play a key role in bone remodelling by regulating the balance between bone formation and resorption by the osteoblasts and osteoclasts respectively. Since they are embedded in the bone matrix, the osteocytes are in a unique position to detect minute deformations and can relay this information through their physical contacts with the osteoblasts and osteoclasts.
Just how the osteocytes detect minute bone deformations and relay this information to the other bone cells is still not clear. These cells are difficult to study because they are embedded within bone. A strain amplification hypothesis proposes that there is an elaborate interconnected strain sensory system running down the length of the long osteocyte processes (Module 7: Figure osteocyte mechanotransduction). The core of these processes contains hexagonally packed actin filaments that are connected to integral membrane proteins, which in turn, are connected to rigid transverse tethering elements that are attached to the bone lining the canals. The basic idea is that bone deformation displaces the pericellular fluid which then deflects the transverse elements to create a ‘hoop strain’ that will be transmitted to the actin core. Amplification might be achieved by virtue of having numerous transduction elements dispersed throughout the interconnected protein network. Just how this molecular deformation creates an intracellular signal is unclear, but one can imagine that tension on the integral membrane proteins might act to generate messengers, perhaps from the opening of Ca2+ channels. Such a mechanotransduction process is very reminiscent of the mechanoelectrical transduction process present on hair cells, where an ion channel is also connected to both the extracellular matrix and an internal actin core (Module 10: Figure tip link).
The nature of the osteocyte signals that are used to communicate with the other bone cells is uncertain. Given the apparent importance of the gap junctions, it is conceivable that small messengers such as Ca2+ or cyclic AMP might be passed directly to the osteoblasts and osteoclasts. Alternatively, the osteocyte might release factors such as prostaglandin E2 (PGE2), ATP or nitric oxide (NO). The osteocytes respond to parathyroid hormone (PTH) by decreasing the release of sclerostin (SOST) that acts to inhibit the binding to the LRP5/6 co-receptors (Module 7: Figure osteoblast function).
Osteoclasts are large multinucleated cells whose function is to resorb bone (Module 7: Figure bone cells). Osteoclasts originate from a haematopoietic stem cell (HSC) line located in the bone marrow by a process of osteoclastogenesis (Module 8: bone cell differentiation). An important determinant of bone remodelling is the balance between the number of osteoblasts and osteoclast. The rate of osteoclast formation is thus a critical factor in determining the rate of bone resorption. Once the mature osteoclasts have been formed, they are activated to begin bone resorption once they attach and spread out on the bone surface. The process of bone resorption by the osteoclast depends upon two major control mechanisms:
An important aspect of bone remodelling is the balance between the activity of the osteoclasts (bone resorption) and osteoblasts (bone formation). One way of enhancing the contribution of the former is to increase the number of osteoclasts by stimulating their differentiation. The process of osteoclastogenesis is responsible for providing a constant supply of osteoclasts (Module 8: bone cell differentiation). The orderly development of a mature osteoclast is tightly regulated by a number of cytokines such as colony-stimulating factor-1 (CSF-1), which acts on the colony-stimulating factor (CSF-1) receptor (CSF-1R). Another key component is the tumour necrosis-related factor called receptor activator of nuclear factor κB (NF-κB) ligand (RANKL), which is located on the surface of the osteoblasts (Module 8: Figure osteoclastogenesis). The section on osteoclastogenesis contains details of the signalling pathways responsible for orchestrating the transcriptional events responsible for carrying out this developmental process (Module 8: Figure osteoclast differentiation). Once the mature osteoclasts are formed, they are highly mobile and can migrate over the bone surface until they find an area where the bone has to be resorbed. The interaction with the bone surface triggers an osteoclast activation process that converts the mobile cell into a sedentary cell that can begin to resorb bone (Module 7: Figure osteoclast function).
The mature osteoclast is unusual in being able to rapidly switch from being a highly mobile cell, which can migrate to different bone regions, to a sedentary cell that is polarized to carry out the process of bone resorption (Module 7: Figure osteoclast podosome). The switch to a polarized cell occurs when osteoclasts attach themselves to the bone surface. The signalling mechanisms have to control both the movement of the migrating cell and the events that occur when the cell attaches and spreads out on the bone to become a sedentary resorbing cell.
The signalling pathways responsible for activating osteoclasts are complex and not fully established. An important feature of osteoclast activation seems to depend on integrin signalling, which can generate a number of intracellular messengers (Module 1: Figure integrin receptor). In the case of osteoclasts, integrin signalling takes place in the podosome, which is a specialized adhesion site that is used by the cell both when it is migrating and as it spreads out to begin resorbing bone (Module 7: Figure osteoclast podosome). The signalling events that occur within this attachment site are described in the section on the osteoclast podosome. Here we will concentrate on the events that occur when the osteoclast begins to settle down on the bone surface to begin the process of resorption as outlined in the following sequence of events Module 7: Figure osteoclast function:
Bone cell coupling
Bone remodelling is a dynamic process where the activity of the bone forming osteoblasts is balanced by the bone resorbing activity of the osteoclasts. There is a positive balance during growth when there is net bone formation and a negative balance during ageing resulting in osteoporosis. During most of adult life, however, these two processes are balanced, and this is achieved through a tight coupling of the activities of the osteoblasts and osteoclasts. Osteopetrosis is a rare inherited disorder caused by mutations that interfere with the osteoclast activation mechanism. Although there is considerable information on osteoblast-to-osteoclast signalling, there is less information on the reverse process of osteoclast-to-osteoblast signalling. These two cell types use a number of mechanisms to communicate with each other.
The osteoblasts exert a profound effect on the process of osteoclastogenesis where they provide the receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). They also release osteoprotegerin (OPG), which is a soluble RANKL-binding decoy receptor that inhibits the action of RANKL. Parathyroid hormone (PTH) promotes bone resorption by inhibiting the release of OPG and enhancing the expression of RANKL (Module 8: Figure osteoclastogenesis).
Osteoclast to osteoblast signalling
There is evidence that once osteoclasts have been activated to resorb bone, there is a simultaneous activation of the osteoblasts to replace the bone that has been resorbed. It appears that the osteoclasts orchestrate this activation of the osteoblasts through mechanisms that are still being worked out. One suggestion is that the osteoclasts may communicate with the osteoblasts through the ephrin (Eph) receptor signalling pathway (Module 1: Eph receptor signalling). The osteoclast expresses ephrin-B2, which then activates forward signalling through the EphB4 receptors to enhance osteoblast differentiation (Module 7: Figure osteoclast function). Since Eph receptor signalling is bidirectional, the reverse signalling received through ephrin-B2 simultaneously switches off the osteoclasts, thus further enhancing the ascendancy of the osteoblasts to replace the previous phase of bone resorption with bone formation. The fact that this cell communication is taking place through close cell contacts necessitated by the nature of Eph signalling means that the coupling between resorption is achieved within a highly localized region.
The podosome is one of the cell adhesion complexes that are used by cells to attach to the extracelluar matrix (ECM) or to interact with other cells. Podosomes contain many of the same components that are found in the focal adhesion complex (Module 6: Figure integrin signalling), but they differ from the latter in that they are much more labile. While the adhesion complexes can last for hours, podosomes turn over within minutes. The osteoclast podosome is a typical example of how this structure can rapidly remodel the actin cytoskeleton to enable both movement of the migrating cell and the formation of the actin ring that contributes to the tight seal that forms as the astrocyte spreads out to form the resorbing cell (Module 7: Figure osteoclast podosome). The podosomes are highly dynamic structures that can assemble and disassemble in a few minutes.
The rapid formation of the podosome is driven by integrin signalling pathways. Of the multiple known combinations of the α- and β-integrin subunits (Module 1: Figure integrin heterodimeric complexes), osteoclasts mainly express the αV/β3 heterodimer. Once the αV subunit recognizes the RGD motif on bone matrix proteins, such as osteopontin and bone sialoprotein, the receptor undergoes a conformational change enabling the cytoplasmic regions to bring together the macromolecular complex that assembles actin filaments (Module 7: Figure osteoclast podosome). One of the earliest events is the activation of non-receptor protein tyrosine kinases such as proline-rich tyrosine kinase 2 (Pyk2) and Src. Following integrin receptor activation, Pyk2 is phosphorylated on Tyr-402, which provides a binding site for the Src homology 2 (SH2) domain of Src. This binding of Src removes its autoinhibition, allowing it to phosphorylate and recruit the adaptor protein Cbl, which has two functions. Firstly, the Cbl down-regulation of signalling components (Module 1: Figure receptor down-regulation) will begin to apply the brakes to the signalling complex by initiating the proteasomal degradation of key transducing elements such as Src. Secondly, Cbl acts together with Src to recruit PtdIns 3-kinase that generates the lipid messenger PtdIns3,4,5P3, which contributes to the processes that control actin assembly.
The monomeric G protein Rho, which is responsible for a number of signalling events related to actin remodelling (Module 2: Figure Rho signalling), is activated by PtdIns3,4,5P3 (Module 7: Figure osteoclast podosome). One of the functions of Rho is to activate the PtdIns4P 5-kinase (PtdIns4P 5-K) that converts PtdIns4P into PtdIns4,5P2. The latter has an important role in the process of PtdIns4,5P2 regulation of actin remodelling that is responsible for the activation of Wiskott–Aldrich syndrome protein (WASP) and the actin-related protein 2/3 complex (Arp2/3 complex) that control actin assembly. The podosomes contain proteins such as talin, paxillin and filamin that function to attach the growing actin filaments to the β-integrin subunit. In the case of the resorbing cell, these filaments form the actin ring that contributes to the tight seal (Module 7: Figure osteoclast function). Another key feature of the podosome is a complex formed between supervillin and the cytoskeletal proteins such as actin, non-muscle myosin II (NMII) and myosin light chain kinase (MLCK). Activation of this contractile unit may result in the dissolution of the podosome.
Chondrocytes are derived from mesenchymal stem cells (MSCs) that split into two cell lineages early in developmental (Module 8: Figure MSC differentiation). The skeletal precursor line then undergoes two separate processes: chondrogenesis, which results in the formation of the chondrocytes and osteoblastogenesis that gives rise to the osteoblasts. Chondrocytes are located in articular cartilage, where they function to maintain the dynamic equilibrium between matrix synthesis and degradation. Alterations in this equilibrium result in rheumatoid arthritis (RA) and osteoarthritis (OA). These pathological changes may depend upon pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumour necrosis factor α (TNF-α), which act on chondrocytes to increase the release of matrix proteinases and to inhibit the synthesis of cartilage proteoglycans and type II collagen. ATP may also play a role because chondrocytes release this nucleotide, which then acts in an autocrine manner to enhance the release of prostaglandin E2 (PGE2).
Skin is the largest organ of the body. Its primary function is to provide a protective barrier against many environmental insults such as temperature changes, UV radiation and pathogens. It is composed of two main layers: the epidermis (composed of keratinocytes) and the dermis that contains the sensory nerve endings and blood vessels (Module 7: Figure skin). In addition, the skin also has a number of specialized cells and structures:
In performing its barrier role, the skin suffers numerous assaults such as harmful radiation, scratches and wounds that can be repaired by a remarkable capacity for self-renewal. The adult processes of skin regeneration and repair have much in common with the process of skin development that occurs when the skin is formed in the embryo.
Keratinocytes are the cells that make up the multi-layered epidermis. The different layers consist of keratinocytes that are in different stages of the differentiation that occurs during the process of skin development and this gives rise to the different layers.
The basal layer that rests on the extracellular matrix is mitotically active and is responsible for generating the continuous supply of keratinocytes that then move outwards and gradually transform into the other layers of the epidermis (Module 7: Figure skin cells).
The spinous layer is formed from spindle-shaped spinous cells that stop growing once they move away from the basal layer. Having withdrawn from the cell cycle, they initiate a terminal differentiation programme resembling that which occurs during skin development.
To form the granule layer, cells of the spinous layer move outwards, they begin to flatten and accumulate numerous granules.
The stratum corneum forms as the granule layers die to form the flattened cornified layers that make up the upper surface of the skin.
The way in which the keratinocytes are constantly being formed and then transformed to form the outer layers of the skin is first seen during skin development and continues to operate during skin regeneration and repair.
The keratinocytes release thymic stromal lymphopoietin (TSLP), which has been linked to the development of atopic dermatitis (AD). The TSLP then acts on sensory neurons in the skin to activate Itch (Module 10: Figure Itch signal transduction mechanism).
The dermis is a connective tissue layer that lies beneath the epidermis (Module 7: Figure skin). The dermis contains the mesenchymal cells that contribute to the control of skin regeneration and repair. The dermis also contains the blood vessels and nerve fibres that serve the skin.
Hairs are produced by the hair follicles that are a self-contained organ, which is formed from a specialized region of the epidermis that invaginates deep into the dermis (Module 7: Figure skin cells). The hair follicle is divided into two discrete regions: a permanent non-cycling upper follicle (sebaceous gland and the bulge region containing the stem cells) and a lower cycling bulb region that produces the hair and undergoes a regular hair follicle cycle (Module 8: Figure hair follicle cycle).
Arrector pilli muscle
The arrector pilli muscle functions to adjust the position of the hair shaft. This bundle of smooth muscle cells forms an attachment between the hair follicle and an infolding of the epidermis (Module 7: Figure skin cells). The neural innervation controls the contraction of this muscle, which causes the hair to 'stand on end’.
The branched sebaceous gland is made up of epithelial cells that secrete an oily secretion called sebum that oozes out through a duct on to the hair shaft before moving out to the skin surface (Module 7: Figure skin cells).
The surface of the body has approximately 20000 small tubular epithelial sweat glands (Module 7: Figure skin). They consist of two parts: about half of the 3–5mm long tube is the secretory coil that is located deep in the dermis. The sweat gland secretory coil cells are responsible for secreting an isotonic NaCl solution (Module 7: Figure sweat gland function). This primary secretion then passes through the straighter sweat gland reabsorptive duct cells, which reabsorbs much of the Na+ and Cl− resulting in the hypotonic sweat that passes out through pores on the surface of the skin.
Sweat glands are innervated by both parasympathetic and sympathetic neurons that function to regulate the formation of sweat. An increase in body temperature induces the anterior hypothalamus to stimulate the sympathetic neurons that release noradrenaline (NA), which is one of the sweat-promoting stimuli. The NA acts through the cyclic AMP signalling pathway to control fluid secretion by the sweat gland secretory coil cells. These cells are also activated by acetylcholine (ACh) released from the parasympathetic neurons.
Sweat gland secretory coil cells
The secretory coil cells, which are responsible for secreting the primary isotonic NaCl solution, are regulated by both the parasympathetic and sympathetic nervous systems (Module 7: Figure sweat gland function).
Fluid secretion by the secretory coil cells is driven by a net flux of Cl− from the plasma into the lumen. Entry of Cl− across the basolateral membrane is carried out by the Na+/K+/2Cl− 1 (NKCC1) cotransporter with the driving force supplied by the Na+ entering the cell down its electrochemical gradient that is maintained by the classical ouabain-sensitive Na+ pump (Na+/K+-ATPase). Any accumulation of K+ is leaked back to the plasma through a K+ channel. The accumulation of Cl− within the cell increases the electrochemical gradient and this enables this anion to pass into the lumen through two channels: the cystic fibrosis transmembrane conductance regulator (CFTR) channel and one of the Ca2+-sensitive chloride channels. The flow of Cl− into the lumen then provides the electrical gradient for a parallel flow of Na+ and this movement of NaCl then provides the osmotic gradient for the flow of water.
This fluid secretion can be regulated by both noradrenaline (NA) released from the sympathetic neurons or by acetylcholine (ACh) coming from the parasympathetic neurons. These two stimuli use different signalling pathways to regulate the mechanism of fluid secretion. NA acts through the cyclic AMP signalling pathway to open the CFTR that gates Cl− across the apical membrane. On the other hand, ACh uses the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette to open the Ca2+-sensitive chloride channels (CLCAs).
In patients with cystic fibrosis, the defect in the CFTR channel results in a marked reduction in the secretory response to NA, but there is no defect in the response to ACh, which uses the Ca2+-sensitive chloride channels instead of CFTR.
Sweat gland reabsorptive duct cells
The reabsorptive duct cells dilute the isotonic solution produced by the secretory coil cells by removing Na+ and Cl− to produce hypotonic sweat (Module 7: Figure sweat gland function). The driving force for this flux of ions depends upon the Na+/K+-ATPase located on the basolateral membrane that extrudes Na+ from the cell and this provides the driving force for the uptake of Na+ through the epithelial Na+ channels (ENaCs) in the apical membrane. The balancing negatively charged ion is Cl−, which enters and leaves the cell through the cystic fibrosis transmembrane conductance regulator (CFTR) channels located in the apical and basolateral membranes.
Melanocytes are dendritic cells located in the basal layer of the skin epidermis (Module 7: Figure skin cells) and are also found in the uvea in the eye and also in the ear. Early in development these melanocytes, which are derived from the neural crest, migrate out into the skin where they comprise about 5–10% of the cells in the basal layer. Melanocytes are responsible for pigmentation of the hair and skin. Skin colour depends upon three main chromophores: melanin, carotenoids and haemoglobin. The red tinge of the skin is provided by haemoglobin in red blood cells circulating in the profuse blood supply in the dermis (Module 7: Figure skin). Dietary carotenoids can also appear in the skin often evident on the palms of the hand. However, the most important chromophore is melanin, which comes in two forms: pheomelanin (red/yellow) and eumelanin (brown/black). The wide variation in pigmentation within the human population is largely dependent on variations in the production of these two major forms of melanin.
