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The transfer of nutrients from the external environment into the internal environment defines the purpose of the Gastrointestinal tract. Motility, digestion, absorption of nutrients and excretion of waste products are all key physiological processes facilitating the function of GI tract which is continuous from the mouth to the anus. This essay will outline the structure and function of the gastrointestinal (GI) tract and the importance of ion channels in its function, describe the structure and function of TRP channels and describe and explain the function and importance of the TRP vanilloid channel in the GI tract and therapeutically.
The GI tract throughout, consists of four separate layers as seen from figure 1a which shows the four main layers: the Mucosa which is the innermost layer, Submucosa, Muscularis Externa and Serosa which is the outermost layer, the epithelium, lamina propria and Muscularis mucosae are all part of the mucosa and together provide a soft lining for the GI tract (1). Figure 1b shows a histological section of how the tissues are arranged in relation to each other.
Figure 1a: Ilusatration showing a generalised organisation of the layers of the alimaentary canal and also shows were specifcic specialed fetures are located within the layers (2.)
Figure 1b: Histology of the GI tract and how the layers are arranged at a microscopic level (2).
Arterial and Venous Blood Supply of the Gastrointestinal Tract
Three major unpaired arteries supply the structures of the GI tract: the celiac trunk, superior mesenteric artery and the inferior mesenteric artery. They all branch to the gastrointestinal viscera from the anterior surface of the abdominal aorta as can be seen from figure 2a below. The celiac trunk supplies the foregut which originates at the abdominal osephagus and terminates at the inferior margin of the major duodenal papilla. The superior mesenteric artery supplies the midgut from the termination of the foregut and terminates in the proximal two-thirds of the transverse colon where the hindgut begins. The hind gut which is supplied by the inferior mesenteric artery comes to an end midway through the anal canal (2). As seen in figure 2b venous drainage of the abdominal viscera occurs through the inferior vena cava (IVC). The celiac trunk, superior and inferior mesentery veins all drain into the Hepatic Portal Vein which joins on to the Inferior Vena Cava (IVC) which drains into the right atrium. Figure 1 below illustrates how the three main regions of the gut are supplied arterially and figure two illustrates how the 3 veins from the different regions all drain into the hepatic portal vein.
Figure 2a: An illustration showing the arterial blood supply to the foregut, midgut and hindgut (2).
Superior Mesenteric Artery
Inferior Mesenteric Artery
Inferior Mesenteric Artery
Inferior Vena Cava
Superior Mesenteric Artery
Figure 2b: A diagram showing venous drainage to the foregut, midgut and hindgut (2).
Nervous Supply to Gastrointestinal Tract
The nervous system innervating the abdominal viscera contains an intrinsic and extrinsic component. The extrinsic innervations involve sensory information being sent through visceral afferent fibres to the central nervous system (CNS) from the viscera which also receive motor impulses being sent from the CNS through visceral efferent fibres (3). Both the sympathetic and parasympathetic nervous system, part of the peripheral nervous system (PNS) are involved in the extrinsic innervations of the abdominal viscera (3). The main neurotransmitters involved in the extrinsic component are noradrenaline and acetylcholine. Noradrenaline is released only from the post ganglionic neurons of the sympathetic nervous system. Acetylcholine however is released from both sympathetic and parasympathetic pre ganglionic neurons, post ganglionic parasympathetic neurons, and postganglionic sympathetic neurons innervating the sweat glands. The Enteric Nervous system (ENS) is a branch of the PNS and consists of interconnected plexuses called the myenteric and submucosal plexuses forming the intrinsic innervations which regulate GI tract activity such as coordination of relaxing and contracting GI smooth muscle (peristalsis), gastric secretions and blood flow (3). The myenteric plexus is involved in the motility of the GI tract and as seen from figure 3 below it is located in-between the longitudinal and circular smooth muscle layers and embedded into the mucosa is the submucous plexus (1). Postganglionic sympathetic and preganglionic parasympathetic neurons within the wall of the GI tract also play a part in the modifications of GI tract activities such as smooth muscle contraction (1).
