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The pharmacology of gastrointestinal nociceptive pathways
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The pharmacology of gastrointestinal nociceptive pathways LA Blackshaw* and GF Gebhart† The principal conscious sensations that arise from the gastrointestinal tract are discomfort and pain. Chronic visceral discomfort and pain are generally managed poorly with currently available pharmacological agents. Receptors and ion channels present on extrinsic visceral primary afferent (sensory) neurons are targets for the development of new pharmacological strategies for control of visceral pain. Addresses *Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia † Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, IA 52242, USA Correspondence: GF Gebhart; e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:642–649 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations 5-HT 5-hydroxytryptamine AMPA α-amino-3-hydroxy-5-methyl-isoxazoleproprionate BDNF brain-derived neurotrophic factor BK bradykinin CCK cholecystokinin CNS central nervous system DRG dorsal root ganglion GABA γ amino butyric acid GI gastrointestinal GRNF glial cell line-derived neurotrophic factor IBS irritable bowel syndrome mGluR metabotrophic glutamate receptor NGF nerve growth factor NMDA N-methyl-D-aspartate NT neurotrophins PG prostaglandin SST somatostatin TTX tetrodotoxin VR1 vanilloid receptor 1
Introduction Although discomfort and pain undeniably arise from the gut, it is only relatively recently that nociceptors have been accepted as innervating the viscera [1]. Somatic nociceptors were defined by Sherrington as signalling damage or threat of damage, but the stimuli that activate somatic nociceptors (e.g. cutting, crushing, burning) are generally ineffective when applied to the viscera. Adequate stimuli for activation of visceral nociceptors include distension of hollow organs, traction on the mesentery, ischaemia and inflammation. Gastrointestinal (GI) mechanonociceptors are best understood; they are located in viscus muscle layers where they function as in-series tension or stretch receptors. Adequate stimuli for other visceral nociceptors are less well studied, but it is clear that mechanonociceptors can be sensitised by prior exposure to chemical or thermal stimuli. Indeed, present evidence supports the notion that much of the visceral sensory apparatus is polymodal in character. That is, visceral
sensory receptors that subserve a nociceptive function also transduce chemical and thermal stimuli into electrical nerve activity. The sensory innervation of the gut, moreover, is complex relative to innervation of somatic structures. Despite general agreement about the existence and role of polymodal nociceptors in the gut, whether they or other nociceptors (e.g. chemonociceptors, silent nociceptors) exist and predominate in different GI organs, convey information to the central nervous system (CNS) through all visceral sensory pathways, or express similar sets of pharmacological targets remains to be determined. We begin this overview with a brief review of the sensory innervation of the gut, following which pharmacological targets on these sensory neurons are considered.
Pathways of primary sensory information to the central nervous system Anatomy
Sensory (afferent) fibres from the GI tract may follow any of several paths to the CNS (Figure 1): • Vagal afferent endings are sparse in the colon but more numerous proximally (particularly the stomach and oesophagus). Their axons project directly into the brainstem and their cell bodies are located in the nodose or jugular ganglia. Viscerofugal neurons have cell bodies in the gastric and oesophageal myenteric plexus and project centrally in the vagal trunks, although their function is not yet understood. • Spinal afferent endings are distributed throughout the gut, and travel in the splanchnic nerves to the thoracic and lumbar spinal cord and in the pelvic nerves to the sacral cord. Spinal afferent axons make synaptic contacts in prevertebral (sympathetic) ganglia and the spinal dorsal horn; their cell bodies are in dorsal root ganglia. Intestinofugal neurons with cell bodies in the myenteric plexus project with spinal afferents from the gut to prevertebral ganglia and occasionally to the spinal cord. These sensory fibres are mainly unmyelinated C-fibres, which are difficult to trace to their endings, but which differ in their transmitter content and embryonic origin. Unlike sensory end organs in other tissues, no precise anatomical specialisation of afferent endings is evident in the GI tract, suggesting that the bare nerve endings rely on features of the innervated tissue for their physiological specialisation. Intra-ganglionic laminar endings of vagal afferents are distributed in the capsule of individual myenteric ganglia between muscle layers. Intramuscular arrays are arranged along smooth muscle bundles within a particular muscle layer. Vagal mucosal endings follow the central
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vascular supply to individual villi in the intestine or branch within the superficial layers of the oesophageal squamous epithelium [2]. Spinal oesophageal mucosal afferents show similar branching [3]. Preliminary data suggest that intra-ganglionic laminar endings exist in the colon [4], but which pathway they follow has not yet been established. Evidence is also emerging for serosal and mesenteric endings that follow splanchnic and pelvic pathways [4]. Sensory modalities
The pathways responsible for the full range of GI sensations have not been well defined, probably because of the difficulty in assessing symptoms in animal models; however, animal models have revealed pathways involved in nociception, aversion, emesis and reflex control of gut motility. Vagal fibres are associated with upper GI sensations such as satiety, nausea and hunger. Spinal afferents are associated with sensations of fullness, bloating, discomfort and pain from the stomach and bloating, urgency, stool, colic and pain from the large intestine, and are also thought to mediate discomfort and pain from the small bowel. The role of intestinofugal neurons is well established in the reflex control of motility, but their involvement and that of vagal viscerofugal neurons in signalling sensation or pain to the CNS is uncertain.
Functional properties of afferent endings Vagal afferents
Vagal afferents may be divided into three classes based on the layer of gut containing the receptive field. Location of endings is pivotal in determining sensitivity, as anatomical and electrophysiological data concur (Figure 2). Mucosal receptors are silent at rest, but develop activity after acute inflammation or damage because of release of mediators (e.g. 5-hydroxytryptamine [5-HT]) [5,6]. They are mechanosensitive to fine mucosal stroking with rapidly adapting responses and are unresponsive to distension. They are also chemosensitive to a range of intra-luminal chemical and osmotic stimuli. They respond directly to several locally released mediators, including cholecystokinin (CCK), 5-HT, noradrenaline, opioids, bradykinin (BK), purines and prostaglandins (PGs) [5–12]. The major role of mucosal receptors in sensation is thought to lie in the generation of satiety, nausea and vomiting, with a minor role in direct generation of reflex responses.