Melanin plays a primary role in protecting the skin against ultraviolet (UV) radiation. This photoprotection is adaptive in that the skin darkens in response to UV radiation. This tanning response depends on a process of melanogenesis, which is controlled by an interaction between the keratinocytes and melanocytes (Module 7: Figure melanogenesis). The keratinocytes respond to UV radiation by releasing various stimuli that diffuse out to activate the melanocytes to increase their synthesis of melanin that is then packaged into melanosomes and transported down the dendrites where it is released from the tips. These melanosomes are then taken up by the keratinocytes and tend to congregate over the top of the nucleus to form an apical cap that protects DNA from harmful UV radiation.
Modifications of the complex series of biochemical events that occur during melanogenesis and their orchestration by various signalling pathways are responsible for a number of disorders:
Melanogenesis is the tanning process that occurs in the skin in response to ultraviolet (UV) irradiation. It depends on an interaction between the keratinocytes, which provide the stimuli to induce the melanocytes to synthesize melanin that is then transferred to the keratinocytes. The following sequence of events summarizes the key events that occur during this two-way communication between the keratinocytes and the melanocytes (Module 7: Figure melanogenesis):
Mutations in MITF are responsible for Waardenburg syndrome 2a (WS2a).
The alimentary canal is a complex organ system responsible for digestion and the absorption of nutrients. It is composed of two main parts: the stomach and the intestine. The arrival of food in the stomach triggers a battery of neural and endocrine functions of the stomach designed to co-ordinate the control of acid secretion by the parietal cell. The intestine is divided into the small intestine and the colon. The small intestine has a major role in the absorption of nutrients, but it also has a role in maintaining the fluid content of the intestinal lumen by adjusting the balance between NaCl absorption and secretion. There is a small intestine neural and endocrine system that regulates ion and water transport across the small intestine. Similar mechanisms operate in the colon to regulate the bidirectional flux of NaCl. The small intestine also contributes to Ca2+ homoeostasis (Module 7: Figure Ca2+ homoeostasis). Ca2+ reabsorption by the intestine is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3].
The stomach functions as a storage organ where food is processed into a liquid form called chyme. Secretion of acid by parietal cells located in the lining of the stomach (Module 7: Figure stomach structure) creates conditions for the early stages of digestion and also plays an important role in sterilizing the upper part of the gastrointestinal tract. The neural and endocrine functions of the stomach are responsible for providing the neural (acetylcholine), endocrine (gastrin) and paracrine (histamine and somatostatin) systems that co-ordinate the control of parietal cell secretion.
The stomach is divided into three parts, the fundus, the body and the antrum, which contain different cell types. In the fundus and body, the gastric epithelium of the stomach invaginates to form gastric glands, which are composed of mucous neck cells that secrete a mucous glycoprotein, chief (zymogen) cells that secrete pepsinogen and the parietal cells that secrete acid. There are no parietal cells in the epithelium of the antrum, but this region does have G cells that synthesize and release gastrin, which is one of the major stimuli of acid secretion.
Secretion of hydrochloric acid (HCl) by the parietal cells maintains the very low pH in the stomach and the gastric mucosa, which has developed special devices for protecting itself against acid. If this defensive system breaks down or if it is overwhelmed by excessive acid production, the outer epithelium begins to leak acid into the underlying interstitial layers causing bleeding and the development of peptic ulcers. There is thus considerable interest in understanding the neural and endocrine functions of the stomach that release the excitatory and inhibitory stimuli that regulate acid secretion.
Neural and endocrine functions of the stomach
Control of acid secretion depends upon neural, endocrine and paracrine mechanisms that are activated by different aspects of feeding (Module 7: Figure stomach structure). There is a cephalic phase (control of acid secretion by the brain) during which the smell of food and the process of ingestion generates neural signals that are conveyed to the stomach by the vagus nerve. These neural signals may be responsible for releasing the gut hormone ghrelin from the X/A-like cells. The vagus does not innervate parietal cells directly, but the signals are relayed through a network of nerve cells in the myenteric and submucous plexus, which contain the postganglionic neurons that then innervate the parietal cells. These cholinergic neurons release acetylcholine (ACh), which is a potent parietal cell stimulus. Postganglionic neurons also innervate the enterochromaffin-like cells (ECLs) to stimulate the release of histamine, which is another major activator of parietal cell secretion.
Another part of the cephalic phase that is mediated through the vagus nerve is the release of gastrin from the G cells located in the pyloric antrum. As described above, the vagus terminates at the myenteric and submucous plexus from which a postganglionic neuron releases gastrin-releasing peptide (GRP) to activate the G cells to release gastrin. This gastrin enters the bloodstream and is carried to the fundic region, where it has multiple functions to activate the parietal cells, the enterochromaffin-like cells (ECLs) and the somatostatin-secreting D cells. Separate populations of D cells, which are located in the antral region, are part of the epithelium and have an apical surface that can sample the level of H+ as part of a negative-feedback loop. If the acid content of the stomach gets too high, there is an increase in the release of somatostatin that then switches off the release of gastrin by the G cells. This cephalic phase is controlled solely by the brain and can occur independently of any food entering the stomach.
In addition to the cephalic phase, there is a gastric phase of secretion that is initiated by the arrival of food in the stomach. Sensory neurons in the wall of the mucosa respond to stretching of the stomach, and this triggers a simple reflex through the submucous plexus to activate the postganglionic neurons that activate both the parietal and G cells as described above. Another aspect of the gastric phase is the direct stimulation of the G cells by the products of digestion such as the amino acids.
G (gastrin) cells are located in the gastric epithelium in the antral region of the stomach (Module 7: Figure stomach structure). These are unusual endocrine cells, as they are part of the gastric epithelium. They have apical microvilli that increase the surface area to enhance their ability to detect food components in the stomach such as amino acids, which act to increase the release of gastrin. The G cell is also activated via the vagus nerve, which makes contact with the myenteric and submucous complex to activate a postganglionic neuron that releases gastrin-releasing peptide (GRP) that functions to stimulate the release of gastrin.
This antral region also contains D cells that are a part of the epithelium and they function to secrete somatostatin (Sst), which inhibits the G cells. The apical membrane has microvilli, whereas the basal region has characteristically long projections that release Sst in the vicinity of the G cell.
Gastrin is an amidated heptapeptide that is synthesized and released by the G cells. Gastrin is synthesized as a large preprogastrin that undergoes a number of post-translational modifications as it passes through the endoplasmic reticulum/Golgi/granule secretory pathway:
Gastrin released from the G cells enters the surrounding blood vessels and is carried to the fundal region where it has three main targets: it stimulates HCl secretion by the parietal cells, it stimulates histamine release by the enterochromaffin-like cells (ECLs) and it stimulates somatostatin release by the D cells.
Infection of the stomach by Helicobacter pylori enhances the secretory activity of these G cells, and this may contribute to the enhanced secretion of acid that causes peptic ulcer.
Zollinger–Ellison syndrome is caused by the overproduction of gastrin.
D cells are endocrine cells that release somatostatin (Sst). They are found in tissues such as the islets of Langerhans, intestine and in different regions of the stomach (Module 7: Figure stomach structure). They have long cytoplasmic processes that make intimate contact with both the parietal and G cells. Such a paracrine arrangement allows Sst to exert its inhibitory effect by being released directly on to the G cells and the parietal cells. In the case of the latter, somatostatin acts to inhibit acid secretion (Module 7: Figure HCl secretion).
Infection of the stomach by Helicobacter pylori inhibits the secretory activity of these D cells and this may contribute to the enhanced secretion of acid that causes peptic ulcers.
Enterochromaffin-like cells (ECLs)
Enterochromaffin-like cells (ECLs), which resemble mast cells, are endocrine cells located within the gastric mucosa, where they contribute to the control of acid secretion by releasing histamine (Module 7: Figure stomach structure). Histamine is synthesized from histidine by a decarboxylation reaction carried out by histidine decarboxylase (HDC). The histamine is stored within large secretory vesicles that are released by a variety of stimuli. The main stimuli are gastrin, which is produced by the antral G cells and reaches the ELCs through the circulation and pituitary adenylyl cyclase-activating peptide (PACAP) that is released from the nerve endings of postganglionic neurons.
Release of histamine is controlled by Ca2+ signalling pathways. Gastrin acts through the cholecystokinin-B (CCKB) receptors that are coupled to the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette. In addition to driving histamine secretion, the increase in Ca2+ also activates transcription of genes for HDC, the vesicular monamine transporter of subtype 2 (VMAT-2), which transfers histamine into the vesicular store, and chromogranin A. The action of PACAP is unclear; the most likely possibility is for it to act through the PACAP type 1 (PAC1) receptor that is coupled to the same signalling pathway used by the gastrin receptor (CCKB).
Prolonged stimulation of these cells can result in hyperplasia and this can contribute to Zollinger–Ellison syndrome. Such a scenario occurs in gastrinoma when there are tumours that cause an excessive secretion of gastrin.
X/A-like cells, which are located mainly in the stomach, are endocrine cells specialized for the synthesis and release of the gut hormone ghrelin. They constitute about 20% of the endocrine cell population in the stomach but are also found in other gut regions (duodenum, ileum and colon). X/A-like cells have a large population of dense-cored vesicles containing stored ghrelin, which is released on anticipation of feeding and plays a major role in the control of food intake and body weight (Module 7: Figure control of food intake).
Parietal cells function to secrete acid into the stomach. They are located in the main body and, to a lesser extent, in the fundic region of the stomach (Module 7: Figure stomach structure). The gastric mucosa contains a large number of gastric glands, which open to the surface through gastric pits (Module 7: Figure parietal cell). The glands are composed of parietal cells interspersed amongst the chief (zymogen) cells that release pepsinogen. The mucous neck cells at the surface secrete mucous, which protects the surface against the harsh environment in the stomach.
The ultrastructure of the parietal cell reflects their function in fluid secretion. The surface area of the luminal surface is greatly enlarged through an extensive system of canaliculi that are lined with microvilli. These canaliculi are highly dynamic, depending on the degree of stimulation. In a resting cell, the canaliculi collapse in on themselves as the membrane is withdrawn into the cell to form tightly packed vesicles and short tubules known as the tubulovesicular system. When cells are stimulated to secrete acid, the collection of tubules and vesicles fuse with the apical membrane to reform the canaliculi. This fusion process seems to occur through the classical exocytotic/endocytotic cycle because the vesicles contain the usual components of the exocytotic machinery such as the N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNAREs), synaptobrevin [also known as vesicle-associated membrane protein 2 (VAMP2)] and syntaxin 3.
The canaliculi are the sites where H+ and Cl− are secreted to provide the osmotic gradient for a parallel flow of water. The control of parietal cell secretion is carried out by neural (acetylcholine), endocrine (gastrin) and paracrine (histamine and somatostatin) stimuli (Module 7: Figure HCl secretion).
Control of parietal cell secretion
Hydrochloric acid (HCl) secretion by the parietal cell is regulated by neural (acetylcholine), endocrine (gastrin) and paracrine (histamine and somatostatin) stimuli (Module 7: Figure HCl secretion). The key component of the ionic mechanisms responsible for acid secretion is the H+/K+-ATPase, which exists in two states. In the resting cell, the inactive enzyme is located on the tubulovesicular system (Module 7: Figure parietal cell). Following stimulation, canaliculi are formed when the vesicles and tubules fuse with the apical membrane and results in the activation of the H+/K+-ATPase that begins to transport H+ into the lumen of the canaliculi in exchange for K+ (Module 7: Figure HCl secretion). The extrusion of H+ provides the driving force for the parallel flux of Cl− and water. A continuous supply of H+ is provided by carbonic anhydrase that combines CO2 and water to form H+ and HCO3−. The latter is then extruded across the basolateral membrane by a Cl−/HCO3− exchanger, which also supplies the influx of Cl− used for acid secretion at the canaliculus. This elegant fluid secretory mechanism is controlled by the fusion of the tubulovesicular system with the canaliculus that results in the activation of the HCO3−/Cl− exchanger.
The stimuli that control acid secretion all activate signalling pathways that converge on this fusion event. The neural signal acetylcholine acts through the muscarinic M3 receptor to induce the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette. Exactly the same signalling mechanism is employed by gastrin acting through cholecystokinin-B (CCKB) receptors. The increase in Ca2+ then acts to stimulate the fusion events that result in the transfer of tubulovesicles to the apical membrane. The role of Ca2+ is still not clear, but one of its actions is to stimulate Ca2+/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate the Ca2+-sensitive phosphoprotein of 28 kDa (CSPP-28).
Histamine, which is one of the major secretagogues, acts on H2 receptors that are coupled to the cyclic AMP signalling pathway. Cyclic AMP then acts through protein kinase A (PKA) to phosphorylate a range of substrates that may contribute to the translocation and fusion of vesicles with the apical membrane. Two of these phosphorylated proteins (ezrin and lasp-1) function in actin remodelling. Lasp-1 appears to be an adaptor protein, which contains an N-terminal LIM domain, two internal nebulin repeats and a C-terminal Src homology 3 (SH3) domain. When lasp-1 is phosphorylated by PKA, it binds to actin and may thus contribute to reorganization of the cytoskeleton during membrane fusion. PKA also phosphorylates parchorin, which then translocates from the cytosol to the apical membrane, where it may contribute to the onset of acid secretion.
Somatostatin (Sst), which is released from the D cells (Module 7: Figure stomach structure), is a paracrine regulator that functions to inhibit acid secretion. Sst binds to an sstR2 receptor that acts through the G protein Gi to inhibit cyclic AMP formation by adenylyl cyclase (Module 7: Figure HCl secretion).
The main function of the small intestine is to absorb nutrients from the chyme that is periodically squirted out of the stomach into the duodenum. As the liquid chyme passes through the intestine, there are very large fluxes of ions and water in both directions. The structure of the small intestine reveals the presence of a highly infolded epithelium that is backed up by a complex interstitium containing layers of smooth muscle cells and a small intestine neural and endocrine system that controls small intestine fluid secretion (Module 7: Figure small intestine).
Structure of the small intestine
The intestine is divided into a number of regions. The chyme that leaves the stomach passes first into the duodenum, which together with the jejunum and ileum constitutes the small intestine. The ileum connects to the colon. The small intestine is a complex tissue consisting of outer layers of longitudinal and circular muscle surrounding the convoluted mucosa, which is highly infolded to produce long villi interspersed between the crypts of Lieberkühn (Module 7: Figure small intestine). Both the crypts and villi are lined with columnar epithelial cells that are responsible for nutrient absorption and the regulation of fluid absorption and secretion.
Each epithelial cell has approximately 1000 microvilli, which serve to increase the surface area available for absorption. This delicate absorptive surface is protected by a dense fluffy mucous coat of fine filaments attached to the outer surface of the microvilli. The regular organization of the microvilli is maintained by an actin cytoskeleton. Each microvillus has a bundle of actin filaments, which is attached to a cap at the tip of the microvillus. The other end of the actin bundle protrudes into the cytoplasm, where it makes contact with the terminal web.
The small intestine neural and endocrine system functions to control both intestinal motility and the transport functions of the mucosa.
Small intestine neural and endocrine system
Ion and water transport by the small intestine is regulated by stimuli originating from both neural and endocrine sources (Module 7: Figure small intestine). The intestine contains a nervous system often referred to as the enteric nervous system that rivals in complexity many parts of the central nervous system. Similar to what is found in the stomach (Module 7: Figure stomach structure), the enteric nervous system consists of a complex network of interconnecting neurons organized into the myenteric plexus and the submucous plexus (Module 7: Figure small intestine). They receive a cholinergic input from the vagal nerve and an adrenergic input from the sympathetic nervous system. This enteric neural complex functions as a local nervous system to control all of the major processes in the intestine. It regulates the activity of the longitudinal and circular layers of smooth muscle that are responsible for intestinal motility. It also contains postganglionic neurons that send out axons to release transmitters such as acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) in the vicinity of the mucosa to regulate ion transport across the epithelium.
The small intestine is also regulated by endocrine cells such as the enterochromaffin cells that synthesize and release 5-hydroxytryptamine (5-HT). These cells are an integral part of the epithelium in that they communicate with both the lumen (mucosal) and plasma (serosal) surface. Release of 5-HT, which is controlled by stimuli arriving at both surfaces, can have a number of actions. It can stimulate the epithelial cells to increase fluid secretion. 5-HT can also provide an input into the nervous system with two important consequences. By activating neurons that are connected to the enteric nervous system, it can induce a local feedback loop resulting in the release of both ACh and VIP. Termination of the 5-HT signal is carried out by a serotonin-selective reuptake transporter (SERT). All of the epithelial cells that line the mucosa express SERT, which means that the intestine has an enormous capacity to remove 5-HT once it has been released.
The small intestine contains a population of enteroendocrine L cells that release various gut hormones such as peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), which contribute to the control of food intake and body weight (Module 7: Figure control of food intake).