Figure 3: An illustration showing how the myenteric and submucous plexus are embedded within the tissue and how it innervates the layers (3).
Embedded in the muscularis externa is unstriated smooth muscle which is under involuntary control containing small spindle shaped cells which don't exist as organized sarcomeres like skeletal muscle. Two types of smooth muscle exist. Rapid contractions and relaxations are produced by phasic muscle, tonic muscle produces sustained and slow manner contractions, both muscle types have their contractions modified due to hormonal and neuronal influence (1). The contractions occur by the formation of a cross bridge cycle involving sliding of the actin and myosin filaments. The excitation wave produced propagates through gap junctions which result in muscle contraction. The excitation wave termed "slow wave" is produced by a group of pacemaker cells called the Interstitial Cells of Cajal (ICC) which are coupled to the smooth muscle (4). Rooted within the tunica muscularis (muscular coating surrounding the sub mucosa) of the gastrointestinal viscera are well defined regions which generate the pacemaker activity (4). Phasic contractions are produced by propagation of the slow wave through the intrinsic network within the ICC which extends to the organ and around it generating a strong contractile response due to the coupling of muscle excitation and contraction. The influx caused by the opening of voltage dependent calcium channels ( a class of ion channels) due to the resting membrane potential changing from -80mV to about -25mV is dependent on the ion channel function and its availability (4) The electrical wave results in the segmental contraction in the small intestine.
Ion channels are integral membrane proteins aiding the flow of ions down their respected electrochemical gradient to maintain the resting membrane potential. Spanning the whole membrane they consist of protein molecules which form a water filled pore which can switch from being open or closed. The membrane potential and the ionic concentration on either side of the membrane is what govern the electrochemical gradient which in turn determine the rate and movement of ions (1). Ion channels can be characterized by their diversity of ionic selectivity, gating properties and molecular architecture (5). Selectivity depends on whether the channel moves cations (Na+/ Ca2+/K+) or moves anions (Cl-) examples of ion channels include the voltage gated ion channels and the ligand gated ion channels.
Voltage Gated Ion Channels
Composed of several subunits, voltage gated ion channels comprise of a single transmembrane domain in which a central pore is formed through the specific arrangement of the subunits. Activation which is induced by a short lasting burst of depolarization causes a conformational change in the channel protein resulting in the opening of the channel allowing ions to flow down their electrochemical gradient. This process is further followed by another conformational change in which the channel becomes inactivated and so ions can no longer flow down the channel (5)(6). The main ion channels are those of sodium, calcium and potassium. Voltage gated ion channels are essential in the formation of action potentials, they open upon membrane depolarization in which they become activated and allow the conduction of the action potential which leads to the contraction of skeletal, smooth and cardiac muscle and can be a target for new drugs.
Ligand Gated Ion Channels
Ligand Gated ion channels (also called ionotropic receptors) open upon the binding of a chemical such as a fast neurotransmitter such as glutamate or acetylcholine. These channels consist of a pentameric structure made from non identical protein subunits in which each has 4 transmembrane helices. Each M2 segment from these helices line the pore in which ions can flow through upon binding of the neurotransmitter to the lignad binding region, which allosterically opens the ion pore due to a conformational change (5) (6). Hydrophobic amino acids wind in and out of the membrane which forms a large extracellular N-terminus in which the ligand binding region is located. Examples of ligand gated binding channels include the nicotinic acetylcholine receptor located within the postsynaptic side of the neuromuscular junction and also the Vanilloid receptor of the TRPV1 found on sensory nerve endings which is activated due to capsaicin resulting in the mediation of pain (5).
Transient Receptor Potential Channels
The evidence of the existence of transient receptor potential channels was first discovered within the fruit fly Drosophila Melanogaster in which membrane cation channels were activated during phototransduction resulting in a depolarizing current. Phospholipase C- beta is stimulated upon activation of the G protein coupled receptor, rhodopsin, which is coupled to the photoreceptors (7). Light induced currents (LIC) are produced upon activation of the rhodopsin which lead to the mutant strain of the fruit fly displaying a transient LIC in response to light compared to the wild type which had a sustained LIC and for this reason was given the name transient receptor potential (trp) (7). Disruptions occurring on the photoreceptors Ca2+ entry channel were produced by mutations in the gene, which pointed out that the protein encoded by the trp gene could the Ca2+ influx channel (7).