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Tension/mucosal receptors have so far been observed only in oesophageal striated muscle and show features of both tension receptors and mucosal receptors [14]. They presumably have a specialised role in detection of rapidly moving boli of food. Spinal afferent
Spinal afferents, like vagal afferents, are similarly located in different layers of organs (Figure 2) and contribute to a variety of reflexes. Importantly, spinal afferents are considered to convey to the CNS the sensations of discomfort and pain [1]. Tonic (or wide dynamic range) mechanoreceptors have tonic levels of resting activity; they respond to contractions and distension with a linear relationship to wall tension [15–17]. It is most likely that these receptors signal filling of the stomach, colon and rectum to give rise to sensations of fullness. Because they encode distending stimuli well into the noxious, non-physiological range, it is considered that they can also contribute to discomfort and pain, particularly in the presence of organ inflammation. High threshold (or phasic) mechanoreceptors have low resting activity and respond only to noxious intensities of organ distension. Accordingly, they are considered mechanonociceptors [1,15–17]. They are also chemosensitive, responding directly to inflammatory mediators, including BK, eicosanoids and free radicals [18]. Their receptive fields have sometimes been identified in the serosa and mesentery. Silent nociceptors (or mechanically insensitive afferents) are silent at rest. They develop activity and mechanosensitivity during and after inflammation through action of inflammatory mediators (BK and eicosanoids demonstrated so far) and nerve growth factor [1,19]. Silent nociceptors have been most extensively studied in somatic tissues, where some have been characterised as chemonociceptors. Mucosal receptors have similar properties to vagal mucosal afferents in their sensitivity to luminal chemicals and selective responses to fine tactile stimulation. So far they have been found only in in vitro preparations of colon [20].
Pharmacological targets on visceral afferents Excitatory receptors
Tension receptors (also known as muscular afferents) have resting activity, often modulated in phase with ongoing contractions. They are mechanosensitive to contractions and distension with a slowly adapting, linear relationship to wall tension within the physiological range [13]. Tension receptors as a population signal the amplitude, pattern and direction of luminal contractions to the CNS, which is important in triggering reflexes controlling GI function. Their responses to distension are also important in signalling food intake and mediating satiety and fullness.
Adenosine is a breakdown product of ATP, which is released by numerous processes (see below), many of which are relevant to healthy and diseased GI function. Kirkup et al. [11] showed that adenosine increased mesenteric afferent nerve activity and intestinal motility in the anesthetised rat through A1 and A2B and/or A2B-like receptors. They found that increased motor activity induced part of the afferent response but did not wholly account for adenosine-evoked excitation, a fact supported by an in vitro study of these afferents [21].
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Figure 1
Nodose ganglion
NTS
Paravertebral ganglia
Greater splanchnic nerve
Lumbar colonic n.
Hypogastric nerve
Prevertebral ganglia
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 1 2 3 4 5
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Vagus nerve
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Representation of sensory innervation of the GI tract. Left: visceral afferent pathways through prevertebral and paravertebral ganglia to the spinal cord; cell bodies are located in dorsal root ganglia (not illustrated). Right: vagal and pelvic nerve afferent input through nodose and dorsal root ganglia, respectively. The innervation of the viscera as
illustrated is overlapping with spinal inputs to specific, but anatomically separated spinal segments. For example, the distal colon is represented in thoracolumbar and lumbosacral spinal segments by least splanchnic and pelvic nerve inputs. Gastric input to the CNS is represented in the brainstem and thoracic spinal cord by vagal and splanchnic inputs.
ATP is released from damaged cells, making it a prime candidate for signalling of nociceptive events. It is also released from sympathetic and myenteric nerve terminals, and from mechanically deformed epithelia and endothelia. Both ionotropic (P2X) and metabotropic (P2Y) receptors have been found on neurons, although P2X receptors are more important in sensory pathways. Each type has seven subtypes. P2X receptors may have two types of effect on vagal and spinal GI primary afferents: direct activation or sensitisation to other stimuli [12,22]. Responses to ATP and its analogues may be prolonged or may desensitise rapidly, depending possibly on the subunit composition of the receptor. Thus a homodimeric receptor composed of two P2X2 subunits desensitises rapidly, whereas a heterodimer of P2X2 and P2X3 does not. Burnstock [23•] has suggested that ATP may be involved in visceral mechanosensory transduction and a recent report by Rong et al. [24] documented that P2X3 receptors modulate sensitivity of pelvic afferents to urinary bladder distension.
BK is formed during tissue damage and is well recognised as a stimulant of somatic nociceptors. In the GI tract, BK activates fibres with high (nociceptive) as well as those with low (non-nociceptive) thresholds to distension. In spinal afferents, the receptor subtype involved in direct activation is the B2 subtype, whereas indirect activation could occur through the B1 subtype following smooth muscle contraction [25]. Blackshaw and Grundy [6] showed an action on vagal mucosal afferents, which suggests that BK in the gut may activate emetic as well as nociceptive pathways. B2 antagonists have been developed for somatic pain indications, and these data would indicate their potential efficacy additionally in visceral pain. B1 receptors have been reported to upregulate (increase) following tissue injury and thus have also been targeted for development of potential analgesics. Because BK receptors activate phospholipase A2, they induce sensitisation of responses by production of PGs, which themselves are potent algogens.