Alterations in the operation of this 5-HT signalling system might be one of the causes of irritable bowel syndrome. In addition, 5-HT can excite 5-HT3 receptors on the endings of vagal afferents that convey information back to the brain to induce nausea and vomiting.
Enterochromaffin cells (ECs) are located in the mucosa of the small intestine (Module 7: Figure small intestine) and colon. The apical surface has microvilli that project out from the mucosal surface and are thought to monitor the chemical composition of the lumen. They synthesize large amounts of 5-hydroxytryptamine (5-HT), which is accumulated in secretory vesicles by a vesicular monoamine transporter I (VMAT-1). The ECs also secrete the peptide guanylin, which functions to control intestinal fluid secretion. These vesicles are located in the basolateral pole. 5-HT is released through a Ca2+-dependent mechanism that is activated by a number of different stimuli acting from both the apical and basolateral surfaces. The latter include mechanical forces, acidification of the lumen and noxious stimuli that cause nausea. The basolateral membrane appears to have muscarinic and nicotinic receptors, suggesting that the ECs are sensitive to the stimulatory action of acetylcholine. However, they also have receptors that may inhibit secretion in response to 5-HT, γ-aminobutyric acid (GABA), adenosine and somatostatin (Sst).
5-HT has an important role in regulating the intestine by acting on the enteric nervous system. It can control peristalsis, secretion and can activate vagal afferents. The latter send signals to the spinal cord that communicate the signals that lead to nausea. In addition, the released 5-HT enters the circulation where it can have peripheral actions such as the inhibition of bone resorption by the osteoblasts (Module 7: Figure osteoblast function).
There is growing evidence that changes in 5-HT signalling, which is an integral part of the small intestine neural and endocrine system, may be responsible for many of the symptoms associated with irritable bowel syndrome.
L cells are enteroendocrine cells that are located in the intestinal tract where they function to synthesize and release various gut hormones such as peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) (Module 7: Figure small intestine). GLP-1 and PYY contribute to the control of food intake and body weight (Module 7: Figure control of food intake). Most of the L cells are located in the lower intestine and colon. L cells are activated by stimuli arriving at either the basolateral or apical membranes (Module 7: Figure L cell). The activation that occurs after feeding takes place in two phases. The first phase is triggered by acetylcholine released from neurons innervating the foregut regions. The second phase is activated by nutrients that begin to appear in the lumen especially in the lower regions of the gut. In both cases, the stimuli induce hormone release by acting on G protein-coupled receptors (GPCRs) to produce the Ca2+ necessary to trigger exocytosis. The nutrient receptors on the apical surface are coupled to α-gustducin (Gαgust) (Module 2: Table heterotimeric G proteins), which also functions in detecting bitter compounds by type II receptor cells (Module 10: Figure taste receptor cells).
Small intestine fluid secretion
The intestine can both absorb and secrete fluid, and the amount of water lost from the intestine depends upon a balance between these two processes. Under normal conditions, the small intestine receives approximately 10 litres of water every 24 h. As this fluid passes down the intestinal lumen, water is absorbed and secreted, but the former predominates, which means that there is a net absorption of 7.5 litres. The remaining 2.5 litres enters the colon, where most of the water is absorbed, leaving only 100 ml in the faeces.
Unlike many other secretory systems, which are either active or quiescent, the intestine is continuously active, during which absorption and secretion occur continuously. The absorptive mechanism predominates and may be considered to be the resting default mode. Originally, it was thought that secretion occurred in the crypts, whereas absorption was restricted to the villi. However, recent work suggests that these two processes occur throughout the epithelium. The absorption of water is linked to the uptake of NaCl, and this absorptive mechanism is similar to that described in more detail in the colon (see Module 7: Figure colon function). Here attention will focus on fluid secretion and how it is controlled by a variety of factors.
Fluid secretion by the small intestine is driven by a net flux of Cl− from the plasma into the lumen (Module 7: Figure intestinal secretion). Entry of Cl− across the basolateral membrane is carried out by the Na+-K+-Cl− cotransporter 1 (NKCC1) with the driving force supplied by the Na+ entering the cell down its electrochemical gradient that is maintained by the classical ouabain-sensitive Na+ pump (Na+/K+-ATPase). Any accumulation of K+ is leaked back to the plasma through a K+ channel. The accumulation of Cl− within the cell increases the electrochemical gradient, and this enables this anion to pass into the lumen through two channels: the cystic fibrosis transmembrane conductance regulator (CFTR) channel and one of the Ca2+-sensitive Cl− channels (CLCAs). The flow of Cl− into the lumen then provides the electrical gradient for a parallel flow of Na+, and this movement of NaCl then provides the osmotic gradient for the flow of water.
This secretion of fluid by the small intestine is controlled by a number of stimuli. Of particular importance are acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) released from the enteric nervous system (Module 7: Figure small intestine). ACh acts through the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette, and the increase in Ca2+ activates the K+ and Cl− channels responsible for fluid secretion (Module 7: Figure intestinal secretion). On the other hand, VIP acts through the cyclic AMP signalling pathway, which acts through protein kinase A (PKA) to enhance both the entry and exit of Cl−. It phosphorylates the Na+-K+-Cl− cotransporter 1 (NKCC1), which is activated to enhance the entry of Cl−. In addition, PKA phosphorylates the R domain of the CFTR channel (Module 3: Figure CFTR channel) to increase Cl− flux into the lumen.
The cyclic GMP signalling pathway is switched on by the intestinal hormone guanylin, which acts on pGC-C, which is one of the particulate guanylyl cyclases (pGCs). Cyclic GMP then acts through cyclic GMP-dependent protein kinase II (cGKII) to phosphorylate CFTR (Module 7: Figure intestinal secretion).
Bacteria such as Escherichia coli and Vibrio cholerae produce toxins that induce diarrhoea by taking over some of the signalling pathways that control intestinal secretion. In the case of cholera, cholera toxin (CT) produced by V. cholerae stimulates intestinal secretion by activating the cyclic AMP signalling pathway.
Each day approximately 2.5 litres of fluid leaves the small intestine to enter the colon, which removes most of the water, leaving only 100 ml in the faeces. The colon is divided into proximal and distal segments that have subtly different uptake mechanisms. The overall structure of the mucosa is similar to that found in the small intestine in that it contains epithelial cells, mucous cells and a small number of enterochromaffin cells (Module 7: Figure small intestine). As for the small intestine, there is a dynamic balance between absorption and secretion, with the system biased towards net absorption to account for the almost complete removal of ions and water as fluid moves down the colon. Pathological conditions such as irritable bowel syndrome or diarrhoea are caused by alterations in this balance between absorption and secretion. For the sake of simplicity, the major ion transport mechanisms responsible for both secretion and absorption will be described in the context of a generic colon cell (Module 7: Figure colon function).
Absorption depends upon the active transport of NaCl from the lumen to the serosal side. The driving force for this flux of ions depends upon the Na+/K+-ATPase located on the basolateral membrane that extrudes Na+ from the cell, and this provides the driving force for the uptake of Na+ across the apical membrane. This uptake of Na+ can occur through two mechanisms. Firstly, there is an electroneutral mechanism based on the type 3 Na+/H+ exchanger (NHE3), which is responsible for the bulk transport of Na+. This electroneutral mechanism is enhanced by an electrogenic mechanism based on the epithelial Na+ channel (ENaC), which is particularly prevalent in the posterior colon. The balancing negatively charged ion is Cl−, which enters the cell through the anion exchanger type 1 (AE1) Cl−/HCO3− exchanger, the Cl−/OH− exchanger or the cystic fibrosis transmembrane conductance regulator (CFTR) channel. A CLC-2 channel located in the basolateral membrane is also responsible for Cl− transport across the serosal surface. In addition, Cl− and water can move passively through the paracellular pathway.
The Cl−/OH− exchanger is thought to be the down-regulated in colonic adenoma (DRA) protein, which is mutated in congenital chloride diarrhoea.
Fluid secretion by the colon is driven by a net flux of Cl− from the plasma into the lumen by mechanisms that are very similar to those in the small intestine (Module 7: Figure intestinal secretion). Entry of Cl− across the basolateral membrane is carried out by the Na+-K+-Cl− cotransporter 1 (NKCC1) with the driving force supplied by Na+ entering the cell down its electrochemical gradient that is maintained by the classical ouabain-sensitive Na+ pump (Na+/K+-ATPase) (Module 7: Figure colon function). Any accumulation of K+ is leaked back to the plasma through a K+ channel. The accumulation of Cl− within the cell increases the electrochemical gradient, and this enables this anion to pass into the lumen through the CFTR. The transepithelial flow of Cl− then provides the electrical gradient for a parallel flow of Na+ through the paracellular pathway, and this movement of NaCl then provides the osmotic gradient for the flow of water.
A variety of signalling mechanism control the absorptive and secretory mechanisms. Aldosterone is the main regulator of Na+ absorption in the colon, where it acts in a manner very similar to that found in the distal convoluted tubule (DCT) of the kidney. It enters the cell and binds to a mineralocorticoid receptor (MR) in the cytosol before translocating into the nucleus, where it acts to increase the expression of the basolateral Na+/K+-ATPase and the luminal amiloride-sensitive ENaC that act together to absorb Na+ from the lumen. Control of fluid secretion is regulated by the cyclic AMP and cyclic GMP signalling pathways through control mechanisms that closely resemble those found in the small intestine (Module 7: Figure intestinal secretion).
The mammalian kidney is a complex organ that is responsible primarily for excretion and osmoregulation. It removes metabolic waste products and foreign compounds taken up in the diet. With regard to osmoregulation, the kidney is responsible for blood Na+ regulation, which is critical for maintaining normal blood pressure (Module 7 blood pressure control). These various functions are carefully regulated by hormones such as vasopressin, parathyroid hormone (PTH), angiotensin II, aldosterone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. For example, water balance is regulated through vasopressin-induced antidiuresis. In addition to these hormonal controls, the kidney is innervated by the sympathetic nervous system, which apparently can modulate certain processes such as blood flow. Another important regulatory system in the kidney is the tubuloglomerular feedback (TGF) mechanism, which is a local control network operated by the juxtaglomerular apparatus (JGA).
In addition to its role in excretion and osmoregulation, the kidney also functions as an endocrine organ by producing various hormones:
The architectural organization of tubules and accompanying blood vessels is a major feature of kidney structure, which determines how it carries out its many functions (Module 7: Figure kidney tubule). Each tubule contains a primary cilium, which functions in mechanotransduction in kidney cells. Mutations in the polycystin channels located on these cilia cause polycystic kidney disease.
Blood Na+ regulation
The kidney tubule is at the centre of a hormonal network that plays a major role in ensuring that the blood Na+ level remains normal. The kidney functions as an endocrine system to release signals that regulates the processes of Na+ filtration and reabsorption through the following sequence of events (Module 7: blood pressure control):
An important component of this regulation of blood Na+ levels is the hormone angiotensin II, which is critical for the control of blood pressure. Abnormal elevation in the levels of this hormone is one of the main contributors to hypertension.
Blood pressure, which provides the driving force to move blood through the vascular system, varies during the cardiac cycle of contraction (systole) and relaxation (diastole). ‘Normal’ blood pressure is considered to be a systolic pressure of 120 mmHg and a diastolic pressure of 80 mmHg (i.e. 120/80). However, blood pressure can vary considerably to meet different demands for oxygen and nutrients, as occur during strenuous exercise or thermogenesis. This physiological regulation of blood pressure is complex and depends upon a number of interacting systems (Module 7: blood pressure control). The pressure within the vascular system can be varied either by altering the volume of fluid or by altering the force exerted on this fluid by the blood vessels. With regard to the former, changes in blood volume result from an alteration in blood Na+ regulation. If the Na+ level rises, it will draw water out of the cells leading to a large blood volume and hypertension. With regard to regulation of blood vessel contractility, baroreceptors located in the aortic arch and carotid sinus send information to the brain to modify the neural input to the smooth muscle cells that surround the blood vessels.
Varying the peripheral resistance of the blood vessels is a particularly important parameter, because their diameter is a major determinant of peripheral vascular resistance, as revealed by Poiseuille's Law:
where R is the resistance to laminar flow down a cylindrical tube of length L, η is the fluid viscosity, and r is the radius of the tube.
Since the resistance is inversely proportional to the fourth power of the tube radius, it is clear that blood pressure is very dependent on the radius of the blood vessels, which can be modified by many factors such as hormones [e.g. angiotensin II and adrenaline (epinephrine)], neurotransmitters [e.g. noradrenaline (norepinephrine), ATP and acetylcholine released from the neuronal innervation] and factors released from the endothelium [e.g. nitric oxide (NO) and endothelin].
The two kidneys in mammals are bean-shaped organs located on the posterior wall on either side of the vertebral column. The indented region is known as the hilum, which marks the point of exit of the ureter. The renal artery and vein are also connected through the hilum. The kidney is subdivided into a series of pyramids whose apices face the calyxes that are fan-shaped collecting funnels that channel urine towards the ureter. The pyramids, which have an outer cortex and an inner medulla, contain the kidney tubules. Each kidney contains approximately one million tubules, which have a fairly consistent structure, which is crucial for their function (Module 7: Figure kidney tubule). Each kidney tubule is an autonomous unit that has its own in-built control system based on the tubuloglomerular feedback (TGF) mechanism.
A critical feature of kidney structure is the relationship of tubules to the renal arterial system. Each tubule receives an afferent arteriole that forms a glomerulus, which is a network of capillaries surrounded by a Bowman's capsule, which marks the beginning of the tubule. Urine formation begins with ultrafiltration into Bowman's capsule to form the primary urine that then flows into the proximal convoluted tubule (PCT), which then straightens out as it passes towards the medulla where it abruptly changes into the thin descending segment of the loop of Henle. This descending segment then loops back on itself and begins to move back towards the cortex where the thin ascending loop changes into the thick ascending loop of Henle (TALH). During its return passage, the TALH comes into close contact with the afferent arteriole (AA) and efferent arteriole (EA) to form the juxtaglomerular apparatus (JGA) (Module 7: Figure juxtaglomerular apparatus). Finally, the TALH leads into the distal convoluted tubule (DCT) that is connected to the collecting duct. A number of ducts drain into these collecting tubules that finally open at the tips of the pyramids.
The different parts of the tubule receive a vascular supply, which is unusual in that each artery is associated with two capillary networks arranged in series (Module 7: Figure kidney tubule). The first capillary network is that found in the glomerulus and is responsible for the process of ultrafiltration that forms the primary urine. The EA that leaves the glomerulus then divides into another capillary network which surrounds different parts of the tubule. Not shown in the figure is the extensive capillary network in the cortex. In addition, efferent vessels dip down into the medulla to form the vasa recta that extend deep into the pyramid to serve the lower parts of the loop of Henle.
Juxtaglomerular apparatus (JGA)
A consistent structural feature of each tubule is the juxtaglomerular apparatus (JGA), which is formed by the close association between the thick ascending loop of Henle (TALH) and the afferent and efferent arterioles in the immediate vicinity of the glomerulus (Module 7: Figure juxtaglomerular apparatus). In this area of close contact, there are considerable modifications in both the vascular and tubular elements. Cells of the TALH that face the JGA have a denser cytoplasm and are known as the macula densa. There are smooth muscle cells surrounding the afferent arteriole that regulate the flow of blood in to the glomerulus. These smooth muscle cells can be transformed into secretory cells and are the origin of the renin-producing granular cells. These typical secretory cells, which surround the afferent arteriole next to the glomerulus, are responsible for synthesizing and secreting renin, which is an essential component of the renin–angiotensin system (RAS). These renin-producing granular cells and the macula densa are connected by a network of extraglomerular mesangial cells, which are also known as lacis cells, which have long ramifying extensions. These extraglomerular mesangial cells are connected to other cells of the JGA (smooth muscle cells, renin-producing granular cells and the intraglomerular mesangial cells) through gap junctions (double-headed red arrows in Module 7: Figure juxtaglomerular apparatus) that may be responsible for an intercellular Ca2+ wave that spreads throughout this community of cells during the operation of the tubuloglomerular feedback (TGF) mechanism.
Urine formation begins at the glomerulus with ultrafiltration of the blood into Bowman's capsule (mauve arrow in Module 7: Figure juxtaglomerular apparatus). The primary filtrate is selectively modified as it passes down the tubule (Module 7: Figure kidney tubule function). It is this modification of the primary filtrate by different regions of the tubule which is particularly important for homoeostasis and represents some of the main sites of action of the various hormones.
The human kidney filters about 125 ml of fluid every 1 min. This ultrafiltrate, which contains all soluble components in the plasma up to a molecular mass of 5000 Da, is formed by the first capillary network located in the glomerulus. Most plasma proteins are too large to pass through the filter and are retained to pass out through the efferent arteriole (EA). The filtration process is passive, deriving its energy from the hydrostatic pressure of blood which forces fluid through the filter. Despite changes in arterial pressure, the kidney maintains a constant rate of glomerular filtration through the operation of an autoregulatory tubuloglomerular feedback (TGF) mechanism. The primary filtrate leaves Bowman's capsule and begins its journey down the tubule by first passing through the proximal convoluted tubule (PCT).