Transient receptor potential (TRP) channels are a diverse set of proteins which are ligand gated channels; they are a multifunctional super family which can be divided into 6 different sub families as seen from figure 4 aside (7).
Figure 4. Phylogenetic tree of the various transient receptor potential (TRP) superfamily in mammals. TRPC (canonical),
TRPM (melastatin), TRPV (vanilloid), TRPA
(Ankyrin), TRPP (polycystin), and TRPML (mucolipin) are
the only identified subfamilies in mammals (7).
The 28 mammalian TRP channels have a common homology within their structures consisting of 6 putative segments (S1-S6) spanning the transmembrane forming an intracellular cation permeable pore between the 5th and 6th segment (7). The S6 segment forms the lower gate which opens and closes regulating influx and efflux of ions, the S6 segment along with the S5 segment is also involved in anchoring an extracellular loop which aids cation selectivity for permeation (8). The TRPV, TRPA, and TRPC families have adaptor proteins called Ankyrin which anchor the channels to the plasma membrane through their N terminus. The N and C terminus of all TRP channels are located intracellular (9). Within the transmembrane domains of the TRP family the amino acid sequence only shows 20% homology, however within each of the sub families there is much greater homology throughout their sequence (9).
Figure 5: An illustration showing the detailed structure of the TRP channels and also shows the specific structural differences between each of the TRP subfamilies (8).
The TRP family is huge consisting of 6 subfamilies which among themselves have their differences in their structural architecture (as seen from figure 5), function and also to the type of stimuli they respond to. Broadly speaking there are two main functions of the TRP channels which can be split into either being Ca2+ entry channels or they can be sensing TRP channels (9). Involved in Ca2+ entry into the cell are the TRPC, TRPP and TRPV channels. The TRPM, TRPA as well as the TRPV channels are involved in sensing different stimuli (9).
Generalized Function of Individual Channels
Muscle contraction, Gene transcription, cell death and many other cellular processes depend upon Ca2+ in order for the function to occur. The regulation of free intracellular cytosolic Ca2+ depends upon a couple of methods such as Ca2+ influx upon membrane depolarization, Ca2+ released from internal stores such as the Golgi apparatus and in muscle the sarcoplasmic reticulum, TRP channels such as TRPV1 and TRPM8 may also act as intracellular Ca2+ release channels according to several studies (7). With the exception of the TRPM4 and the TRPM5 channels all TRP channels are permeable to Ca2+ which consist of a poor permeability ratio between 0.3 and 10 in relation to Na+ (PCa / PNa) except TRPV5 and TRPV6 which are highly selective Ca2+ channels (7). TRP channels respond to a range of stimuli including the binding of intra/extra-cellular messengers, temperature changes and stress (mechanically or chemically).
An important location in which the function of the TRP channels is vital is within the GI tract. TRP channels as mentioned previously are regulators of free intracellular Ca2+ which is essential for smooth muscle contraction within the GI tract to aid peristalsis, members of the TRPV family are also expressed within the intestines which are stimulated by pain. Table 1 below highlights the different functions of the individual channels as well as the different stimuli which activate them, their permeability ratio and also the proteins in which they interact with in order to carry out their function.
Table 1. Summarizing the main properties of the TRP channels (8).
Transient Receptor Potential Vanilloid Channels
The TRP Vanilloid subfamily consists of 6 different mammalian channels and like all other TRP channels have 6 transmembrane domains in which a pore exists between the S5 and S6 segment and have intracellular N- and C- termini. On their cytosolic NH2 termini they contain 3 to 5 ankyrin repeats, these domains help to localise and anchor the TRPV1 channel to the nerve terminals, and used in the interaction with other proteins (7). Figure 1 shows how these ankyrin repeats are distributed on the N-termini.