The pharmacology of gastrointestinal nociceptive pathways Blackshaw and Gebhart
CCK is released by products of fat digestion from the small intestinal mucosa. CCK is associated with initiation of satiety from the upper GI tract and reflex inhibition of gastric motility. At high doses, CCK may induce nausea and vomiting. The mechanism by which this occurs is through a vagal reflex, but controversy exists as to the selectivity of mucosal or muscular vagal afferents to CCK. Some evidence supports a circulating action on muscular afferents [26], whereas other data suggest a paracrine action of locally released CCK on mucosal afferents [27,28]. These actions are mediated through CCK1 receptors. No reports exist so far of CCK actions on spinal afferents, but the action of CCK on a mixed population of afferents in the mesenteric nerves was abolished by chronic degeneration of vagal fibres, suggesting that these are the only target for CCK [28]. 5-HT, like CCK, is released from the GI mucosa after a meal; however, 5-HT release may occur in response to mechanical or chemical stimuli throughout the GI tract. There are seven 5-HT receptors that exist in the GI tract and numerous subtypes of these. So far only the ionotropic 5-HT3 receptor has been conclusively demonstrated to mediate actions on extrinsic GI afferents [6,29], which are selective to a distinct subpopulation; however, there is still controversy as to the role of 5-HT4 receptors, and the role of many subtypes remains uninvestigated [30,31]. In humans, 5-HT3 receptor antagonists have been used successfully to reduce nausea and vomiting evoked from the upper GI tract and to reduce pain, discomfort and urgency in diarrhoea-predominant irritable bowel syndrome (IBS) [32•]. Major side effects include constipation and ischaemic colitis, which have so far prevented extensive use in IBS. Ionotropic glutamate receptors include N-methyl D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-isoxazoleproprionate (AMPA) receptors and kainate receptors. Glutamate may be derived from the diet, from intrinsic or extrinsic neurotransmission in the gut, or released from epithelial cells. Although evidence for direct responses to glutamate is lacking, NMDA receptor antagonists have been shown to reduce responses to mechanical stimuli in pelvic and splanchnic afferents from the colon and in vagal oesophageal afferents [33,34•,35], suggesting either involvement of endogenous glutamate in mechanotransduction or an action of antagonists on mechanosensitive channels. The evidence for NMDA receptor involvement in mechanosensitivity so far relies mainly on the action of the open channel blocker, memantine, which has actions at other ionotropic receptors; therefore, this interesting and potentially exploitable area is open for further investigation.
µ and δ Opioids, despite having mostly inhibitory actions on neurons in the CNS, cause excitation of vagal afferents from the small intestine [9]. These actions were not seen in a study of mechanosensitive vagal afferents from the stomach [36] or pelvic afferents from the colon [37•], suggesting a region-specific effect.
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Figure 2
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Schematic representing current anatomical and electrophysiological evidence for the location of sensory endings in the wall of the GI tract. Fibres in red represent serosal/mesenteric high threshold afferents, those in blue represent muscular low threshold afferents (which terminate in muscle layers or around myenteric ganglia), and those in green represent mucosal afferents sensitive to tactile luminal stimuli.
Proteases (e.g. thrombin, trypsin and mast cell tryptase) are likely to be present on GI afferent nerve terminals when the intestine is ulcerated or when mast cells degranulate following inflammation. Until recently the role of mast cell proteases was considered to be restricted to the destruction of pathogens; however, somatic and pancreatic nociceptive afferents were shown to express specific protease-activated receptors. The ligand is tethered to the receptor and allowed to bind following proteolytic cleavage. These receptors have recently been demonstrated on small intestinal afferents [38], although whether vagal or spinal in origin remains to be established. PGs are produced in a wide range of cell types, and are important mediators of pain. PGs are often produced in response to other stimuli, and they in turn potentiate responses of GI afferents to other stimuli [39], constituting a positive feedback system. Blockade of PG synthesis is a common means to reduce pain and inflammation. Five classes of prostanoid receptors (DP, EP, FP, IP and TP receptors) have been identified and distinguished by rank order of agonist potency (e.g. PGE2 acts at EP receptors). EP1 receptors were recently shown to mediate rapid excitation of small intestinal afferents and EP2 receptors to cause longer term activation [10]. Because blockade of PG synthesis compromises other processes, such as mucosal defence, approaches to reducing visceral pain based on receptor antagonism may prove to be more selectively targeted.
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Vanilloids, such as the chilli extract capsaicin, are well-established excitants of somatic nociceptors, giving rise to a burning sensation mediated through the vanilloid receptor 1 (VR1 or TRPV1), which is also a natural sensor for heat and low pH [40•]. Capsaicin activates a proportion of all classes of vagal and spinal afferents throughout the GI tract, but the sensations evoked are not obvious. An early report found that the majority of GI afferents were activated by capsaicin [41]. Recent reports found that as few as 30% of vagal afferents [42], 42% of nodose ganglion cells [43] and 46% of colon dorsal root ganglion cells [44] were activated. There is no correlation between expression of VR1 and other properties of primary afferents, such as their peptide content, location or pathway; however, recent preliminary evidence points to a co-expression of VR1 with purinergic and bradykinin receptors on colon afferents in the pelvic pathway [45]. In addition, VR1 are expressed in bladder epithelial cells, suggesting a functional association of urothelial cells with afferent fibre nerve endings [46]. Endogenous activators of VR1 have recently been identified, suggesting contributions to physiological and/or pathophysiological processes [40•]. This endogenous pathway is yet to be investigated in the GI tract.
peripheral innervation of the gut a novel, κ opioid-like receptor that could be targeted to develop an analgesic for visceral discomfort and pain [49]. Recently, however, it was determined that these κ opioid agonists, which share structural similarity, significantly reduce voltage-activated sodium currents in colon sensory neurons [50]. This effect was shown not to be opioid receptor-mediated and to mimic the effects of other sodium channel blockers (e.g. effects were tonic and use-dependent). Metabotropic glutamate receptors (mGluR) may be divided into eight molecular subtypes, which currently fall into three pharmacological and functional groups: group I (mGlu 1 and 5 receptors) are excitatory, whereas group II (mGlu 2 and 3 receptors) and group III (mGlu 4, 6, 7 and 8 receptors) are inhibitory to neuronal function. Preliminary data from in vitro studies in ferret and mouse indicates that group I mGluR do not affect vagal afferents, but both group II and III inhibit mechanical sensitivity in a similar manner to GABAB receptors [51]. mRNA and protein for all of the mGluR subtypes are found in the nodose ganglia [52]. Pharmacological tools for mGluR are currently limited in selectivity but, as this hopefully improves, the role of individual subtypes may become more apparent.