Proximal convoluted tubule (PCT)
As described earlier, the tubules filter about 125 ml/min, which means that during the course of a day 180 litres of water will have entered the kidney tubules. Since the total blood volume is approximately 3 litres, the entire blood volume would be lost in less than 30 min, unless most of the fluid is returned to the blood. The proximal convoluted tubule (PCT) is responsible for reabsorbing almost 99% of the water in the primary filtrate. Proximal convoluted tubule cells are ideally suited for isotonic fluid absorption (Module 7: Figure kidney tubule). There is an extensive brush border, which greatly facilitates the entry of solutes and water by enlarging the surface area of the mucosal surface.
The permeability to water depends on aquaporin 1 (AQP1) (Module 3: Table aquaporin family), which is expressed in large amounts in both the apical and basolateral membranes of the PCT (Module 7: Figure proximal tubule function). The movement of water through these AQP1 channels is linked to the active transport of Na+ (Module 7: Figure kidney tubule function). The latter enters passively across the brush border and is then extruded by the Na+/K+-ATPase located on the basolateral membranes. As this extrusion of Na+ is carried out in exchange for K+, the cell will gradually fill up with K+ and this will begin to reduce the trans-epithelial flux of Na+ unless this K+ is recycled back across the basolateral membrane by the TASK-2 channel.
Some of the Na+ that enters through the luminal membrane drives the Na+/H+ exchanger 3 (NHE3). The Na+ is exchanged for H+ that is extruded into the lumen as part of the mechanism for reabsorbing bicarbonate (HCO3−). Once in the lumen, the H+ interacts with HCO3− to form CO2 through an interaction catalysed by a luminal carbonic anhhdrase IV (AC IV) (Module 7: Figure proximal tubule function). An intracellular carbonic anhydrase II (AC II) converts this CO2 into HCO3− and H+. The latter is then available to carry out another HCO3− transport cycle across the luminal membrane. The intracellular HCO3− then leaves the cell via a Na+/HCO3− cotransporter (KNBC1). The alkalinisation of the fluid outside the cell will activate the TASK-2 channel to increase the efflux of K+.
Loop of Henle
The transition between the proximal convoluted tubule and the descending thin limb of the loop of Henle is usually abrupt. The cells are very thin and have little membrane elaboration (Module 7: Figure kidney tubule). Cells in this descending portion of the loop of Henle play little active role in modifying the urine, but they allow passive equilibration with the interstitial fluid. Water is pulled out of the fluid, thus greatly concentrating the luminal contents (Module 7: Figure kidney tubule function). The cells in this descending portion have a high water permeability due to the expression of aquaporin 1 (AQP1) (Module 3: Table aquaporin family), which facilitates this equilibration.
As the fluid rounds the U-bend and begins to move up the ascending thin limb, it comes into contact with cells that have a much lower water permeability because they lack the aquaporin channels. The thin cells are tightly linked together by numerous tight junctions further increasing their impermeability. This thin segment then connects to the thick ascending loop of Henle (TALH).
Thick ascending loop of Henle (TALH)
Cells of the thick ascending loop of Henle (TALH) are much larger than those in the thin segment (Module 7: Figure kidney tubule). There is little change in the structure of the apical surface, but the basal surface is much more elaborate, with numerous very-long infoldings, of which many are formed by interdigitation with cytoplasmic processes from neighbouring cells. The TALH is impermeable to water, but can absorb Na+ and Cl−, and this serves to dilute the urine that is passed on to the distal convoluted tubule (DCT).
The apical surface of the TALH contains the electroneutral Na+-K+-2Cl− cotransporter 2 (NKCC2), which is the target of the loop diuretics and transports Na+, K+ and 2Cl− from the lumen into the cell (Module 7: Figure kidney salt reabsorption). The K+ is returned to the lumen by the Kir1.1 (ROMK) channel and perhaps also through the TWIK-1 channel. This recycling of K+ is essential in order to maintain the activity of NKCC2. The Na+ leaves the cell via the Na+/K+-ATPase whereas the Cl− passes out through the CLC-Kb channel. The Ca2+-sensing receptor (CaR), which is strongly expressed on the basolateral membrane of the TALH cells, responds to an increase in extracellular Ca2+ to produce an intracellular signal, either an increase in Ca2+ or a decline in the level of cyclic AMP that then inhibits NKCC2 and ROMK.
Mutations in different components of this TALH mechanism of salt reabsorption results in Bartter's disease.
A small section of the TALH comes into contact with glomerulus, where some of the tubule cells are modified to form the macula densa, which is part of the juxtaglomerular apparatus (JGA) (Module 7: Figure juxtaglomerular apparatus).
Distal convoluted tubule (DCT)
Cells of the distal convoluted tubule (DCT) resemble those of the thick ascending loop of Henle (TALH) (Module 7: Figure kidney tubule), but the infoldings of the basal membrane are less elaborate. The DCT is the site of K+ secretion, and it also continues the process of Na+ and Cl− reabsorption, thus diluting the urine even further. The mechanism of salt reabsorption by cells of the DCT differs from that found in the thick ascending loop of Henle (TALH) (Module 7: Figure kidney salt reabsorption). The Na+-Cl− cotransporter (NCC), which is inhibited by the thiazides, transports Na+ and Cl− from the lumen into the DCT cell. Na+ also enters through the amiloride-sensitive epithelial Na+ channel (ENaC) especially in the lower reaches of the DCT and in the collecting duct. The K+ that is secreted into the lumen crosses the apical membrane through both the Kir1.1 (ROMK) channel and the K+-Cl− cotransporter 1 (KCC1). Na+ leaves the cell via the Na+/K+-ATPase, which also provides some of the K+ that is secreted into the lumen. The WNK protein kinases control the activity of both NCC and ROMK. This regulation seems to depend on the WNK4 isoform controlling the expression of these carriers in the apical membrane. For example WNK4 reduces the activity of NCC by activating its internalization and trafficking to the lysosome. WNK1 is part of the signalling cascade because it acts to inhibit the activity of WNK4.
Mutations in WNK1 and WNK4 result in the increased NCC activity responsible for the hypotension seen in Gordon's disease. On the other hand, mutations in NCC that result in a decrease in salt absorption cause the hypertension seen in Gitelman's disease.
The steroid hormone aldosterone, which is released from the adrenal cortex, regulates this uptake of Na+ and is critical for regulating Na+ balance. Aldosterone acts through a genomic pathway (Module 1: Figure steroid stimuli). It enters the cell and binds to a mineralocorticoid receptor (MR) in the cytosol before translocating into the nucleus, where it acts to increase the expression of the basolateral Na+/K+-ATPase and the luminal amiloride-sensitive epithelial Na+ channel (ENaC) that act together to absorb Na+ from the lumen.
The DCT is the main site for Ca2+ reabsorption by the kidney, which is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (Module 7: Figure kidney Ca2+ reabsorption). Fluid leaving the DCT is then passed into the collecting duct.
Mutations in proline-rich segments of ENaC cause Liddle's disease, which is a salt-sensitive form of hypertension.
Epithelial Na+ channel (ENaC)
The epithelial Na+ channel (ENaC) is expressed in a number of epithelia such as the kidney, intestine, pancreas, testis, lung and sweat glands. In the kidney, ENaC is expressed mainly in the cortical and distal collecting ducts. ENaC consists of three subunits (α, β and γ). The α-subunit is responsible for conducting Na+ whereas the β and γ subunits have a regulatory function. ENAC expression is regulated by oestrogen.
The activity of ENaC depends upon its rate of membrane insertion. ENaC is ubiquitinylated by the ubiguitin ligase Nedd4-2, which binds to a PPPXY motif on the cytoplasmic tail of the α-subunit, which is then degraded by proteasomes. In Liddle's disease, this cytoplasmic region is removed thus reducing the ENaC degradation that then builds up in the membrane resulting in enhanced Na+ reabsorption and hypertension.
The collecting ducts, which are connected to a number of distal convoluted tubules (DCTs), are responsible for passing fluid down to the pyramid. The apical surface has numerous short microvilli. The basal membrane is also slightly enlarged by having short infoldings, while the lateral borders are fairly tortuous (Module 7: Figure kidney tubule). The upper regions continue the process of Na+ reabsorption, thus contributing further to Na+ retention and dilution of the urine. This hypotonic urine then passes through the lower regions, where water may or may not be absorbed depending on the water balance.
If there is an excess of water, the collecting duct cells are almost totally impermeable, thus preventing reabsorption and water is lost in the urine. During dehydration, vasopressin-induced antidiuresis conserves water by increasing the permeability of the apical membrane to draw water in from the final urine (Module 7: Figure collecting duct function). The driving force for water movement is the high concentration of solutes maintained in the interstitial space through the operation of the countercurrent mechanism.
Water excretion is regulated by the antidiuretic hormone vasopressin, which is released from the posterior pituitary. The urine entering the collecting duct is hypotonic as a result of Na+ and Cl− absorption by the distal convoluted tubule (DCT) and the upper regions of the collecting duct, which are impermeable to water (Module 7: Figure collecting duct function). In the absence of vasopressin, this dilute urine passes down the collecting duct, resulting in rapid diuresis. An increase in the circulating level of vasopressin reverses this diuresis by increasing the permeability of the apical membrane of the collecting duct, which is the rate-limiting barrier to water movement.
Vasopressin acts through the V2 vasopressin receptor, which is a G protein-coupled receptor (GPCR) that acts through the cyclic AMP signalling pathway (Module 1: Table G protein-coupled receptors). The messenger cyclic AMP acts through protein kinase A (PKA) to phosphorylate Ser-256 on the C-terminal cytoplasmic tail of aquaporin 2 (AQP2) (Module 3: Figure aquaporin structure). The vesicles containing phosphorylated AQP2 molecules are linked through the dynein adaptor dynactin to the molecular motor dynein, which propels them along microtubules to the apical membrane. Once they reach the apical surface, they fuse to allow water to enter the cell. Water then leaves the cell through both AQP3 and AQP4 channels in the basolateral membrane (Module 7: Figure collecting duct function). Just how the fusion process is carried out is not clear. Many of the classical fusion proteins seem to be involved, and there also is some suggestion that there might be a localized Ca2+ signal to induce the fusion event. Alterations in the terminal web of actin filaments may also play a role.
Vasopressin can also increase the transcription of the AQP2 and AQP3 channels through stimulation of the transcription factor cyclic AMP response element-binding protein (CREB), which is phosphorylated on Ser-133 by protein kinase A (PKA).
Diabetes insipidus (DI), which can have multiple causes, results from a defect in this role of the collecting ducts to reabsorb water.
Ca2+ reabsorption by the kidney
On a daily basis, the kidney filters about 8 g of Ca2+, and all but 200 mg are reabsorbed by the kidney (Module 7: Figure Ca2+ homoeostasis). Reabsorption takes place in different regions down the length of the kidney tubule (Module 7: Figure kidney tubule function). Approximately 70% of this Ca2+ is taken back across the proximal convoluted tubule (PCT). Most of this Ca2+ is reabsorbed passively along a paracellular pathway (i.e. moves between the cells) driven by the reabsorption of Na+ (Module 7: Figure kidney Ca2+ reabsorption). There is no absorption in the thin limbs of Henle, but reabsorption through the paracellular pathway resumes along the thick ascending limb of Henle (TALH). In these regions where paracellular transport occurs, the tight junctions are fairly permeable to enable this passive flux to occur. A protein called paracellin 1 (PCLN-1) may play a role in regulating this passive permeability to both Ca2+ and Mg2+. Mutations in PCLN-1 may be responsible for hypomagnesaemia hypercalciuria syndrome.
The final stages of Ca2+ reabsorption are completed in the distal convoluted tubule (DCT). This reabsorption occurs against the electrochemical gradient, and this active transport is carried out by the transcellular pathway, which has three main stages: Ca2+ entry from the lumen, diffusion across the cell and then active extrusion by pumps in the basolateral membrane. The main entry channel on the apical membrane is TRPV5, which is a member of the vanilloid transient receptor potential (TRP) ion channel family (Module 3: Figure TRP channel phylogeny). In these kidney tubules, the Ca2+-dependent inactivation of TRPV5 appears to be alleviated by the Ca2+ buffer calbindin D-28k (CB). When the internal Ca2+ concentration is low, calbindin D-28k associates with TRPV5 and is thus positioned to rapidly buffer Ca2+ to keep the channel open. As it charges up with Ca2+, the calbindin D-28k leaves the channel to diffuse across the cell to the basolateral membrane, where it gives up its cargo to the pumps that extrude Ca2+ thus completing the transcellular flux of Ca2+ (Module 7: Figure kidney Ca2+ reabsorption). The active transport step is the extrusion of Ca2+ by the Na+/Ca2+ exchanger (NCX) and the plasma membrane Ca2+-ATPase 1b (PMCA1b). NCX1 and PMCA1b seem to be the main isoforms in these kidney cells.
The transcellular reabsorption of Ca2+ is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which acts by increasing the expression of all the main components responsible for transporting Ca2+ across the cell. The 1,25(OH)2D3 binds to the vitamin D receptor (VDR), which then acts on the VDR-response element (VDRE) to increase the expression of TRPV5, CB, NCX1 and PMCA1b.
Tubuloglomerular feedback (TGF) mechanism
Each kidney tubule has a built-in tubuloglomerular feedback (TGF) mechanism that functions to balance glomerular filtration rate to the reabsorptive capacity of the tubule. The feedback mechanism is located within the juxtaglomerular apparatus (JGA) (Module 7: Figure juxtaglomerular apparatus) where a portion of the thick ascending loop of Henle (TALH) comes into close contact with the afferent arteriole. The composition of the fluid passing down the TALH is monitored by the macula densa cells, and any change is converted into signals that are sent back to the glomerular region to adjust the rate of fluid filtration and renal haemodynamics. As such, the TGF mechanism is an important component of blood Na+ regulation (Module 7: Figure blood pressure control). For example, if the filtration rate is too high, the flow of solutes down the tubule overwhelms the reabsorptive capacity of the tubule resulting in a large loss of solutes, particularly Na+ and Cl−. The high concentration of Na+ and Cl− in the JGA stimulates the macula densa cells to release signals, such as ATP, to contract the arteriole smooth muscle cells, which then reduces the flow of blood and the filtration rate is reduced to match the reabsorptive capacity of the tubule. In addition, the macula densa cells also release prostaglandin E2 (PGE2), which regulates the activity of the renin-producing granular cells (Module 7: Figure renin secretion).
The role of the extraglomerular mesangial cells, which face the macula densa (Module 7: Figure juxtaglomerular apparatus) is not absolutely clear. They may play a critical role by initiating an intercellular Ca2+ wave that relays information to other cells within the JGA such as the smooth muscle cells, the renin-producing granular cells and intraglomerular mesangial cells.
The macula densa is made up of a specialized group of cells within the thick ascending loop of Henle (TALH) that is a part of the juxtaglomerular apparatus (JGA) (Module 7: Figure juxtaglomerular apparatus). There are about 20 macula densa cells, which are part of the tubuloglomerular feedback (TGF) mechanism that respond to an increase in the concentration of Na+ and Cl− in the lumen of the TALH by releasing ATP that then activates the other cells of the JGA (Module 7: Figure macula densa). The signal transduction mechanism is unusual because the cells have to respond to a change in the level of ions within the lumen by stimulating the release of ATP across the opposite surface of the cell. There is general agreement that transduction begins with the furosemide-sensitive Na+-K+-2Cl− cotransporter 2 (NKCC2) located within the apical plasma membrane facing the lumen. In order for this transporter to continue to operate, K+ is returned to the lumen through an inwardly rectifying K+ channel, which seems to be a renal outer medullary K+ channel (ROMK). The removal of Na+ is somewhat more problematical because these macula densa cells are not very effective at extruding Na+. They have a very weak conventional Na+ pump (Na+/K+-ATPase) activity, but they do have a ouabain-sensitive colonic form of H+/K+-ATPase. The latter is able to extrude some Na+ together with H+. However, the decreased capacity to extrude Na+ means that the intracellular level is very sensitive to fluctuations in the luminal level of this ion.
In the region of the TALH where it forms part of the JGA, the concentration of NaCl fluctuates around a value of about 25mM,which is close to the equilibrium value of the Na+/K+/2Cl− co-transporter. This means that the NaCl uptake mechanism is poised at a value that optimizes its ability to respond to fluctuations in the luminal level of NaCl. If the level of NaCl increases, there is an immediate increase in the uptake of Na+ and Cl−, resulting in an increase in the intracellular level of these two ions. Just how these increases in the intracellular levels of Na+ and Cl− trigger the release of ATP is still debated, but there does appear to be a requirement for an increase in the level of Ca2+. One possibility is that the increase in Cl− enhances its efflux through a basolateral Cl− channel to cause membrane depolarization (ΔV) and that this then triggers an influx of Ca2+ through a channel that remains to be identified. Another possibility is that the accumulation of Na+ slows down the Na+/Ca2+ exchanger, and this will reduce Ca+ extrusion and allow the intracellular level of this ion to increase. It remains to be seen whether or not this increase in Ca2+ is responsible for stimulating the release of ATP through the maxi-anion channel.
The increase in Ca+ may also function to activate phospholipase A2 (PLA2) to release arachidonic acid, which is converted into prostaglandin E2 (PGE2) (Module 1: Figure eicosanoids). PGE2 is released from the macula densa and is one of the stimuli responsible for activating the renin-producing granular cells.