TRPV2 is 50% identical to TRPV1. Through lightly mylinated A delta nociceptors it mediates a sensation of high threshold heat (>52oC) in the sensory ganglia. TRPV2 channels translocates into the cell membrane in response to stimulation of cells which contain insulin growth factor resulting in an increase in intracellular calcium concentrations (8). TRPV2 has also been said to be activated by changes in osmolarity and also membrane stretch (10).
Highly expressed in the skin, tongue, and also the nervous system the TRPV3 channel is activated by high temperatures (> 31oC), its distribution through sensory neurons is similar to that of TRPV1 (8).
TRPV4 is another thermosensitive channel which becomes stimulated by temperatures above 25oC (7). The Ca2+ is potentiated by the channel by hypertonicity; this is enhanced by prostaglandins which is a hyperalgesic inflammatory mediator, this is in turn enhances the TRPV4 mediated current in primary afferent nociceptive nerve fibres (8).
The remainder two channels, TRPV5 and TRPV6 slightly vary in function compared to the other four channels. Found in transporting epithelia, kidney and intestine these highly permeable Ca2+ selective channels respond to changes in intracellular Ca2+ levels (7). There is evidence that both TRPV6 and TRPV5 also mediate uptake of Ca2+ as they both show strong inwardly rectifying currents (8).
The TRPV1 channel is a Ca2+ permeate channel which in particular is sensitive to noxious heat (>43oC) and capsaicin. It's mainly involved in the transmission and modulation of pain (8). The TRPV1 channel can be activated by a wide range of endogenous and exogenous stimuli such as decreased pH. These particular features interest me and how they may be involved in the function of a particular organ system. I will therefore mainly concentrate on the function within the GI tract of TRPV1 and they are associated with GI tract diseases. I will also pay close attention to the various agonists and antagonists of TRPV1 and how they can be used therapeutically in pathological conditions associated with the GI tract.
Transient Receptor Potential Vanilloid 1
Transient receptor potential vanilloid 1 (TRPV1) channel is a ligand gated channel first discovered through the use of capsaicin a hot pepper derivative used as a vanilloid ligand in expression cloning (8) carried out by caterina et al. Painful burning sensations are produced upon the binding of capsaicin which leads to the activation of the TRPV1 channel after phosphorylation has taken place, this results in the production of action potentials as the channel opens allowing the influx of Ca2+ and Na+ (5) The influx of Ca2+ into the nerve endings results in the local release of Calcitonin Gene Related Peptide (CGRP), substance P (SP), tachykinins and neurokinin A (NKA) (14). TRPV1 is not only stimulated by noxious heat and capsaicin but also by other endogenous stimulants such as decreased pH, N-arachidonoyldopamine (NADA), lipoxygenase products of arachadonic acid, polyamines, ethanol and anandamide (11), the effects that are produced by these variable stimuli is illustrated in figure 6. Should I describe general activation?
Figure 6: Shows how the TRPV1 channel produces different intracellular pathways in response to different types of stimuli which contribute to TRPV1 sensitization in terminals of the primary sensory neurones (12).
The 426 amino acid channel (12) has been expressed in many areas of primary sensory neurones in the dorsal root ganglion (DRG) and the trigeminal ganglion (TG) mainly within A-delta and C fibres of the PNS and CNS which respond to chemical, mechanical and thermal stimuli. These polymodal nociceptors have been expressed in vast areas of the CNS such as the limbic system, hypothalamus, substantia nigra, the cerebellum and also the spinal cord (12). TRPPV1 immunoposotive fibres are also expressed in the dermis of the skin and visceral layers of the mucous membrane, submucous, and also the muscular layer (13). They accompany blood vessels in all layers of the viscera such as the bladder, in the myenteric and submucous plexus of the GI tract, and also been found in the inner ear, airways, urinary tract and densely within the heart (13).