Inhibitory receptors
γ-Amino butyric acid (GABA) activates ionotropic (GABAA and GABAC) receptors and metabotropic (GABAB) receptors on neurons of the central and enteric nervous systems. In these systems GABA functions as a direct inhibitor of neuron excitability and an inhibitory modulator of synaptic transmission. The role of endogenous GABA in modulation of vagal and spinal primary afferents is considered to be minor, but exogenous GABAB receptor agonists (but not GABAA agonists) are potent inhibitors of mechanosensitivity in a subpopulation of vagal afferents. This has stimulated clinical interest in the use of these drugs to reduce signalling of gastric distension to the brainstem. This in turn reduces triggering of motor patterns that lead to gastroesophageal acid reflux [47•]. The potential for GABAB receptors agonists in reducing signalling through spinal pathways is yet to be explored. κ Opioids Interest in κ opioids was stimulated by initial reports that a compound (fedotozine) with κ opioid efficacy was effective in reducing visceral pain in humans [48]. Subsequent electrophysiological studies of pelvic afferent responses to colon distension and vagal afferent responses to gastric distension confirmed dose-dependent attenuation of responses by κ, but not either µ or δ opioid agonists [37•], suggesting a potentially useful strategy to modulate visceral pain; however, several features of the κ opioid effects were unusual. The effects were not fully reversed or prevented by opioid receptor antagonists, including κ opioid receptor selective antagonists. In addition, the effective concentrations of different κ opioids for reduction of pelvic afferent responses to colon distension were similar and overlapping, despite wide divergence of potencies in other models. Initially it was considered that there existed in the
Somatostatin Several clinical studies concluded that the somatostatin (SST) analogue, octreotide, inhibited visceral afferent fibres and by so doing reduced discomfort and pain produced by balloon distension of the colon in normal and IBS patients [53,54]; however, two recent tests of this hypothesis produced divergent outcomes, leaving unclear the site of action of octreotide. In one study, octreotide was ineffective in the rat in reducing responses of pelvic afferents to distension in either normal or acetic acid inflamed colon, but did reduce behavioural responses when given intrathecally, suggesting a central site of action of octreotide as a visceral analgesic [55]. Conversely, in a study of mesenteric afferents innervating the rat jejunum, octreotide and a selective SST2 receptor agonist (BIM 23027) both attenuated baseline discharge and responses to ramp distension, suggesting that SST2 receptors inhibit populations of mesenteric afferents likely to be involved in nociceptive transmission [56]. Neurotrophins
There are five main endogenous neurotrophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT) 3 and 4, and glial cell linederived NF (GDNF). Others besides these may prove important in the GI tract as the field grows rapidly. mRNAs for NGF, BDNF, GDNF, NT3 and NT4, and protein content for NGF are significantly increased after acute and chronic cyclophosphamide-induced cystitis [57]. NGF is induced within 3 hours of chemical inflammation of the urinary bladder, and leads to lowering of pelvic bladder afferent mechanical thresholds [19]. These changes may underlie persistent symptoms of cystitis. NGF is also required for development and survival of many populations
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of spinal afferents. Therefore a balance must be struck to maintain normal sensory function. An imbalance in NGF secretion by the gut may be a mechanism for clinical hypersensitivity (following inflammation or not). Other neurotrophins are required for development and survival of other, non-nociceptive afferents, including vagal afferents sensitive primarily to BDNF [58].
cell bodies of colonic afferents in the DRG [64], but little information is available about the potentially important roles of voltage-gated K+ (Kv) channels in visceral sensation. A recent report suggests that reduction in the expression of Kv channel subunits in small diameter DRG cells after peripheral nerve injury leads to increased excitability and contributes to alterations in pain sensitivity [65].
Ion channels
Effects of inflammation
This is an area studied extensively in cell bodies of sensory neurons generally, but hitherto inaccessible in GI afferent endings (where transduction occurs). It addresses the fundamentals of sensory transduction. In addition to being themselves therapeutic targets, ion channels also constitute the final pathway underlying many drug actions on sensory endings. The gut is endowed with afferents showing different rates of adaptation to mechanical deformation that are almost certain to depend on expression of different channels. The coupling of G protein receptors is often direct to cation channels (e.g. [59]). In sensory neurons these are assumed to be N-type calcium channels because of studies of cell somata and central transmitter release, but this is questionable in peripheral endings where potassium channels may be more important.
GI infections or inflammatory diseases are not necessarily associated with visceral hypersensitivity; however, in subgroups of IBS and functional dyspepsia patients, symptoms do not occur until after an episode of GI infection or inflammation. The sensitisation of somatic afferents, principally if not exclusively nociceptors, by inflammatory and immune mediators is well documented and many of the established principles apply to GI afferents. Release of many of the excitatory substances listed above is increased in inflammatory conditions, and their effects on GI afferents are therefore relevant. That virtually all mechanosensitive spinal afferents that innervate the GI tract exhibit the ability to sensitise suggests that, unlike afferents in the somatic realm, all such GI afferents can contribute to discomfort and pain in circumstances where the chemical milieu at the receptive ending changes receptor excitability. Accordingly, GI afferents that encode into the noxious range generally show increased responses to mechanical stimuli following inflammation; the effect of inflammation on chemoreceptive function is relatively unexplored experimentally.