The ATP released from the macula densa cells can function as an extracellular messenger, but it can also be converted by various ecto-ATPases into AMP and then to adenosine, which can also function as an extracelullar signal to regulate other components of the JGA such as the mesangial cells, renin-producing granular cells and the afferent arterial smooth muscle cells (Module 7: Figure renin secretion).
Renin–angiotensin system (RAS)
The renin–angiotensin system (RAS) plays a major role in blood Na+ regulation (Step 5 in Module 7: Figure blood pressure control). The renin-producing granular cells secrete the enzyme renin that acts on angiotensinogen in the plasma to release angiotensin I. The latter is then converted into the hormone angiotensin II by the angiotensin-converting enzyme (ACE), which is particularly rich in the lung. Angiotensin II is a multifunctional hormone capable of stimulating a number of different cellular processes (Module 7: Figure blood pressure control):
Renin-producing granular cells
The synthesis and release of renin is a major component of the renin–angiotensin system (RAS) (Module 7: Figure blood pressure control). These renin-producing granular cells are also known as juxtaglomerular cells, but the latter term can be confusing because it could apply equally as well to the other cells within the juxtaglomerular apparatus (JGA) (Module 7: Figure juxtaglomerular apparatus). Therefore the more functional terminology will be used, as it relates directly to their role in synthesizing and releasing renin. The cells that release renin are modified smooth muscle cells located in the media of the afferent arteriole that feeds blood into the glomerulus. The phenotypic transformation of smooth muscle cells into the renin-secreting cells is a gradual process, and there are intermediate cells that retain the ability to contract as they begin to assemble the large granules containing renin. These granules are unlike most other Golgi-derived secretory granules in that they are modified lysosomal vesicles. The fact that these renin-containing granules are derived from lysosomes may explain there unusual secretory mechanism, which occurs independently of the classical Ca2+-dependent exocytosis mechanism (Module 4: Figure Ca2+-induced membrane fusion). Not only is secretion independent of Ca2+, but also it is inhibited by elevations in Ca2+, which is a most unusual situation. Another example of such an inverse arrangement operates in parathyroid hormone (PTH) synthesis and release (Module 7: Figure PTH secretion).
In the case of the renin-producing granular cells, this Ca2+ paradox seems to have been resolved by finding that the secretion of renin is controlled by the cyclic AMP signalling pathway (Module 7: Figure renin secretion). A large number of external signals regulate renin secretion and many of these interact in one way or another with cyclic AMP signalling. Noradrenaline (norepinephrine) released from the renal nerves (Module 7: Figure juxtaglomerular apparatus) and prostaglandin E2 (PGE2) released from the macula densa cells (Module 7: Figure macula densa) act through the heterotrimeric G protein GαS to stimulate the type 5 adenylyl cyclase (AC5) (Module 2: Table adenylyl cyclases). By contrast, the adenosine derived from the ATP released from the macula densa cells (Module 7: Figure macula densa) acts through Gαi to inhibit AC5.
A number of stimuli (angiotensin II, endothelin and vasopressin), which act through the G protein-coupled receptors (GPCRs) that induce the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette (Module 1: Figure stimuli for InsP3/DAG signalling) all function to increase the level of Ca2+, which then acts to reduce the activity of the Ca2+-inhibitable AC5 (Module 7: Figure renin secretion). This Ca2+-dependent inhibition of cyclic AMP formation can explain why an increase in intracellular Ca2+ reduces the secretion of renin. The inhibitory action of angiotensin II is part of a negative-feedback loop because the released renin is part of the renin–angiotensin system (RAS) responsible for generating angiotensin II, which then feeds back to inhibit the renin-producing granular cells (Step 8 in Module 7: Figure blood pressure control).
The nitric oxide (NO)/cyclic GMP signalling pathway has a bimodal effect on renin secretion. At low levels of stimulation, cyclic GMP inhibits phosphodiesterase PDE3, which enhances the level of cyclic AMP to promote renin secretion (Module 7: Figure renin secretion). On the other hand, high levels of cyclic GMP result in the activation of cyclic GMP-dependent protein kinase II (cGKII), which then inhibits the action of cyclic AMP resulting in a reduction in renin secretion.
The cyclic AMP signalling pathway acting through the cyclic AMP response element-binding protein (CREB) transcription factor also plays an important role in regulating transcription of the preprorenin gene.
There are two types of mesangial cells. The extraglomerular mesangial cells, which are also known as lacis cells, are located below the macula densa in the cleft formed by the afferent and efferent arterioles (Module 7: Figure juxtaglomerular apparatus). The function of these mesangial cells is not clear, but they may function to relay information to the afferent arteriole smooth muscle cells and to the renin-producing granular cells. The other mesangial cell type is the intraglomerular mesangial cells, which are specialized pericytes that function to control the flow of blood through the glomerular capillaries and thus can modulate glomerular filtration rate. They closely resemble vascular smooth muscle cells in both their morphology and embryological origins. These intraglomerular mesangial cells are highly versatile cells that regulate the flow of blood, can proliferate and also play a structural role as one of the major contributors to the formation and degradation of the extracellular matrix proteins, such as fibronectin, type IV collagen, percalan and laminin, that make up the renal filter (broken line in Module 7: Figure juxtaglomerular apparatus).
The different mesanglial cell functions are regulated by a number of signalling pathways (Module 7: Figure mesangial cell). Their contractile tone, which determines the glomerular filtration rate, depends upon a balance between contractile and relaxing hormones. The major contractile hormones are angiotensin II and endothelin-1 that act through the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette. Release of Ca2+ from the internal stores provides an initial burst of Ca2+ that can initiate contraction. However, this initial release of Ca2+ may trigger a subsequent entry of Ca2+ to maintain contractile activity for longer periods. The Ca2+ can activate a Ca2+-sensitive Cl− channel (CLCA), which then depolarizes the membrane sufficiently to activate a voltage-operated channel (VOC) to allow Ca2+ to enter from the outside. Excessive increases in Ca2+ are avoided through a negative-feedback loop whereby Ca2+ activates the large-conductance (BK) channel, which increases the efflux of K+ to hyperpolarize the membrane and thus reduce entry through the VOC. Depletion of stored Ca2+ can also activate store-operated channels (SOCs) that remain to be properly defined in mesangial cells.
The major relaxing stimuli are atrial natriuretic factor (ANF) and nitric oxide (NO), which act through the nitric oxide (NO)/cyclic GMP signalling pathway (Module 2: Figure NO and cyclic GMP signalling). The increase in cyclic GMP appears to reduce Ca2+ signalling in mesangial cells as it does in smooth muscle cells (Module 7: Figure smooth muscle cell cGMP signalling). One of the actions of cyclic GMP is to activate cyclic GMP-dependent protein kinase I (cGKI), which phosphorylates the BK channel to enhance its sensitivity to Ca2+ and this increases the negative-feedback loop.
An important feature of the mesangial cells is that they are readily induced to proliferate following stimulation with either hormones such as angiotensin II and endothelin-1 or growth factors such as platelet-derived growth factor (PDGF) (Module 7: Figure mesangial cell). Such mesangial proliferation plays an important function in maintaining the cell population and hence the integrity of the glomerulus. For example, such proliferation is an important adaptive response to restore the glomeruli that are damaged after streptococcal infections or lupus nephritis.
Mesangial cell proliferation is curbed by the local release of transforming growth factor β1 (TGF-β1), which acts through the Smad signalling pathway (Module 2: Figure Smad signalling). Activation of Smad signalling also acts to increase the expression of various extracellular matrix components such as collagen IV, which are components of the filtration filter (Module 7: Figure mesangial cell).
The propensity of the mesangial cell to proliferate does have serious pathological consequences because it is one of the major causes of a number of kidney diseases such as diabetic nephropathy that then lead to end-stage renal disease (ESRD).
Mechanotransduction in kidney cells
All of the cells of the kidney tubule, except the collecting duct, contain a single primary cilium about 2–3 μm long, which functions in mechanotransduction in kidney cells. The single primary cilium on each kidney cell is located on the apical surface and extends into the lumen. The suggestion is that this cilium has a sensory role in monitoring the flow of fluid in the tubule lumen, and this information is used to control the rate of ion transport. Whatever its function turns out to be, it is clear that mechanical deformation of the cilium will generate a Ca2+ signal (Module 7: Figure cilia deformation and Ca2+ signalling). The increase in Ca2+ seems to depend on the activation of the polycystins using mechanisms 1 and 2 in Module 3: Figure polycystin channel location.
The Ca2+ that enters through the polycystin channel may also be responsible for maintaining phenotypic stability, especially with regard to the expression of the mitogen-activated protein kinase (MAPK) signalling pathway. Polycystic kidney disease results from mutations of these channels, which decrease Ca2+ entry and may result in kidney cell proliferation and cyst formation through alterations in MAPK signalling (Module 12: Figure polycystins and polycystic kidney disease).
Mammalian salivary glands elaborate fluid that moistens the oral cavity between meals and also prepares food for ingestion and digestion. The first function is carried out by numerous small labial, buccal and palatal glands, which secrete continuously to moisten the mouth. The copious flow of saliva secreted during feeding originates from three large glands (submandibular, parotid and sublingual), which secrete in response to mechanical, thermal or chemical stimuli. In humans, as much as 0.75 litres of fluid is secreted by the salivary glands each day. In addition to producing the watery saliva, these glands also synthesize and secrete amylase, which functions in the digestion of starch and glycogen (Module 7: salivary gland structure). Salivary gland structure reflects this dual role of fluid and enzyme secretion. This dual function is also reflected in the salivary gland control mechanisms in that the parasympathetic system controls fluid secretion, whereas the sympathetic system regulates the release of amylase.
Salivary gland structure
Salivary glands have a racemose organization consisting of numerous bulbous acini connected to a central collecting duct by a variety of tubular elements (Module 7: salivary gland structure). The organization of the ducts can vary between the different glands. In the case of rat submandibular glands, the terminal acini drain into the main tubular system via short thin intercalated ducts. The more proximal parts of the main duct system, which often contains numerous granules, merge into a region characterized by elaborate striations. These striated ducts then connect to the main ducts that carry fluid into the oral cavity.
The acini have a pyramidal structure with their apical surfaces clustered around a narrow central lumen. The basal plasma membrane has numerous short infoldings, and the lateral membranes have similar infoldings to form intercellular canaliculi that function in fluid secretion. The acini also have all the structural features associated with protein secretion. There is an elaborate system of rough endoplasmic reticulum, Golgi and a number of large amylase-containing granules, which take up a large volume of the cell.
The granular and striated ducts, which function in ion reabsorption, have elaborate infoldings of the basal plasma membrane including an elaborate interdigitation of the infoldings of neighbouring cells. These basal structures are responsible for the striations of the striated ducts. By contrast, the apical surface is almost completely unspecialized with occasional short bulbous microvilli. The simplicity of the apical membrane may enable these duct cells to reabsorb ions without a concomitant flow of water, thus leading to dilute saliva.
Each acinus has a single contractile myoepithelial cell, which has a starfish-shaped structure with the long arms forming a basket around the acinus. Numerous bipolar spindle-shaped myoepithelial cells are associated with the intercalated ducts. These myoepithelial cells are thought to facilitate the flow of saliva from the acini into the main ducts. Contraction of the single basket-shaped cell squeezes the acinus, whereas contractions of the longitudinal cells of the intercalated ducts shorten and widen the lumen to allow secreted products access to the main ducts.
Salivary glands have a rich blood supply. Fenestrated capillaries ramify throughout the gland and come into close contact with both the secretory acini as well as the reabsorptive cells of the ducts. The presence of fenestrae permits ions and water to leave the capillary lumen rapidly enough to maintain high rates of salivary secretion.
Salivary secretion is controlled by transmitters released from both the parasympathetic and sympathetic nervous systems. Nerve bundles ramify throughout the gland and single neurons come to lie close to the surface. In some cases, neurons lie in grooves between two epithelial cells enabling transmitter to be released directly on to the surface of the epithelial cells (white arrows in Module 7: salivary gland structure). Transmitters such as acetylcholine (ACh) and noradrenaline (norepinephrine) activate the salivary gland control mechanisms responsible for regulating fluid and enzyme secretion.
Salivary gland control mechanisms
A characteristic of salivary gland function is that long periods of quiescence are interrupted with short periods of intense secretory activity. These short bursts of secretion are regulated by both components of the autonomic nervous system (Module 7: Figure salivary gland function). The parasympathetic nervous system primarily regulates fluid secretion, whereas the sympathetic system controls the release of amylase. The distinction between these two control mechanisms is not completely clear-cut in that there is some enzyme release during parasympathetic stimulation. Similarly, some fluid is stimulated by the sympathetic system.
Fluid secretion depends upon a transepithelial flux of Cl− (Module 7: Figure salivary gland secretion). Entry across the basolateral membrane is carried out by the Na+-K+-Cl− cotransporter 1 (NKCC1) with the driving force supplied by the Na+ entering the cell down its electrochemical gradient that is maintained by the classical ouabain-sensitive Na+ pump (Na+/K+-ATPase). Any accumulation of K+ is leaked back to the plasma through a Ca2+-sensitive large-conductance (BK) channel. Another mechanism for Cl− entry depends on the paired Cl−/HCO3− and Na+/H+ exchangers. Fluid secretion is very dependent on the activity of carbonic anhydrase (CA), which catalyses a reaction between water and CO2 to give the HCO3− that is exchanged for Cl−. The other product of this reaction is expelled by the Na+/H+ exchanger. The accumulation of Cl− within the cell increases the electrochemical gradient and this enables this anion to pass into the lumen through Ca2+-sensitive Cl− channels (CLCAs). The flow of Cl− into the lumen then provides the electrical gradient for a parallel flow of Na+, and this movement of NaCl then provides the osmotic gradient for the flow of water. Water flow into the lumen occurs through two pathways: by passing either through the cell (transcellular) or via the intercellular junctions (paracellular). The transcellular flow is carried out by aquaporins with AQP3 facilitating entry into the cell and AQP5 in the apical membrane enabling water to flow into the lumen.
Control of fluid secretion by the parasympathetic neurons depends on the release of acetylcholine (ACh) that acts through muscarinic (M3) receptors that are coupled to the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette (Module 7: Figure salivary gland secretion). The increase in Ca2+, which depends both on the release from internal stores and the entry through a store-operated channel (SOC), has two main functions. Firstly, Ca2+ activates the Ca2+-sensitive Cl− channels (CLCAs) that pass Cl− into the lumen, which then provides the driving force for Na+ and water movement. Secondly, Ca2+ activates the BK channel on the basolateral membrane and thus acts to hyperpolarize the membrane to maintain the electrochemical gradient that drives Cl− efflux into the lumen.
The control of amylase release by the adrenergic pathway is not fully established. The cyclic AMP pathway clearly has a major role, but exactly how it functions and whether there is a role for Ca2+ is still not clear.
The main function of the exocrine pancreas is to synthesize and secrete a range of digestive enzymes. These enter the duodenum in a flow of pancreatic juice that is rich in HCO3−, which plays an essential role in the control of duodenal pH by neutralizing the H+ ions coming from the stomach. The latter periodically releases some of its contents into the duodenum, and this gastric emptying is co-ordinated with pancreatic secretion by a complex interplay between various hormonal and neural pathways.
The pancreas consists of numerous lobules held together with connective tissue containing blood vessels and nerves (Module 7: Figure exocrine pancreas). The exocrine pancreatic acinar cells, which are most conspicuous because of their profuse rough endoplasmic reticulum and large accumulation of zymogen granules, have a pyramidal shape, with their apices facing the lumen where the zymogen granules are released. The exocrine pancreatic centroacinar cells, which merge into the ducts, are much lighter due to the low density of intracellular organelles. These exocrine pancreatic centroacinar cells are responsible for much of the fluid produced by the pancreas. Their activity is controlled by secretin, which acts to stimulate the secretion of ions such as Na+ and HCO3−.
Exocrine pancreatic acinar cells
Most attention has been focused on the pancreatic acinar cells, which are typical secretory cells designed to release both proteins and fluid. The former are the digestive enzymes stored in the zymogen granules located in the apical part of the cell. The human pancreas secretes a wide range of proteins such as lipases, ribonuclease, deoxyribonuclease, proelastase, chymotrypsinogen and trypsinogen. This secretory activity is regulated by secretagogues such as acetylcholine (ACh) and cholecystokinin (CCK), which generate Ca2+ signals with complex spatial and temporal profiles. At low doses, both ACh and CCK generate rapid Ca2+ transients localized to the apical region. A mitochondrial firewall that surrounds this apical zone may help to restrict this apical microdomain. At higher doses, these stimuli can also produce stronger Ca2+ signals that start in the apical zone, but then breach the mitochondrial firewall to propagate outwards into the basal region of the cell in the form of an intracellular Ca2+ wave. The generation of these Ca2+ signals is very dependent on release from the endoplasmic reticulum (ER), which is very extensive in these pancreatic cells. Most of the rough ER that synthesizes proteins is located in the basal region, but there are thin strands that percolate down into the apical region, where it plays a critical role in producing the localized Ca2+ signals that regulate both exocytosis and fluid secretion (Module 7: Figure control of pancreatic secretion). Another source of Ca2+ is the bafilomycin-sensitive acid compartment that consists of the zymogen granules, endosomes and granules that are located exclusively in the apical granular pole.