One of the pivotal roles that TRPV1 channels have in the GI tract is gastroprotection in which the dense capsaicin sensitive sensory neurones maintain the integrity of the mucosa against injurious interventions (14). There are two proposed mechanisms in which TRPV1 positive sensory nerves are involved in gastroprotection: One in which hyperaemia induced by capsaicin results in vasorelaxation through the release of CGRP from capsaicin sensitive primary afferents increasing the metabolic activity of cells (13). The other also involves CGRP release from TRPV1 positive sensory nerves resulting in the production of Prostaglandin E2 by activating the cyclooxygenase-1 enzyme (13) (14). A thick viscous mucus layer is secreted onto the gastric mucosa as the prostaglandins activate secretory cells (14). Figure 6 illustrates just how prostaglandin E2 activates TRPV1.
In non neuronal cells such as the gastric epithelial cells and gastrin cells, TRPV1 expression is also present (14) as well as in nerves throughout the mucosa, the muscular layers and throughout the blood vessels within the wall of the GI tract in which the TRPV1 plays an important role in the different physiological processes related to acid, intestinal motility, visceral and gastrin secretion (14). Damage of gastric epithelial cells induced by protons and ethanol can be prevented by the use of TRPV1 agonists such as capsaicin, vanilloids and resinferatoxin which activate the TRPV1 channel showing that it has some defensive properties (13)(14). A study carried out by Bielefeldt and Davis (2008) showed that luminal acidification didn't activate mechanosensory neurones in TRPV1 knockout mice, however in isolated human antral glands activation of TRPV1 by capsaicin produced stimulation of the gastrin secretion emphasizing how important TRPV1 is in acid sensation (14).
Agonists and Antagonists of TRPV1
TRPV1 channels are involved in many gastrointestinal diseases such inflammatory bowel disease (IMD), pancreatitis and Gastro Oesophageal Reflux Disease (GORD) (7). Many agonists and antagonists of the TRPV1 channel are used as methods of therapeutic interventions to help relieve patients of symptoms associated with these diseases.
As mentioned previously agonists such as capsaicin, anandamide, NADA, arachadonic acid derivatives and lipoxygenase products all produce stimulatory effect due to their high potency. Other agonists of TRPV1 include N-oleoyldopamine (OLDA), Resinferatoxin, Rutaecarpin (14), ethanol can also be considered as a possible TRPV1 agonist (12). NADA has a 20 more fold potency than capsaicin and anandamide and produces thermal hyperalgesia, however OLDA a TRPV1 selective agonist can produce TRPV1 mediated thermal hyperalgesia and having a stronger potency than NADA it shows that OLDA is the most powerful agonist for TRPV1 up to date (12). Capsaicin a homovanillic derivative is another potent exogenous agonist acting upon nociception fibres the nerve endings it acts upon depending upon dose can either be stimulated or destroyed (12). In low concentrations of ligands such as capsaicin beneficial effects on gastrointestinal function such as resistance of the gastric mucosa against chemical injury can be increased however in high concentrations chemical induced mucosa damage in the stomach can be aggravated, the same can be said for acid and alcohol based ligands (13). Capsaicin is hazardous if ingested, inhaled, or if it comes into contact with the skin or the cornea, severe over exposure can prove to be fatal which results in death (14).
In rat DRG, exposure to 0.3 - 3% ethanol and other alcohols produce a TRPPV1 mediated responses such as mobilisation of intracellular Ca2+ and also the release of SP and CGRP (12) Figure 6 illustrates the intracellular mechanisms involved in TRPV1 activation. TRPV1 has a threshold temperature of around 43oC before it gets activated but ethanol reduces the threshold to a much lower temperature of 37oC providing a mechanistic explanation into why a burning pain sensation is caused in patients with GORD or when the mucosal surface is exposed to alcohol or hot foods or liquids (12). The burning sensation, a result of TRPV1 activation caused by the stimuli could be enhanced if the stimuli all synergise together (12). This is true for alcohol, because when ethanol is applied to oesophageal wall of a rat there is a rise in plasma extravasation brought upon by sensory nerve activation which can be prevented by the TRPV1 antagonist capsazepine (12). Capsazepine prevents stimulation of the TRPV1 channel by inhibiting capsaicin and heat activation but doesn't prevent proton activation (11). Being an antagonist to the TRPV1 channel, capsazepine will inhibit any of the pain or burning sensations meaning it can attenuate thermal hyperalgesia. Capsazepine can be used therapeutically and provide beneficial effects in systems such as the GI tract. I will concentrate on three pathological conditions Inflammatory Bowel Disease (IBD), pancreatitis and Faecal urgency and incontinence.