Sodium channels of the Deg/ENaC family, which include acid-sensing ion channels, have very recently been shown to be involved in visceral and cutaneous mechanosensitivity [60•]. This has been demonstrated using pharmacological blockade with the lanthanide ion gadolinium and with amiloride and analogues, and most recently by gene targeting. In addition, voltage-gated Na+ (NaV) channels, which are principally responsible for the rising phase of neuron action potentials, have become a target of interest. NaV1.3 is a tetrodotoxin (TTX)-sensitive sodium channel present principally in the CNS in adults; however, when peripheral nerves are injured, NaV1.3 is expressed in peripheral sensory neurons where it is considered to contribute to increased excitability. Two TTX-resistant channels, NaV1.8 and NaV1.9, are present principally in a subset of small dorsal root ganglion (DRG) neurons, those commonly considered to be the cell bodies of nociceptors. Recent reports show that NaV1.8 is upregulated in sacral DRG after urinary bladder inflammation [61] and that the increase in peak sodium current recorded in nodose and thoracic DRG cells after gastric insult is contributed to principally by the TTX-resistant component of the current [62,63]. Because the sodium current is important to generation of action potentials, these outcomes suggest that increased excitability of visceral sensory neurons contributes to visceral hypersensitivity. It is important to recognise that cell bodies of vagal afferents also exhibited changes in excitability, suggesting that in circumstances of organ insult vagal afferents could contribute to altered sensations, including discomfort and pain that arise from the gut. Mechanosensitive potassium channels may be important in regulating activity of GI afferents so that intense mechanical activation does not lead to toxic effects of cation imbalance. Their role as mechanosensors has been demonstrated at the
Conclusions Primary afferent neurons are among promising targets for future pharmacotherapy of visceral pain, as they are the direct link from the GI tract to the CNS, before processing and convergence have made the targets more distant from the circulation and more alike pharmacologically. We have outlined how GI afferent neurons express a variety of molecular targets, many of which are present on only a subpopulation of neurons, providing an opportunity for selective modulation of particular afferent modalities, and possibly therefore reducing particular sensations. Superimposed on the choice of which afferents to target is the choice of excitatory and inhibitory mechanisms. Thus, we may antagonise the excitatory action of an endogenous mediator that excites afferent endings, or we may activate inhibitory receptors whose endogenous ligand is rarely encountered. There are further choices provided by developing those drugs that may act directly on ion channels. These channels may be those that normally transduce the signal from membrane receptors, those that are regulatory for cellular excitability or those that transduce mechanical stimuli directly into membrane depolarisation.
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29. Hillsley K, Kirkup AJ, Grundy D: Direct and indirect actions of 5-hydroxytryptamine on the discharge of mesenteric afferent fibres innervating the rat jejunum. J Physiol 1998, 506:551-561. 30. Hicks G, Clayton J, Gaskin P: 5-HT4 receptor agonists stimulate small intestinal transit but do not have direct visceral antinociceptive effects in the rat. Gastroenterology 2001, 120:A6. 31. Schikowski A, Thewissen M, Mathis C, Ross HG, Enck P: Serotonin type-4 receptors modulate the sensitivity of intramural mechanoreceptive afferents of the cat rectum. Neurogastroenterol Motil 2002, 14:221-227. 32. Camilleri M, Northcutt AR, Kong S, Dukes GE, McSorley D, • Mangel AW: Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial. Lancet 2000, 355:1035-1040. One of the first demonstrations of the clinical efficacy of 5-HT3 receptor antagonists in visceral pain. 33. Sengupta J, Medda B, Petersen J, Shaker R: Mechanical and chemical properties of distension-sensitive vagal afferents innervating the esophagus of rats. Gastroenterology 2002, 122:A155. 34. McRoberts JA, Coutinho SV, Marvizon JC, Grady EF, Tognetto M, • Sengupta JN, Ennes HS, Chaban VV, Amadesi S, Creminon C et al.: Role of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral nociception in rats. Gastroenterology 2001, 120:1737-1748. Showed the localisation of NMDA receptors on primary afferents, and effects on transmitter release from sensory fibres in the gut, and effects of an antagonist (memantine) on responses of primary afferent fibres. 35. Wang YH, Wei J, Mayer E, McRoberts J: The glutamate receptor agonist NMDA dose-dependently increases the basal activity and alters the colorectal distension responsiveness of rat inferior splanchnic afferents in vitro. Gastroenterology 2002, 122:A408. 36. Ozaki N, Sengupta JN, Gebhart GF: Differential effects of mu-, delta-, and kappa-opioid receptor agonists on mechanosensitive gastric vagal afferent fibers in the rat. J Neurophysiol 2000, 83:2209-2216. 37. •
Gebhart G, Su X, Joshi S, Ozaki N, Sengupta J: Opioid modulation of visceral pain. In Opioid Sensitivity of Chronic Noncancer Pain. Edited by Kalso E, McQuay H, Wiesenfeld-Hallin Z: IASP Press; 1999:225-235. Pain Research and Management, vol 14. A summary of kappa opioid agonist modulation of visceral nociception. 38. Kirkup A, Bunnett N, Grundy D: A proteinase-activated receptor subtype 2 (PAR-2) agonist stimulates mesenteric afferent nerve discharge. Gastroenterology 2000, 118:A173. 39. Maubach KA, Grundy D: The role of prostaglandins in the bradykinin-induced activation of serosal afferents of the rat jejunum in vitro. J Physiol 1999, 515:277-285.
21. Brunsden AM, Grundy D: Sensitization of visceral afferents to bradykinin in rat jejunum in vitro. J Physiol 1999, 521:517-527.
40. Caterina MJ, Julius D: The vanilloid receptor: a molecular gateway • to the pain pathway. Annu Rev Neurosci 2001, 24:487-517. A review of VR1 and related receptors and their role in pain
22. Page AJ, O’Donnell TA, Blackshaw LA: P2X purinoceptor-induced sensitization of ferret vagal mechanoreceptors in oesophageal inflammation. J Physiol 2000, 523:403-411.
41. Longhurst JC, Kaufman MP, Ordway GA, Musch TI: Effects of bradykinin and capsaicin on endings of afferent fibers from abdominal visceral organs. Am J Physiol 1984, 247:R552-559.
23. Burnstock G: Purine-mediated signalling in pain and visceral • perception. Trends Pharmacol Sci 2001, 22:182-188. An overview and rationale for the involvement of P2X receptors in visceral nociception, with a focus on P2X3 receptors and their involvement in mechanotransduction in viscera.
42. Blackshaw LA, Page AJ, Partosoedarso ER: Acute effects of capsaicin on gastrointestinal vagal afferents. Neuroscience 2000, 96:407-416. 43. Bielefeldt K: Differential effects of capsaicin on rat visceral sensory neurons. Neuroscience 2000, 101:727-736.