Release of Ca2+ from the ER is replenished by uptake of external Ca2+ carried out by a store-operated entry mechanism. As the store empties, the decrease in Ca2+ is detected by the ER Ca2+ sensor STIM that activates the Orai channels located in the baso-lateral membrane through conformational coupling mechanism as outlined in Module 3: Figure conformational coupling hypothesis. The Ca2+ that enters is then pumped into the ER by sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA) pumps.
The localized transients of Ca2+ in the apical region not only function to trigger the exocytosis of the zymogen granules, but also are responsible for stimulating fluid secretion. The latter depends on Ca2+ activating a Ca2+-sensitive Cl− channel (CLCA) located in the apical membrane, and this allows Cl− to enter the lumen. This influx of Cl− makes the lumen negative, thus providing a driving force to allow Na+ to flow through junctions to create the osmotic gradient for a parallel flow of water. The increase in Ca2+ is also important for activating the large-conductance (BK) K+ channel, which keeps the membrane hyperpolarized and thus maintains the driving force for Cl− to flow into the lumen. In addition, the efflux of K+ from the cell helps to maintain the operation of the Na+-K+-Cl− cotransporter 1 (NKCC1) that is responsible for providing the ionic gradient that maintains this mechanism of fluid secretion. With regard to control, both exocytosis and the two Ca2+-sensitive channels are driven by Ca2+ signals localized to the apical region. A number of intracellular Ca2+ release channels contribute to the apical microdomain of Ca2+. The mechanisms for acetylcholine-induced pancreatic secretion differ from that of cholecystokinin (CCK)-induced pancreatic secretion, and are thus considered separately.
Non-oxidative fatty acid ethyl ester (FAEE) metabolites of ethanol, which release Ca2+ from both the ER and acidic Ca2+ stores, have been implicated in pancreatitis.
Acetylcholine-induced pancreatic secretion
Acetylcholine (ACh) acts on muscarinic receptors to produce inositol 1,4,5-trisphosphate (InsP3), which diffuses into the cell to stimulate release of Ca2+ by InsP3 receptors located in the apical region of the cell. The Ca2+ signal may be amplified by recruiting neighbouring ryanodine receptors (RYRs) to create a wave that spreads to the basal region (Module 7: Figure control of pancreatic secretion). During continuous stimulation, ACh can set up regular Ca2+ oscillations (Module 6: Figure pancreatic Ca2+ oscillations).
Cholecystokinin (CCK)-induced pancreatic secretion
The action of cholecystokinin (CCK)-induced pancreatic secretion seems to depend on nicotinic acid–adenine dinucleotide phosphate (NAADP) signalling (Module 2: Figure cADPR/NAADP function). Low doses of CCK stimulate the formation of NAADP by a mechanism that remains to be fully characterized. This NAADP then acts on a putative receptor located on a lysosomal-related organelle to provide a small amount of trigger Ca2+, which is amplified by stimulating the inositol 1,4,5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RYRs) (Module 7: Figure control of pancreatic secretion).
Exocrine pancreatic centroacinar cells
The exocrine pancreatic centroacinar cells and duct cells are responsible for secreting the bicarbonate-rich fluid that helps to carry the enzymes secreted by the acinar cells down into the intestine (/1Module 7: Figure exocrine pancreas). The prime mover for fluid secretion is the trans-epithelial transport of HCO3−, which enters across the basolateral membrane through the pancreatic Na+- HCO3− cotransporter (pNBC1) and is secreted across the apical membrane into the lumen through two members of the solute carrier family 26 (SLC26): SLC26A3 and SLC26A6, which are coupled Cl−/HCO3− exchangers. The secretion of HCO3− may be maintained by the /1cystic fibrosis transmembrane conductance regulator (CFTR) channel that provides the Cl− in the lumen that is exchanged for HCO3−. The entry of HCO3− across the basolateral membrane and its exit from the cell into the lumen appears to be coordinated by /1InsP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) acting to enhance the activity of both pNBC1 and CFTR.
The liver carries out a number of important functions. It maintains the level of circulating metabolites, particularly glucose (Module 7: Figure metabolic energy network). There is a constant ebb and flow of oxidative substrates between the plasma and liver cells. They contain a major store of glycogen, and the process of glycogen synthesis during periods when glucose is plentiful is controlled by insulin, whereas glucagon activates a process of glycogenolysis to release glucose back into the plasma when glucose levels are low. Liver cells thus occupy a critical position in the overall metabolic energy network. In addition to this role in glucose metabolism, the liver has a number of other functions:
The organization of the liver reveals how the liver cells receive a constant flow of blood from both the hepatic artery cells and hepatic portal vein (Module 7: Figure liver lobule). About 70% of the blood enters from the portal vein, which comes from the digestive tract, whereas the remainder enters via the hepatic artery. Most of the nutrients absorbed by the intestine pass directly to the liver. Immediately after a meal, there is a rapid rise in the level of glucose in the hepatic portal vein, whereas the level in the central hepatic venous system changes very little. This is because much of the glucose is taken up from the plasma as it percolates along the hepatic sinusoid. Liver cells are arranged in epithelial sheets, with one surface facing the space of Disse that is open to the sinusoid where the metabolic exchanges occur, whereas the other side is responsible for secreting bile (Module 7: Figure liver cell structure). Liver cells, which are arranged as cell sheets radiating out from the central vein, comprise about 85% of the total cell mass of the liver. The other main cells in the liver are the endothelial cells that line the sinusoids, the Kupffer cells that are resident macrophage-like cells and the hepatic stellate cells that have a variety of functions.
Most attention will be focused on the regulation of glucose metabolism, which is the main function of liver cells.
Regulation of glucose metabolism
The regulation of glucose metabolism by liver cells is dictated by the energy balance within the metabolic energy network (Module 7: Figure metabolic energy network). If energy is required during fasting or stress, catabolic hormones such as glucagon and adrenaline (epinephrine) stimulate the release of glucose (Step 11 in Module 7: Figure metabolic energy network). However, when glucose levels are high after feeding, the hormone insulin shifts the emphasis from catabolism to anabolism and the liver begins to synthesize and store glycogen (Step 8 in Module 7: Figure metabolic energy network). Control of the two processes of liver cell glycogenolysis and liver cell gluconeogenesis are at the centre of the regulation of glucose metabolism.
Liver cell glycogenolysis
Liver cell glycogenolysis is the process whereby the cell regulates its glycogen store. The level of glycogen is controlled by a balance between glycogen hydrolysis carried out by the enzyme phosphorylase and glycogen synthesis carried out by the enzyme glycogen synthase (Module 7: Figure glycogenolysis and gluconeogenesis). Phosphorylase exists in two states: an active phosphorylase a form and an inactive phosphorylase b form. Conversion into the active state is carried out by phosphorylation through another enzyme, phosphorylase kinase, which in turn exists in an inactive b form and an active a form. Phosphorylase kinase is a Ca2+-sensitive enzyme that is controlled by Ca2+ acting through a resident calmodulin (CaM) molecule. The enzyme is also stimulated by the cyclic AMP signalling pathway, which acts through protein kinase A (PKA) to phosphorylate the enzyme to enhance its sensitivity to Ca2+. Cyclic AMP also promotes glycogen breakdown by simultaneously phosphorylating glycogen synthase to convert it into the inactive b form. The phosphorylation events are reversed by protein phosphatase 1 (PP1), which is targeted to the surface of glycogen particles by the glycogen targeting subunit GL (Module 5: Figure PP1 targeting to glycogen).
Hydrolysis of glycogen gives glucose 1-phosphate (G1P), which is converted by a phosphoglucomutase into glucose 6-phosphate (G6P), which is then hydrolysed by the enzyme glucose-6-phosphatase (G6Pase) to form glucose. G6Pase is an enzyme unique to liver cells, where it also serves as the final step in the process of liver cell gluconeogenesis.
Liver cell gluconeogenesis
The liver cell is also capable of synthesizing glucose from various precursors, such as the lactate it receives from skeletal muscle and the free fatty acids and glycerol that come from the white fat cells (Module 7: Figure metabolic energy network). It is also able to use amino acids, and this source becomes particularly relevant during extreme starvation, when autophagy begins to degrade structural proteins to maintain glucose supplies. These precursors enter the mitochondrion, where they are converted into oxaloacetic acid (OAA), which is the common precursor used during gluconeogenesis (Module 7: Figure glycogenolysis and gluconeogenesis). This process of gluconeogenesis is particularly important during periods of fasting when glycogen reserves have been exhausted. In humans, glycogen reserves can satisfy the demand for glucose for about 12 h; thereafter, the liver has to switch on gluconeogenesis. This is a remarkably efficient process, because it can maintain the normal blood glucose levels that are essential for the energy-consuming tissues, such as the brain, kidney, testis and red blood cells, as occurs during calorie restriction (CR) or during periods of starvation.
Many of the reactions of gluconeogenesis are simply a reversal of glycolysis. Most of the glycolytic reactions are close to equilibrium, so the whole process can be reversed simply by varying the input of substrate at either end. However, there are several non-equilibrium reactions that are not easily reversed and have to be bypassed. Two of these steps, namely those mediated by hexokinase and phosphofructokinase (PFK-2), are bypassed by glucose-6-phosphatase (G6Pase) and fructose bisphosphatase. The interconversion of phosphoenolpyruvate (PEP) and pyruvate by pyruvate kinase is also by passed by two separate reactions, which are important sites for the hormonal control of gluconeogenesis. In the first reaction, pyruvate is converted into OAA by a carboxylation step that requires ATP and is catalysed by pyruvate carboxylase. OAA is then decarboxylated and phosphorylated to PEP by phosphoenolpyruvate carboxykinase (PEPCK). The significant point about some of these bypass enzymes is that their expression is regulated by liver cell signalling mechanisms.
Liver cell signalling mechanisms
The liver cell signalling mechanisms are responsible for switching the liver cell from its energy storage mode into its role of delivering energy in the form of glucose during fasting (Module 7: Figure metabolic energy network). The former is controlled by insulin, whereas the release of glucose is a somewhat more complicated process, as it is sensitive to a number of factors, including hormones [e.g. adrenaline (epinephrine), glucagon and vasopressin], lipid signals such as the fatty acids derived from white fat cells, or the energy status of the cell (Module 7: Figure liver cell signalling). Vasopressin and noradrenaline (norepinephrine) act through the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette, whereas glucagon and adrenaline act through the cyclic AMP signalling pathway. The way in which these two pathways regulate glycogenolysis is shown in more detail in Module 7: Figure glycogenolysis and gluconeogenesis. They both act to stimulate the enzyme phosphorylase kinase, which is responsible for switching on the enzyme phosphorylase that hydrolyses glycogen to glucose. Cyclic AMP enhances this breakdown of glycogen by simultaneously inhibiting glycogen synthesis.
In addition to activating this process of liver cell glycogenolysis, these signalling mechanisms are also able to activate liver cell gluconeogenesis, which is a somewhat more complicated process because it depends upon a phenotypic remodelling of the liver cell signalsome. The different signalling pathways activate a transcriptional cascade that results in an increase in the expression of some of the key rate-limiting steps in glycogenolysis (Module 7: Figure liver cell signalling). Control of this cascade has two main components. Firstly, there is activation of transducer of regulated CREB 2 (TORC2) and the transcription factor cyclic AMP response element-binding protein (CREB), which act to increase the expression of peroxisome-proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α). Secondly, the PGC-1α then acts together with PPARα to induce the transcription of some of the rate-limiting enzymes and transporters, such as glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyltransferase 1 (CPT1), which are responsible for liver cell gluconeogenesis (Module 7: Figure glycogenolysis and gluconeogenesis). In addition, this transcriptional cascade can also activate genes that increase fatty acid oxidation or reduce the expression of genes that control glycolysis. An increase in the activity of all these components accelerates the formation of glucose.
The activity of PGC-1α is regulated by acetylation: the histone acetylase GCN5 inactivates PGC-1α, whereas its activation depends on the deacetylase sirtuin SIRT1. This activation by SIRT1 plays an important role in the maintenance of energy metabolism and antioxidant defences (Module 12: Figure ageing mechanisms) and has been implicated in the process of ageing.
The key event in this transcriptional cascade is the increased expression of PGC-1α induced by the CREB transcription factor. A number of signalling pathways are known to activate CREB (Module 4: Figure CREB activation), and two of these are evident in liver cells. Cyclic AMP has two actions. It acts not only by phosphorylating CREB in the nucleus, but also by regulating the activity of the CREB cofactor TORC2. A number of control mechanisms regulate the shuttling of TORC2 between the nucleus and cytoplasm.
Glucagon or fasting stimulates a rapid translocation of TORC2 into the nucleus (within about 10 min), whereas it remains inactive in a phosphorylated state during the action of insulin or when energy reserves are high. An increase in cyclic AMP inhibits the salt-inducible kinase 2 (SIK2), which normally acts to phosphorylate TORC2, thereby preventing it from entering the nucleus (Module 7: Figure liver cell signalling). Ca2+ may also inhibit TORC2 by activating calcineurin (CaN) to dephosphorylate TORC2 so that it can enter the nucleus to carry out its role as a CREB coactivator.
The AMP signalling pathway also plays a role in regulating the CREB/TORC transcriptional mechanism. The AMP-activated protein kinase (AMPK), which is activated during energy stress when the level of AMP rises, phosphorylates TORC2, thereby preventing it from activating the transcriptional cascade responsible for switching on the gluconeogenic pathway. By shutting down gluconeogenesis, the cell is able to conserve energy instead of sending it off to the energy-consuming cells.
Insulin promotes energy storage by counteracting many of the glycolytic and gluconeogenic signalling pathways. Insulin stimulates the PtdIns 3-kinase signalling pathway, which uses the lipid second messenger PtdIns3,4,5P3 (PIP3) to activate protein kinase B (PKB), which then stimulates the phosphodiesterase (PDE3B) that hydrolyses cyclic AMP, thus reducing the stimulatory action of this second messenger on both glycogen breakdown and glucose synthesis (Module 7: Figure liver cell signalling). The PKB can exert some additional inhibitory effects. PKB is one of the protein kinases that can phosphorylate the inositol 1,4,5-trisphosphate receptor (InsP3R) to reduce Ca2+ signalling. In addition, it can inactivate the CREB-binding protein (CBP), thereby reducing the action of CREB in inducing the transcriptional cascade.
Hepatic stellate cell
The hepatic stellate cells, which make up about 15% of the cells in the liver, have had many names such as perisinusoidal cell, Ito cell, lipocyte and fat-storing cell. They are located in the subendothelial space between the hepatocytes and the endothelial cells (Module 7: Figure liver cell structure) and are characterized by their long processes and by their numerous lipid droplets that store large amounts of vitamin A. They are the main storage site for retinoids, which are vitamin A compounds.
Apart from their role in storing the retinoids, these stellate cells are normally quiescent. However, following injury to the liver they undergo a remarkable transformation that enables them to help in organ repair (Module 7: Figure hepatic stellate cell). The hepatic stellate cells respond to stimuli released from the other liver cells. Kupffer cells release transforming growth factor β (TGF-β) and lipid peroxides; hepatocytes release lipid peroxides; endothelial cells release fibronectin and the blood platelets release platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). These stimuli induce a process of phenotypic remodelling whereby the hepatic stellate cells switch from a resting vitamin A-rich cell to a much more active cell capable of carrying out process such as proliferation, contraction, fibrogenesis and inflammation (Module 7: Figure hepatic stellate cell).
The PDGF released from blood platelets is one of the main growth factors responsible for activating hepatic stellate cell proliferation. PDGF acts through the PDGFR to induce many of the typical growth factor signalling pathways such as the Ca2+ signalling and MAP kinase signalling pathways, which are responsible for inducing cell proliferation (Module 9: Figure growth factor signalling).
A diagnostic feature of the transformation of hepatic stellate cells into myofibroblast-like cells is the expression of α-smooth muscle cell actin (α-SMA). Contraction of these cells, which is activated by endothelin-1, appears to be regulated by two mechanism that resemble the smooth muscle Ca2+ signalling cascade and the smooth muscle Rho/Rho kinase signalling pathway. This contraction constricts the sinusoids and thus increases the portal resistance and contributes to the portal hypertension associated with cirrhosis of the liver.
One of the major consequences of hepatic stellate cell activation is the onset of fibrogenesis to form hepatic scars that are mainly caused by the secretion of collagen type I, which is responsible for scar matrix formation. The appearance of these scars results from a complex process of extracellular matrix remodelling that disrupts the normal liver matrix that is then replaced by the collagen-based scar matrix (Module 7: Figure hepatic stellate cell). This matrix degradation and synthesis, which is orchestrated by the fibroblasts, is stimulated mainly by TGF-β. Degradation of the normal liver matrix is carried out by secretion of matrix metalloproteinases (MMPs) such as MMP2, MMP9 and stromelysins (Module 1: Table MMPs and their inhibitors). The formation of the scar matrix begins with the synthesis and secretion of collagen type I. TGF-β acts through the Smad signalling pathway to induce the transcription of collagen type I. The fibroblasts also function to stabilize the scar matrix by secreting TIMP-1 and TIMP-2, which are the tissue inhibitors of metalloproteinases. If the formation of the scar matrix becomes excessive it can disrupt liver cytoarchitecture resulting in cirrhosis of the liver.