TRPV1 in Gastrointestinal pathological conditions
IBD is the name given to describe two chronic diseases: Crohns Disease which results in inflammation of the GI tract but mostly occurs in the small intestine and the other being Ulcerative Colitis in which the inflammation only occurs in the large intestine and the rectum. Patients with IBD experience chronic abdominal pain which occurs from mechanical hypersensitivity of the colon (7). Immunoblotting and immunostaining of intestinal specimens taken from patients with Crohns disease and Ulcerative colitis revealed a significant increase in TRPV1 density in comparison to specimens taken from control subjects (12). Endogenous inflammatory substances which activate TRPV1 in colonic fibres in active IBD patient could be antagonised providing new therapies for the GI pain and dysmotility (12)
Pancreatitis a severe inflammatory condition of the pancreas in which there are two types acute and chronic. Symptoms associated with pancreatitis are mid and upper abdominal pains radiating to the back, nausea and vomiting are common symptoms along with pancreatic bleeding (7). Local vasodilatation and plasma extravasation lead to large amounts of fluid loss due to ischemia, free radical production and release of inflammatory mediators such as SP and CGRP from sensory neurones (7). Mediators such as protons, heat, leukotrienes, arachadonic acid metabolites and bradykinin all act on A delta fibres and C fibres, they also act on TRPV1 causing depolarisation resulting in an action potential, Ca2+ influx, and exocytosis of SP and CGRP which all exacerbate the pain and disease causing factors (7). As a result TRPV1 antagonists such as capsazepine can help improve the symptoms and progression of pancreatitis which helps protects against tissue damage and also reduces release of SP (7).
Faecal urgency and incontinence is an inadequately treated condition related to rectal hypersensitivity which can cause distress to patients. Patients who had rectal hypersensitivity their colon specimens showed that there was increased number of TRPV1 expression fibres in the muscle , submucosal and mucosal layers but also the threshold for heat and distension correlated with number of TRPV1 positive fibres present (12). This suggested that increased TRPV1 polymodal sensory nerves could be the cause for faecal urgency and rectal hypersensitivity; this again shows that the use of TRPV1 antagonists could be used to help control the symptoms of the condition (12).
TRPV1 channels within the GI tract play a major role in the function of the GI tract, pathological conditions related to the GI and also in providing protection of the GI tract. The fact that the TRPV1 is involved in pathological conditions in the GI tract and beyond makes it an attractive target for the treatment of certain pathological conditions. The vast arrays of TRPV1 agonists in existence today show their importance in gastroprotection by maintaining the gastric mucosa and providing protection against various chemical interventions. Although beneficial effects have been mediated by TRPV1 agonists on gastrointestinal function, agonists that can be used in the treatment for gastrointestinal diseases are not yet in existence. The disadvantages with the use of agonists already in existence is the low bioavailability, low selectivity and high toxicity (14) that they posses. For this reason development of future agonists need to overcome the drawback which could lead to alternative treatment methods for various gastrointestinal diseases, however due to the role played by TRPV1 in inflammatory conditions the potential effects produced by TRPV1 agonists cannot be ignored. As well as agonists further research needs to be carried out to exploit TRPV1 antagonists and its therapeutic role in gastrointestinal conditions which could help ameliorate certain pathological conditions. However the rapidly increasing developments on recently discovered TRPV1 channels as well as other TRP channels indicate the progress of future developments of therapeutically feasible agonists and antagonists of these channels will be very soon.