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44. Su X, Wachtel RE, Gebhart GF: Capsaicin sensitivity and voltagegated sodium currents in colon sensory neurons from rat dorsal root ganglia. Am J Physiol 1999, 277:G1180-G1188. 45. Brierley S, Cooper N, Blackshaw LA: Pelvic colonic primary afferents: mechanosensory and pharmacological subtypes. Gastroenterology 2002, 122:A527. 46. Birder LA, Kanai AJ, deGroat WC, Kiss S, Nealen JL, Burke NE, Dineley KE, Watkins S, Reynolds IJ, Caterina MJ: Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 2001, 98:13396-13401. 47. •
Blackshaw LA: Receptors and transmission in the brain-gut axis: potential for novel therapies. IV. GABA(B) receptors in the braingastroesophageal axis. Am J Physiol Gastrointest Liver Physiol 2001, 281:G311-G315. Describes recent evidence on the points along the brain–gut axis where GABAB receptors may modulate sensory input, and the therapeutic benefits of this action in gastro-oesophageal reflux disease. 48. Delvaux M: Pharmacology and clinical experience with fedotozine. Expert Opin Investig Drugs 2001, 10:97-110. 49. Joshi SK, Su X, Porreca F, Gebhart GF: kappa -opioid receptor agonists modulate visceral nociception at a novel, peripheral site of action. J Neurosci 2000, 20:5874-5879. 50. Su X, Joshi SK, Kardos S, Gebhart GF: Sodium channel blocking actions of the kappa-opioid receptor agonist U50,488 contribute to its visceral antinociceptive effects. J Neurophysiol 2002, 87:1271-1279. 51. Young R, Page A, Cooper N, Martin C, O’Donnell T, Coldwell J, Scott C, Blackshaw L: Metabotropic glutamate receptors inhibit vagal primary afferent mechanosensitivity. Gastroenterology 2002, 122:A381-A382. 52. Chen CY, Ling Eh EH, Horowitz JM, Bonham AC: Synaptic transmission in nucleus tractus solitarius is depressed by Group II and III but not Group I presynaptic metabotropic glutamate receptors in rats. J Physiol 2002, 538:773-786.
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55. Su X, Burton MB, Gebhart GF: Effects of octreotide on responses to colorectal distension in the rat. Gut 2001, 48:676-682. 56. Booth CE, Kirkup AJ, Hicks GA, Humphrey PP, Grundy D: Somatostatin sst(2) receptor-mediated inhibition of mesenteric afferent nerves of the jejunum in the anesthetized rat. Gastroenterology 2001, 121:358-369. 57.
Vizzard MA: Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol 2000, 161:273-284.
58. Winter J: Brain derived neurotrophic factor, but not nerve growth factor, regulates capsaicin sensitivity of rat vagal ganglion neurones. Neurosci Lett 1998, 241:21-24. 59. Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne MC, Hoshi T, Hell JW: A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science 2001, 293:98-101. 60. Welsh MJ, Price MP, Xie J: Biochemical basis of touch perception: • mechanosensory function of degenerin/epithelial Na+ channels. J Biol Chem 2002, 277:2369-2372. An overview of DEG/ENaC channels, including the acid sensing ion channels series. It describes the localisation, structure, molecular biology and electrophysiological properties of these channels, their role in mechanotransduction in different systems, and other functions they may subserve. 61. Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, de Groat WC: The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 2001, 21:8690-8696. 62. Bielefeldt K, Ozaki N, Gebhart GF: Mild gastritis alters voltagesensitive sodium currents in gastric sensory neurons in rats. Gastroenterology 2002, 122:752-761. 63. Bielefeldt K, Ozaki N, Gebhart GF: Experimental ulcers alter voltage-sensitive sodium currents in rat gastric sensory neurons. Gastroenterology 2002, 122:394-405.
53. Plourde V, Lembo T, Shui Z, Parker J, Mertz H, Tache Y, Sytnik B, Mayer E: Effects of the somatostatin analogue octreotide on rectal afferent nerves in humans. Am J Physiol 1993, 265:G742-751.
64. Su X, Wachtel RE, Gebhart GF: Mechanosensitive potassium channels in rat colon sensory neurons. J Neurophysiol 2000, 84:836-843.
54. Chey WD, Beydoun A, Roberts DJ, Hasler WL, Owyang C: Octreotide reduces perception of rectal electrical stimulation by spinal afferent pathway inhibition. Am J Physiol 1995, 269:G821-826.
65. Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, Trimmer JS: Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci USA 2001, 98:13373-13378.
The pharmacology of gastrointestinal nociceptive pathways LA Blackshaw* and GF Gebhart† The principal conscious sensations that arise from the gastrointestinal tract are discomfort and pain. Chronic visceral discomfort and pain are generally managed poorly with currently available pharmacological agents. Receptors and ion channels present on extrinsic visceral primary afferent (sensory) neurons are targets for the development of new pharmacological strategies for control of visceral pain. Addresses *Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia † Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, IA 52242, USA Correspondence: GF Gebhart; e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:642–649 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations 5-HT 5-hydroxytryptamine AMPA α-amino-3-hydroxy-5-methyl-isoxazoleproprionate BDNF brain-derived neurotrophic factor BK bradykinin CCK cholecystokinin CNS central nervous system DRG dorsal root ganglion GABA γ amino butyric acid GI gastrointestinal GRNF glial cell line-derived neurotrophic factor IBS irritable bowel syndrome mGluR metabotrophic glutamate receptor NGF nerve growth factor NMDA N-methyl-D-aspartate NT neurotrophins PG prostaglandin SST somatostatin TTX tetrodotoxin VR1 vanilloid receptor 1
Introduction Although discomfort and pain undeniably arise from the gut, it is only relatively recently that nociceptors have been accepted as innervating the viscera [1]. Somatic nociceptors were defined by Sherrington as signalling damage or threat of damage, but the stimuli that activate somatic nociceptors (e.g. cutting, crushing, burning) are generally ineffective when applied to the viscera. Adequate stimuli for activation of visceral nociceptors include distension of hollow organs, traction on the mesentery, ischaemia and inflammation. Gastrointestinal (GI) mechanonociceptors are best understood; they are located in viscus muscle layers where they function as in-series tension or stretch receptors. Adequate stimuli for other visceral nociceptors are less well studied, but it is clear that mechanonociceptors can be sensitised by prior exposure to chemical or thermal stimuli. Indeed, present evidence supports the notion that much of the visceral sensory apparatus is polymodal in character. That is, visceral
sensory receptors that subserve a nociceptive function also transduce chemical and thermal stimuli into electrical nerve activity. The sensory innervation of the gut, moreover, is complex relative to innervation of somatic structures. Despite general agreement about the existence and role of polymodal nociceptors in the gut, whether they or other nociceptors (e.g. chemonociceptors, silent nociceptors) exist and predominate in different GI organs, convey information to the central nervous system (CNS) through all visceral sensory pathways, or express similar sets of pharmacological targets remains to be determined. We begin this overview with a brief review of the sensory innervation of the gut, following which pharmacological targets on these sensory neurons are considered.