Hepatic stellate cells play an active role in immunoregulation where they contribute to inflammatory responses through a number of mechanisms. Like other immune cells, they express the Toll-like receptors (TLRs) that respond to the pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), which are derived from various pathogens. These TLRs then act through the Toll receptor signalling pathway that induces the activation of the NF-κB and p38 pathway to stimulate the formation of inflammatory cytokines and chemokines (Module 2: Figure Toll receptor signalling). The latter provide a chemotactic gradient to attract various inflammatory cells such as neutrophils (Module 7: Figure hepatic stellate cell).
Once the infection or injury has been resolved, the activity of the hepatic stellate cells is curtailed by various processes that seem to begin with a process of stress-induced senescence whereby the activated cells cease proliferating and withdraw from their various activities such as the formation of the scar matrix. The senescent cells then attract natural killer cells resulting in the hepatic stellate cells being eliminated from the liver. The onset of senescence thus helps to protect the liver from the excessive fibrogenesis that can progress to cirrhosis of the liver
The two adrenal glands are compact, endocrine organs, which lie immediately above each kidney (Module 7: Figure adrenal gland). They consist of two main parts, which differ both in function and embryological origin. The outer adrenal cortex, which arises from the mesoderm and is part of the hypothalamic–pituitary–adrenal axis, makes up most of the gland and secretes both mineralocorticoids (e.g. aldosterone) and glucocorticoids (e.g. corticosterone). The inner adrenal medulla, which is derived from the neural crest, contains chromaffin cells that secrete catecholamines such as adrenaline (epinephrine) and noradrenaline (norepinephrine), especially under stressful situations.
The adrenal gland is richly supplied with blood from vessels arising from the inferior phrenic artery, aorta and renal artery. These incoming arteries subdivide into smaller branches that enter the capsule. There are long parallel vessels running down the length of the cortex that join a reticular plexus in the region where the cortex and medulla meet. Vessels that leave the reticular plexus ramify throughout the medulla before draining into the large central veins, which then unite to form the adrenal vein. Each hormone-secreting cell is intimately associated with a blood vessel, and this ensures that released hormones are rapidly transferred into the circulatory system. This rapid transfer is facilitated further by the fact that the endothelial cells that line the blood vessels have numerous fenestrae.
The outer cortical region of the adrenal gland is divided into three main regions: zona glomerulosa, zona fasciculata and the zona reticularis (Module 7: Figure adrenal gland). The cells in all three regions share many structural similarities. The cells are arranged in radial cords running alongside the parallel blood capillaries. The well-developed system of tightly packed smooth endoplasmic reticulum (ER) is closely associated with a large population of mitochondria. There also are large lipid droplets, which contain a store of cholesterol that is the precursor for hormone synthesis. During aldosterone and cortisol biosynthesis, the synthetic intermediates alternate between the ER and the mitochondria and co-operate with each other during steroid biosynthesis (Module 1: Figure aldosterone and cortisol biosynthesis).
The three cortical regions are specialized to secrete separate steroid hormones in response to different stimuli. Most attention has focussed on the zona glomerulosa cells that secrete aldosterone and the zona fasciculata cells that secrete the corticosteroids such as cortisol (hydrocortisone) and corticosterone. The zona reticularis secretes androgen hormones.
Zona glomerulosa cells
Zona glomerulosa cells, which are located in the outer region of the adrenal cortex, function to synthesize and release aldosterone (Module 7: Figure adrenal gland). These cells contain large amounts of smooth endoplasmic reticulum (ER) that function in aldosterone and cortisol biosynthesis (Module 1: Figure aldosterone and cortisol biosynthesis). The various synthetic steps are located on either the inner membrane of the mitochondria or on the surface of the smooth ER (Module 7: Figure glomerulosa cell signalling). The major physiological stimuli of glomerulosa cells are angiotensin II and small elevations in the plasma level of K+, both of which seem to act by increasing the intracellular level of Ca2+.
Angiotensin II stimulates the AT1 receptor to induce the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette. Following the formation of InsP3, Ca2+ is released from the internal ER store to provide the initial signal to stimulate aldosterone synthesis and release. During prolonged periods of stimulation, the cell has to switch to an external source of Ca2+ as the internal store loses its Ca2+. There appear to be a number of channels responsible for Ca2+ entry, but just how they are activated has been a matter of considerable debate. Glomerulosa cells appear to have a capacitative Ca2+ entry mechanism based on the opening of store-operated channels (SOCs).
Glomerulosa cells express voltage-operated channels (VOCs) such as the CaV1 family of L-type channels and the CaV3 family of T-type channels (Module 3: Table VOC classification). The CaV1.2 and CaV1.3 L-type and the CaV 3.2 T-type channels seem to be the major isoforms used to generate the prolonged Ca2+ signal in response to both angiotensin II and high K+. There has been a problem in understanding how these channels are activated by such small elevations in the plasma level of K+ (secretion is maximally stimulated between 8 and 10 mM), which causes relatively small membrane depolarizations. All the evidence seems to point to the T-type channel being particularly important since they have activation thresholds in the −60 to −70 mV range (Module 3: Table VOC classification). They are thus ideally suited to respond to the relatively small elevations in serum K+ that will produce correspondingly small membrane depolarizations. The L-type channels have also been shown to contribute to Ca2+ signalling, but they may only come into play during more intense bouts of stimulation.
In addition to responding to fluctuations in external K+, the VOCs are also recruited by the angiotensin II AT1 receptors to maintain Ca2+ signalling during prolonged stimulation (Module 7: Figure glomerulosa cell signalling). Several mechanisms have been proposed for how the AT1 receptors activate these Ca2+ entry channels. The most likely is an indirect mechanism that depends upon depolarization of the membrane by switching off various K+ channels using a variety of regulatory signalling pathways. The TREK-1 channels are inhibited by cyclic AMP. The glomerulosa cells express TASK channels, both TASK-1 and TASK-3 isoforms, which may function as heterodimers. Finally, there are Kv7.1 channels. These TASK and KV7.1 channels appear to be under the PtdIns4,5P2 regulation of ion channels and exchanger mechanism (Module 2: Figure PtdIns4,5P2 regulation of K+ channels) whereby this inositol lipid keeps the channel open, but when PtdIns4,5P2 is hydrolysed by angiotensin II, the channel closes (Module 7: Figure glomerulosa cell signalling).
Glomerulosa cells are also sensitive to the high levels of adrenocorticotrophic hormone (ACTH) [or corticotropin] that occur during severe Na+ or fluid loss. ACTH acts through the cyclic AMP signalling pathway and its action appears to be closely integrated with that of Ca2+. L-type Ca2+ channels are known to be phosphorylated and activated by cyclic AMP (Module 3: Figure CaV1.2 L-type channel), and such a mechanism may explain how ACTH can bring about an increase in the level of Ca2+ in glomerulosa cells. Another control system is carried out by atrial natriuretic factor (ANF) that inhibits the secretion of aldosterone by using cyclic GMP to increase the activity of PDE2 to reduce the level of cyclic AMP, which drives the release of this steroid
All of the evidence on the control of glomerulosa cells thus indicates a central role for Ca2+ in regulating aldosterone synthesis and release (Module 7: Figure glomerulosa cell signalling). The primary action of Ca2+ seems to be directed towards an early step in steroid biosynthesis, since it activates the steroidogenic acute regulator (StAR) protein located in the outer mitochondrial membrane. StAR appears to be a rate-limiting step in steroid synthesis because it controls the access of cholesterol to the first enzyme CYP11A1 located on the inner mitochondrial membrane where it functions in aldosterone and cortisol biosynthesis. There is a longer term action of Ca2+ to stimulate the transcription of genes encoding components of the biosynthetic pathway such as StAR and the aldosterone synthase CYP11B2
Mutation of the StAR protein has been linked to congenital lipoid adrenal hyperplasia.
Zona fasciculata cells
The zona fasciculata cells, together with the zona reticularis cells, are responsible for secreting the glucocorticoid cortisol (Module 7: Figure adrenal gland). These cells contain large amounts of smooth endoplasmic reticulum that function in aldosterone and cortisol biosynthesis (Module 1: aldosterone and cortisol biosynthesis). The fasciculata cells differ from the glomerulosa cells by expressing the enzyme CYP17, which is a steroid 17α-hydroxylase and 17,20-lyase that converts progesterone into 17α-progesterone that begins the synthetic sequence that results in the formation of cortisol. Cortisol synthesis is stimulated by adrenocorticotrophic hormone (ACTH) that acts through receptors that are coupled to the cyclic AMP signalling pathway.
Excessive release of cortisol causes Cushing's syndrome.
The region of the adrenal gland known as the adrenal medulla contains the chromaffin cells that function to release catecholamines (Module 7: Figure adrenal gland). The most common cells, which have 10 000–20 000 round or oval opaque granules, secrete adrenaline (epinephrine). The cells are polarized in that most of the granules are located in the apical region of the cell facing the blood vessels. Exocytosis can occur from both the apical and lateral surfaces. The scarcer cell type, which either can form aggregates or are dispersed among the predominant adrenaline-secreting cells, releases noradrenaline (norepinephrine) contained in granules with a higher electron density. This medullary region is richly innervated with preganglionic sympathetic nerves, of which most originate from the thoracic splanchnic nerve that passes through the cortex before dividing repeatedly to form individual axons that innervate the chromaffin cells. From a signalling perspective, both cell types seem to be regulated in much the same way: an action potential travelling down the splanchnic neurons activate the chromaffin cell to release their stored catecholamines.
Chromaffin cells located within the adrenal medulla are arranged in a compact epithelial fashion surrounding the blood vessels that permeate through the adrenal gland (Module 7: Figure adrenal gland). The intercellular membranes are held together by desmosomes in the basal region of the cell. There do not appear to be any gap junctions or tight junctions. Where there are no desmosomes, the intercellular space can be quite open and can contain a primary cilium or a nerve ending. It has been proposed that the cilium may stir up the contents of this intercellular space. Since the chromaffin cells do not secrete fluid, there is no flow of fluid to carry secreted products towards the blood vessels. Having a cilium thrashing about in this confined space would serve to waft secreted products towards the blood vessels. The endothelial cells lining these blood vessels contain numerous fenestrae that will also enhance the movement of secreted catecholamines into the blood vessel.
Chromaffin cells are innervated by branches of the splanchnic nerve and single axons pass through the basement membrane to lie within the intercellular space where they form typical synaptic endings capable of activating both cells (Module 7: Figure chromaffin cell secretion). The splanchnic nerve endings release acetylcholine that acts on nicotinic acetylcholine receptors (nAChRs) to initiate the process of stimulus–secretion coupling in chromaffin cells. The mechanism of nicotinic acetylcholine receptor (nAChR) signalling through membrane depolarization sets up action potentials that spread around the cell surface to recruit voltage-operated channels (VOCs) such as the P/Q-type (55%), N-type (25%) and L-type (20%) (Module 3: Table VOC classification). The proportion of these channels expressed in chromaffin cells is species-specific, and the ratios shown above are for human chromaffin cells. The opening of these channels during the course of the action potentials creates microdomains of Ca2+, which occur immediately below the cell surface. These microdomains can be enhanced further by recruitment of Ca2+ from the internal stores by activation of the ryanodine receptors (RYRs) through a process of Ca2+-induced Ca2+ release (CICR) (Module 2: Figure Ca2+-induced Ca2+ release). The localized high intensity Ca2+ microdomains immediately below the surface of the plasma membrane are responsible for activating two processes. Firstly, it activates adseverin, which is also known as scinderin (SCIN), that severs the cortical actin filaments thus allowing the chromaffin granules to access the membrane. Secondly, the increase in Ca2+ triggers chromaffin granule exocytosis (Module 7: Figure chromaffin cell secretion).
There are two type of exocytosis in chromaffin cells that depend on the intensity of stimulation (Module 7: Figure chromaffin cell exocytosis). Under resting or low intensity stimulus conditions, kiss-and-run vesicle fusion occurs. The brief narrow pore that forms during this exocytotic process allows a small part of the adrenaline (epinephrine) to be released, whereas the remaining adrenaline and larger molecules are retained within the granule that can then participate in multiple kiss-and-run vesicle fusion events. During intense stimulation, when there are higher levels of Ca2+, the classical exocytotic/endocytotic cycle is activated where a complete fusion event occurs, resulting in the release of the entire contents of the granule. Once fusion is complete, the process of endocytosis retrieves the membrane as part of the vesicle cycle (Module 4: Figure vesicle cycle).
The increased release of opiates and perhaps also ATP that occur during intense stimulation contribute to an autoreceptor negative-feedback modulation of the VOCs (Module 7: Figure chromaffin cell exocytosis). For example, the purinergic P2Y and opioid μ and δ receptors inhibit the predominant P/Q-type channels through the G protein-dependent modulation mechanisms (Module 3: Figure CaV2 channel family).
Carbohydrate metabolism is regulated through the antagonistic action of two polypeptide hormones: glucagon and insulin (Module 7: Figure metabolic energy network). The former stimulates the hydrolysis of glycogen, particularly in the liver, resulting in elevated plasma glucose levels, whereas insulin has the opposite effect by increasing the conversion of glucose into glycogen in liver and muscle. A number of external signals can release insulin, including an increase in plasma glucose. There is considerable clinical interest in the mechanism and control of insulin secretion because it often declines during the onset of insulin resistance (Module 12: Figure insulin resistance) that leads to the widespread and increasing occurrence of diabetes.
β-Cells, which are located together with the glucagon-secreting α-cells in the islets of Langerhans, have a structure that reflects their specialized role in protein secretion. The primary stimulus for insulin secretion from pancreatic β-cells is glucose and is part of a feedback loop between the metabolite and the hormone that regulates its metabolism. Glucose has a bifunctional role in that it controls both insulin release and biosynthesis.
β-Cells are also sensitive to circulating hormones and to neural influences that function to adjust their responsiveness to glucose. For example, glucagon appears to sensitize β-cells, whereas noradrenaline (norepinephrine) released from the autonomic nervous system inhibits insulin release. Therefore, the main control features that must be considered are the primary action of glucose, together with the way in which release is modulated by hormonal (glucagon, somatostatin) and neural (adrenergic and cholinergic) pathways.
With regard to control mechanisms, the main problem is therefore to understand how glucose functions in both insulin release and biosynthesis.
Islets of Langerhans
The islets of Langerhans are small groups of glucagon-secreting α-cells (30–40%) and insulin-secreting β-cells (60–70%) (Module 7: Figure pancreas).
Insulin release and biosynthesis
In response to an elevation in blood glucose, there is an oscillatory release of insulin. In addition to triggering the release of insulin, glucose also regulates the biosynthesis of insulin (Module 7: Figure β-cell signalling). The oscillatory response to glucose is driven by slow Ca2+ oscillations with a periodicity of 4–6 minutes. On the crest of each Ca2+ elevation there are faster Ca2+ spikes with a period of 10—20 seconds.
The generation of these two oscillatory modes can be explained by the ‘dual oscillator model’ consisting of a glycolytic oscillator operating together with a Ca2+ oscillator (-cell">Module 7: Figure β-cell signalling).
The glycolytic oscillator is driven by glucose entering the cell through the glucose transporter 2 (GLUT2) and is then rapidly phosphorylated by hexokinase IV (also known as glucokinase) to form glucose-6-phosphate (G-6-P), which is then converted into fructose-6-phosphate (F-6-P). The latter may play a role in the oscillatory mechanism by accelerating glycolysis as part of a feed forward mechanism whereby it is converted into fructose-2,6-P2 (F-2,6-P2) by phosphofructose kinase-2 (PFK2). The F-2,6-P2 then stimulates PFK1 to convert F-6-P into fructose-1,6-P2 (F-1-6-P2). The latter then feeds into glycolysis and the tricarboxylic acid (TCA) cycle to increase the level of ATP while reducing the level of AMP, which function as a metabolic messenger to control insulin biosynthesis. The F-1-6-P2 also has a role in that it acts to stimulate pyruvate kinase M2 (PKM2) to increase the formation of pyruvate that enters the mitochondrion to fuel the formation of ATP. The existence of such a glycolytic oscillator is supported by oscillations in many of the components and related metabolic variables such as oxygen consumption, mitochondrial membrane potential, NAD(P)H and ATP levels. The most important variable is the level of ATP that provides the link to the faster Ca2+ oscillator.
The increase in ATP acts to inhibit an ATP-sensitive K+ (KATP) channel, which then depolarizes the membrane, resulting in the opening of L-type voltage-operated channels (VOCs) that provide the localized pulses of Ca2+ to trigger exocytosis of the preformed insulin vesicles. The membrane potential of β-cells is very sensitive to glucose (the membrane depolarizes by 8.5 mV for each 10-fold increase in glucose concentration). This opening of the Ca2+ channels appears as frequent bursts of action potentials, which constitutes the Ca2+ oscillator, that depend on a rapid feedback effect operating on a Ca2+-sensitive K+ channels, mostly likely to be BK channels, that rapidly terminates each action potential. The oscillatory spiking depends on a rapid switching between the L-type Ca2+ channels and the Ca2+-sensitive K+ channels (Module 7: Figure β-cell signalling). The intense activity of the Ca2+-induced events may begin to deplete ATP and this may then slow down the glycolytic oscillator to terminate each oscillatory phase.