Pathways of primary sensory information to the central nervous system Anatomy
Sensory (afferent) fibres from the GI tract may follow any of several paths to the CNS (Figure 1): • Vagal afferent endings are sparse in the colon but more numerous proximally (particularly the stomach and oesophagus). Their axons project directly into the brainstem and their cell bodies are located in the nodose or jugular ganglia. Viscerofugal neurons have cell bodies in the gastric and oesophageal myenteric plexus and project centrally in the vagal trunks, although their function is not yet understood. • Spinal afferent endings are distributed throughout the gut, and travel in the splanchnic nerves to the thoracic and lumbar spinal cord and in the pelvic nerves to the sacral cord. Spinal afferent axons make synaptic contacts in prevertebral (sympathetic) ganglia and the spinal dorsal horn; their cell bodies are in dorsal root ganglia. Intestinofugal neurons with cell bodies in the myenteric plexus project with spinal afferents from the gut to prevertebral ganglia and occasionally to the spinal cord. These sensory fibres are mainly unmyelinated C-fibres, which are difficult to trace to their endings, but which differ in their transmitter content and embryonic origin. Unlike sensory end organs in other tissues, no precise anatomical specialisation of afferent endings is evident in the GI tract, suggesting that the bare nerve endings rely on features of the innervated tissue for their physiological specialisation. Intra-ganglionic laminar endings of vagal afferents are distributed in the capsule of individual myenteric ganglia between muscle layers. Intramuscular arrays are arranged along smooth muscle bundles within a particular muscle layer. Vagal mucosal endings follow the central
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vascular supply to individual villi in the intestine or branch within the superficial layers of the oesophageal squamous epithelium [2]. Spinal oesophageal mucosal afferents show similar branching [3]. Preliminary data suggest that intra-ganglionic laminar endings exist in the colon [4], but which pathway they follow has not yet been established. Evidence is also emerging for serosal and mesenteric endings that follow splanchnic and pelvic pathways [4]. Sensory modalities
The pathways responsible for the full range of GI sensations have not been well defined, probably because of the difficulty in assessing symptoms in animal models; however, animal models have revealed pathways involved in nociception, aversion, emesis and reflex control of gut motility. Vagal fibres are associated with upper GI sensations such as satiety, nausea and hunger. Spinal afferents are associated with sensations of fullness, bloating, discomfort and pain from the stomach and bloating, urgency, stool, colic and pain from the large intestine, and are also thought to mediate discomfort and pain from the small bowel. The role of intestinofugal neurons is well established in the reflex control of motility, but their involvement and that of vagal viscerofugal neurons in signalling sensation or pain to the CNS is uncertain.
Functional properties of afferent endings Vagal afferents
Vagal afferents may be divided into three classes based on the layer of gut containing the receptive field. Location of endings is pivotal in determining sensitivity, as anatomical and electrophysiological data concur (Figure 2). Mucosal receptors are silent at rest, but develop activity after acute inflammation or damage because of release of mediators (e.g. 5-hydroxytryptamine [5-HT]) [5,6]. They are mechanosensitive to fine mucosal stroking with rapidly adapting responses and are unresponsive to distension. They are also chemosensitive to a range of intra-luminal chemical and osmotic stimuli. They respond directly to several locally released mediators, including cholecystokinin (CCK), 5-HT, noradrenaline, opioids, bradykinin (BK), purines and prostaglandins (PGs) [5–12]. The major role of mucosal receptors in sensation is thought to lie in the generation of satiety, nausea and vomiting, with a minor role in direct generation of reflex responses.
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Tension/mucosal receptors have so far been observed only in oesophageal striated muscle and show features of both tension receptors and mucosal receptors [14]. They presumably have a specialised role in detection of rapidly moving boli of food. Spinal afferent
Spinal afferents, like vagal afferents, are similarly located in different layers of organs (Figure 2) and contribute to a variety of reflexes. Importantly, spinal afferents are considered to convey to the CNS the sensations of discomfort and pain [1]. Tonic (or wide dynamic range) mechanoreceptors have tonic levels of resting activity; they respond to contractions and distension with a linear relationship to wall tension [15–17]. It is most likely that these receptors signal filling of the stomach, colon and rectum to give rise to sensations of fullness. Because they encode distending stimuli well into the noxious, non-physiological range, it is considered that they can also contribute to discomfort and pain, particularly in the presence of organ inflammation. High threshold (or phasic) mechanoreceptors have low resting activity and respond only to noxious intensities of organ distension. Accordingly, they are considered mechanonociceptors [1,15–17]. They are also chemosensitive, responding directly to inflammatory mediators, including BK, eicosanoids and free radicals [18]. Their receptive fields have sometimes been identified in the serosa and mesentery. Silent nociceptors (or mechanically insensitive afferents) are silent at rest. They develop activity and mechanosensitivity during and after inflammation through action of inflammatory mediators (BK and eicosanoids demonstrated so far) and nerve growth factor [1,19]. Silent nociceptors have been most extensively studied in somatic tissues, where some have been characterised as chemonociceptors. Mucosal receptors have similar properties to vagal mucosal afferents in their sensitivity to luminal chemicals and selective responses to fine tactile stimulation. So far they have been found only in in vitro preparations of colon [20].
Pharmacological targets on visceral afferents Excitatory receptors
Tension receptors (also known as muscular afferents) have resting activity, often modulated in phase with ongoing contractions. They are mechanosensitive to contractions and distension with a slowly adapting, linear relationship to wall tension within the physiological range [13]. Tension receptors as a population signal the amplitude, pattern and direction of luminal contractions to the CNS, which is important in triggering reflexes controlling GI function. Their responses to distension are also important in signalling food intake and mediating satiety and fullness.