A second action of ATP may occur through the cyclic ADP-ribose (cADPR) signalling or nicotinic acid–adenine dinucleotide phosphate (NAADP) signalling pathways to release Ca2+ from internal stores. Ca2+ derived from internal stores via the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette has also been implicated in controlling release, and this can account for the way in which secretagogues such as acetylcholine (ACh) activate release.
Control of insulin biosynthesis depends on the ability of the AMP signalling pathway to activate gene transcription. Under resting conditions, a high level of the metabolic messenger AMP normally represses gene transcription by phosphorylating and inactivating the hepatocyte nuclear factor 4α (HNF4α). When glucose is present and metabolism is enhanced, the decline in the level of AMP removes this inhibition, resulting in transcription of preproinsulin, which results in an increase in the synthesis of insulin. Ca2+ acting through the Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) also provides an important stimulus for increasing insulin transcription (-cell">Module 7: Figure β-cell signalling).
The survival and proliferation of β-cells is also regulated by glucose and by various hormones, such as glucagon-like peptide-1 (GLP-1), which appear to act synergistically to activate gene transcription of factors such as cyclic AMP response element-binding protein (CREB) (Module 4: Figure CREB activation). The basis of this synergism seems to depend upon the CREB co-activator called transducer of regulated CREB (TORC). When phosphorylated, TORC remains in the cytoplasm, but upon dephosphorylation, it enters the nucleus, where it acts together with CREB to promote transcription (Module 7: Figure β-cell signalling). TORC phosphorylation depends upon a Ca2+-dependent dephosphorylation mediated by calcineurin (CaN) and the cyclic AMP/protein kinase A (PKA)-dependent inhibition of the salt-inducible kinase 2 (SIK2) that phosphorylates TORC.
Insulin-secreting β-cell proliferation is also regulated by glucose and by various hormones, such as GLP-1. The signalling pathways responsible for activating proliferation are unclear, but seem to depend upon TORC, and perhaps also on Ca2+ signalling mechanisms. There is evidence that Ca2+ activates proliferation by stimulating the Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT).
Autoimmune destruction of these insulin-secreting β-cells is the cause of Type 1 diabetes.
Glucagon-secreting α-cells found within the islets of Langerhans (Module 7: Figure pancreas) play an essential role in maintaining blood glucose levels. In addition to releasing glucagon, they also have a sophisticated signalling system to ensure that the synthesis of glucagon is maintained at a level to satisfy the demands of the secretory system. These two processes of release and biosynthesis are synchronized by being coupled to the same Ca2+ signalling pathway (Module 7: Figure α-cell signalling). One of the important actions of Ca2+ is to activate transcription factors such as nuclear factor of activated T cells (NFAT) (Module 4: Figure NFAT activation) and cyclic AMP response element-binding protein (CREB), which bind to the glucagon gene promoters.
Airway epithelial cells
The cilia lining the airway epithelia are responsible for removing mucus from the lung. The operation of this mucociliary transport mechanism is determined by the tip velocity of the cilia and their co-ordinated activity to set up metachronal waves. The latter develop because successive cilia start their beat in sequence so that each one is slightly more advanced in its cycle, thereby setting up waves that can sweep over the epithelial surface to expel mucus from the lung.
The cilia beat very quickly with a basal frequency of about 14 Hz, but this ciliary beat frequency (CBF) can be increased to rates as high as 35 Hz following stimulation of the cells by ATP (Module 7: Figure airway cell oscillations). The ATP acts by binding to cell-surface P2Y receptors, which recruit the inositol 1,4,5-trisphosphate (InsP3)/Ca2+ signalling cassette to generate the Ca2+ signal responsible for accelerating the CBF. The Ca2+ signal appears as oscillations whose frequency changes with agonist concentration, indicating that they use a frequency modulation (FM) mode of information coding (Module 6: Figure encoding oscillatory information). The very tight coupling between the Ca2+ oscillations and CBF is an example of a digital tracking mechanism for decoding FM Ca2+ signals (Module 6: Figure decoding oscillatory information), which must mean that Ca2+ has a direct action on the ciliary axoneme.
White fat cells
White fat cells are a key component of the metabolic energy network (Module 7: Figure metabolic energy network). They are found in white adipose tissue, which makes up most of the body fat distributed throughout the subcutaneous tissue, the omentum, mesenteries and retroperitoneal regions. They are characterized by having a large fat droplet made up of triacylglycerols; the constituent fatty acids and glycerol can be either removed or added to the droplet, depending on nutritional status. Lipolysis occurs during fasting when the white fat cell mobilizes its triacylglycerol store to release fatty acids and glycerol into the blood. During lipogenesis, free fatty acids are removed from the blood and converted back into triacylglycerol to reconstitute the lipid reservoir (Module 7: Figure white fat cells). The metabolism of white fat cells is thus poised between anabolism and catabolism, depending on the energy requirements of the organism. A number of signalling mechanisms contribute to the hormonal control of white fat metabolism. In addition to its role in lipid storage, there also is an important white fat cell endocrine function.
Lipogenesis is the process of conjugating three free fatty acid (FFA) molecules to a glycerol backbone to form triacylglycerol (TGA) that is stored in the large lipid droplets (Module 7: Figure white fat cell metabolism). There are two sources of the FFA precursor. Firstly, it can enter across the plasma membrane through entry pathways such as fatty acid transport protein 1 (FATP1) or CD36, which is a fatty acid translocase (FAT) also known as FAT/CD36. Secondly, a process of FFA synthesis using pyruvate derived from glycolysis as a precursor. The pyruvate is formed from the glucose that enters through the GLUT4 channel that is regulated by insulin. When it enters the mitochondrion, pyruvate is converted into acetyl-CoA and then to citrate that enters the cytoplasm where an ATP citrate lyase converts it back into acetyl-CoA, which is then carboxylated to malonyl CoA by an acetyl-CoA carboxylase (ACC). The malonyl CoA is then converted into the fatty acid palmitate by a fatty acid synthase (FAS) complex. A variety of enzymes then extends the length of this palmitate and a stearoyl CoA desaturase 1 (SCD-1) introduces double bonds. The resulting fatty acylCoA precursors are now conjugated to glycerol 3-phosphate through a series of steps: glycerol 3-phosphate acyltransferase (GPAT) combines the two precursors to form lysophosphatidic acid (LPA), which is converted into phosphatidic acid (PA) by acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase (AGPAT) and diacylglycerol:acyl-CoA acyltransferase (DGAT) completes the process by converting DAG into TAG (Module 7: Figure white fat cell metabolism). The movement of fatty acids about the cell during these metabolic processes is facilitated by adipocyte protein 2 (aP2).
Most of the stored lipid is composed of neutral triacylglycerols, which are non-polar and aggregate by van der Waals forces. Triacylglycerols consist of three fatty acids (R-COOH, R'-COOH and R''-COOH) connected by ester linkages to glycerol. The nature of the fatty acid components shows considerable variation from species to species. Palmitic and oleic acids are the major components in human fat, which also contains appreciable quantities of linoleic, stearic, myristic and palmitoleic acids. These fatty acids are a very efficient way of storing energy. The carbon atoms are fully reduced (-CH2-) and, because they are hydrophobic, they attract each other, and by excluding water, they thus occupy much less space than other energy stores such as glycogen.
During lipolysis, the triacylglycerol (TAG) stored in lipid droplet is hydrolysed to glycerol and fatty acids that then leave the cell through specific transporters (Module 7: Figure white fat cell metabolism). Glycerol is transported out of the cell following lipolysis by aquaporin 7, which is strongly expressed in white fat cells. The fatty acid leaves through the fatty acid transport protein 1 (FATP1) or CD36, which is a fatty acid translocase (FAT) also known as FAT/CD36. The TAG is hydrolysed by both hormone-sensitive lipase (HLS) and by adipose triglyceride lipase (ATGL).
Hormonal control of white fat cell metabolism
The dynamic nature of this lipid reservoir depends on hormones such as insulin, which drives lipogenesis, and catecholamine, adrenocorticotrophic hormone (ACTH) and glucagon, which act through the cyclic AMP signalling pathway to promote lipolysis (Module 7: Figure lipolysis and lipogenesis).
The activation of lipolysis depends on receptors that are coupled to the formation of cyclic AMP (cAMP) that acts on protein kinase A (PKA) to phosphorylate the hormone-sensitive lipase (HLS) and the adipose triglyceride lipase (ATGL) that initiates the hydrolysis of triacylglycerol to free fatty acids (FFA) and glycerol. These hydrolytic products then diffuse out of the fat cell and enter the blood vessels, where they bind to albumin (Ab).
Insulin opposes this lipolytic process by activating the PtdIns 3-kinase signalling pathway to form the lipid second messenger PtdIns3,4,5P3 (PIP3), which activates phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB). The latter induces lipogenesis through a number of mechanisms. It can phosphorylate phosphodiesterase 3B (PDE3B), and thus attenuates the lipolytic response by enhancing the hydrolysis of the pro-lipolytic second messenger cyclic AMP. The insulin signalling pathway can also enhance lipogenesis by activating the entry of glucose through the GLUT4 transporter and by facilitating the entry of fatty acids by increasing the insertion of the fatty acid transporters FATP1 and FAT/CD36. In addition, insulin enhances the activation of peroxisome-proliferator-activated receptor γ (PPARγ), which acts to enhance the expression of those genes that function in lipid synthesis (Module 4: Figure PPARγ activation).
White fat cell endocrine function
In addition to its role in lipid metabolism and storage, the white fat cell also has an endocrine function in that it releases various adipokine hormones such as leptin and adiponectin (Module 7: Figure lipolysis and lipogenesis). The release of adiponectin is enhanced by the osteocalcin released from osteoblasts. The release of leptin contributes to the mechanisms that control food intake and body weight (Module 7: Figure control of food intake).
Brown fat cells
Brown adipose tissue is much less abundant than white adipose tissue. It is restricted to specific areas such as the back between the shoulder blades, along blood vessels in the neck and surrounding the adrenals and kidney. The brown colour results from the cytochromes packed in to the large number of mitochondria, which are specialized to produce heat. Heat can be generated by animals either through shivering (muscular activity) or by an increase in metabolic rate that has been called nonshivering thermogenesis and is largely confined to the brown fat cells. Unlike white adipose tissue, brown fat cells are organized to form distinct lobules, which are richly supplied with blood vessels and by nerves originating from the sympathetic nervous system. These nerves are responsible for releasing the noradrenaline that regulates heat production (Module 7: Figure brown fat cell). Information regarding temperature, feeding states and energy reserves are thought to be collated by the ventromedial hypothalamic nucleus (VMN), which then send information through to the sympathetic nerves to release the noradrenaline that stimulates heat production by the brown fat cells.
The fuel used for heat production is stored in the form of triacylglycerols within numerous small lipid droplets that are coated with the protein perilipin that protects the lipid from hydrolysis by hormone-sensitive lipase (HLS). Activation of heat production depends upon the cyclic AMP signalling pathway that is induced when noradrenaline binds to a β-adrenoceptor (β3) (Module 1: Table G protein-coupled receptors) that is coupled through GS to adenylyl cyclase. The resulting formation of cyclic AMP then has various actions. It acts through protein kinase A (PKA) to phosphorylate and activate HLS. In addition, PKA phosphorylates perilipin causing it to dissociate from the surface of the lipid that then allows HLS access to its substrate.
Hydrolysis of the triacylglycerols by HLS liberates free fatty acids that enter the mitochondria on the carnitine shuttle and then undergo β-oxidation to release acetyl CoA that enters the tricarboxylic acid (TCA) cycle to fuel heat production. Under normal circumstances, cells exercise a tight control over mitochondrial respiration such that oxidation of fuel reserves is curtailed when energy demands are low. The mitochondria in brown fat cells have evolved a mechanism to overcome this control by developing a proton leak that enables fuels to be oxidized continuously thereby releasing large amounts of heat (Module 7: Figure brown fat cell). This leak depends upon the expression of one of the uncoupling proteins, which in the case of the brown fat cells is uncoupling protein 1 (UCP1) that enables H+ to return to the mitochondrial matrix without producing ATP. During normal mitochondrial metabolism, the generation of ATP depends on fuels such as the FFA entering the tricarboxylic acid (TCA) cycle to produce NADH and FADH, which then provides the electrons that travel down the electron transport chain, during which protons are ejected into the cytoplasm (Module 5: mitochondrial Ca2+ signalling). The inner mitochondrial membrane is exceedingly impermeable to H+ so the removal of H+ creates a very large membrane potential of–180 mV. There are a few components in the inner mitochondrial membrane that are capable of dissipating this energy reservoir. Protons flowing through the ATP synthase energize ATP synthesis. Mitochondrial Ca2+ uptake is driven by this energy gradient. Finally, protons can leak back into the matrix through UCP1 and enables the mitochondria to generate heat (Module 7: Figure brown fat cells).
Just how noradrenaline activates heat production is still somewhat uncertain but all the evidence suggests that the mechanism is indirect and depends on the release of free fatty acid, which then functions as a typical metabolic messenger to activate UCP1.
Uncoupling proteins (UCPs) are integral membrane proteins located in the inner mitochondrial membrane. The UCP family contains five homologues with most information available for uncoupling protein 1 (UCP1), uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3), uncoupling protein 4 (UCP4) and uncoupling protein 5 (UCP5). These three UCPs functions as channels to transport H+ that flow out of the mitochondrial matrix to the cytoplasm. In the case of UCP1, which is strongly expressed in the mitochondria of brown fat cells, this leak serves to dissipate the proton gradient that normally functions to produce ATP through oxidative phosphorylation and the energy is lost as heat (Module 7: Figure brown fat cells).
Uncoupling protein 1 (UCP1)
Uncoupling protein 1 (UCP1) is restricted to brown fat cells where it functions in thermogenesis (Module 7: Figure brown fat cells). The expression of UCP1 during the differentiation of brown fat cells is controlled by noradrenaline operating through the cyclic AMP signalling pathway (Module 8: Figure brown fat cell differentiation).
Uncoupling protein 2 (UCP2)
Uncoupling protein 2 (UCP2) is fairly widely expressed and its function has been studied in some detail in insulin-secreting β-cells. The function of UCP2 remains somewhat problematic. It seems clear that UCP2 can transport H+ to provide a proton leak across the mitochondrial membrane. However, this leak is not as large as that caused by UCP1 so there is less uncoupling of oxidative phosphorylation and less heat production. Nevertheless, any leak will reduce the efficiency of energy production. There is considerable interest in the finding that the activity of UCP2 is increased by superoxide (O2−•), which is one of the reactive oxygen species (ROS) that can function as intracellular messengers (Module 2: Figure summary of redox signalling).
The mitochondrial reactive oxygen species (ROS) formation of superoxide (O2−•) will thus activate UCP2 and this will serve to dissipate the proton gradient and reduce the formation of ATP. In effect, this sets up a negative-feedback loop because the activation of UCP2 by superoxide (O2−•) will reduce the mitochondrial membrane potentials that drive ROS formation. This negative-feedback loop may have serious consequences for the insulin-secreting β-cells because the decrease in ATP production attenuates the main stimulus responsible for insulin secretion (-cell">Module 7: Figure β-cell signalling). A polymorphism in the promoter region of the gene encoding UCP2, which results in an increased expression of this coupling protein, results in defective insulin secretion and contributes to the onset of Type 2 diabetes.
Uncoupling protein 3 (UCP3)
Uncoupling protein 3 (UCP3) is expressed mainly in skeletal and heart muscle. It is highly homologous with uncoupling protein 2 (UCP2) and these two proteins share similar properties. The function of the UCP3-dependent leak of protons across the inner mitochondrial membrane is still not known. Like UCP2, UCP3 activity can be enhanced by superoxide (O2−•), which will reduce the mitochondrial membrane potential, ATP production and the formation of reactive oxygen species (ROS). In effect, this control through superoxide (O2−•) will establish a negative-feedback loop that could protect cells against excessive ROS formation by the mitochondria.
Uncoupling protein 4 (UCP4)
Uncoupling protein 4 (UCP4) is expressed in the dopaminergic neurons of the substantia nigra pars compacta. Like the other UCPs, it can leak protons across the inner mitochondrial membrane and this may provide a mechanism to protect these neurons from oxidative stress (Module 10: Figure tonic oscillation in DA neurons). An increase in mitochondrial ROS stimulates the opening of uncoupling proteins 4 to leak H+ that will depolarize the inner mitochondrial membrane potential thus reducing respiration and the entry of Ca2+.
Uncoupling protein 5 (UCP5)
Uncoupling protein 5 (UCP5) is expressed in the dopaminergic neurons of the substantia nigra pars compacta. Like the other UCPs, it can leak protons across the inner mitochondrial membrane and this may provide a mechanism to protect these neurons from oxidative stress (Module 10: Figure tonic oscillation in DA neurons). An increase in mitochondrial ROS stimulates the opening of uncoupling proteins 5 to leak H+ that will depolarize the inner mitochondrial membrane potential thus reducing respiration and the entry of Ca2+.
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Published online 1 October 2014
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