Adenosine is a breakdown product of ATP, which is released by numerous processes (see below), many of which are relevant to healthy and diseased GI function. Kirkup et al. [11] showed that adenosine increased mesenteric afferent nerve activity and intestinal motility in the anesthetised rat through A1 and A2B and/or A2B-like receptors. They found that increased motor activity induced part of the afferent response but did not wholly account for adenosine-evoked excitation, a fact supported by an in vitro study of these afferents [21].
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Figure 1
Nodose ganglion
NTS
Paravertebral ganglia
Greater splanchnic nerve
Lumbar colonic n.
Hypogastric nerve
Prevertebral ganglia
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 1 2 3 4 5
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Representation of sensory innervation of the GI tract. Left: visceral afferent pathways through prevertebral and paravertebral ganglia to the spinal cord; cell bodies are located in dorsal root ganglia (not illustrated). Right: vagal and pelvic nerve afferent input through nodose and dorsal root ganglia, respectively. The innervation of the viscera as
illustrated is overlapping with spinal inputs to specific, but anatomically separated spinal segments. For example, the distal colon is represented in thoracolumbar and lumbosacral spinal segments by least splanchnic and pelvic nerve inputs. Gastric input to the CNS is represented in the brainstem and thoracic spinal cord by vagal and splanchnic inputs.
ATP is released from damaged cells, making it a prime candidate for signalling of nociceptive events. It is also released from sympathetic and myenteric nerve terminals, and from mechanically deformed epithelia and endothelia. Both ionotropic (P2X) and metabotropic (P2Y) receptors have been found on neurons, although P2X receptors are more important in sensory pathways. Each type has seven subtypes. P2X receptors may have two types of effect on vagal and spinal GI primary afferents: direct activation or sensitisation to other stimuli [12,22]. Responses to ATP and its analogues may be prolonged or may desensitise rapidly, depending possibly on the subunit composition of the receptor. Thus a homodimeric receptor composed of two P2X2 subunits desensitises rapidly, whereas a heterodimer of P2X2 and P2X3 does not. Burnstock [23•] has suggested that ATP may be involved in visceral mechanosensory transduction and a recent report by Rong et al. [24] documented that P2X3 receptors modulate sensitivity of pelvic afferents to urinary bladder distension.
BK is formed during tissue damage and is well recognised as a stimulant of somatic nociceptors. In the GI tract, BK activates fibres with high (nociceptive) as well as those with low (non-nociceptive) thresholds to distension. In spinal afferents, the receptor subtype involved in direct activation is the B2 subtype, whereas indirect activation could occur through the B1 subtype following smooth muscle contraction [25]. Blackshaw and Grundy [6] showed an action on vagal mucosal afferents, which suggests that BK in the gut may activate emetic as well as nociceptive pathways. B2 antagonists have been developed for somatic pain indications, and these data would indicate their potential efficacy additionally in visceral pain. B1 receptors have been reported to upregulate (increase) following tissue injury and thus have also been targeted for development of potential analgesics. Because BK receptors activate phospholipase A2, they induce sensitisation of responses by production of PGs, which themselves are potent algogens.
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CCK is released by products of fat digestion from the small intestinal mucosa. CCK is associated with initiation of satiety from the upper GI tract and reflex inhibition of gastric motility. At high doses, CCK may induce nausea and vomiting. The mechanism by which this occurs is through a vagal reflex, but controversy exists as to the selectivity of mucosal or muscular vagal afferents to CCK. Some evidence supports a circulating action on muscular afferents [26], whereas other data suggest a paracrine action of locally released CCK on mucosal afferents [27,28]. These actions are mediated through CCK1 receptors. No reports exist so far of CCK actions on spinal afferents, but the action of CCK on a mixed population of afferents in the mesenteric nerves was abolished by chronic degeneration of vagal fibres, suggesting that these are the only target for CCK [28]. 5-HT, like CCK, is released from the GI mucosa after a meal; however, 5-HT release may occur in response to mechanical or chemical stimuli throughout the GI tract. There are seven 5-HT receptors that exist in the GI tract and numerous subtypes of these. So far only the ionotropic 5-HT3 receptor has been conclusively demonstrated to mediate actions on extrinsic GI afferents [6,29], which are selective to a distinct subpopulation; however, there is still controversy as to the role of 5-HT4 receptors, and the role of many subtypes remains uninvestigated [30,31]. In humans, 5-HT3 receptor antagonists have been used successfully to reduce nausea and vomiting evoked from the upper GI tract and to reduce pain, discomfort and urgency in diarrhoea-predominant irritable bowel syndrome (IBS) [32•]. Major side effects include constipation and ischaemic colitis, which have so far prevented extensive use in IBS. Ionotropic glutamate receptors include N-methyl D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-isoxazoleproprionate (AMPA) receptors and kainate receptors. Glutamate may be derived from the diet, from intrinsic or extrinsic neurotransmission in the gut, or released from epithelial cells. Although evidence for direct responses to glutamate is lacking, NMDA receptor antagonists have been shown to reduce responses to mechanical stimuli in pelvic and splanchnic afferents from the colon and in vagal oesophageal afferents [33,34•,35], suggesting either involvement of endogenous glutamate in mechanotransduction or an action of antagonists on mechanosensitive channels. The evidence for NMDA receptor involvement in mechanosensitivity so far relies mainly on the action of the open channel blocker, memantine, which has actions at other ionotropic receptors; therefore, this interesting and potentially exploitable area is open for further investigation.
µ and δ Opioids, despite having mostly inhibitory actions on neurons in the CNS, cause excitation of vagal afferents from the small intestine [9]. These actions were not seen in a study of mechanosensitive vagal afferents from the stomach [36] or pelvic afferents from the colon [37•], suggesting a region-specific effect.
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Figure 2
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Schematic representing current anatomical and electrophysiological evidence for the location of sensory endings in the wall of the GI tract. Fibres in red represent serosal/mesenteric high threshold afferents, those in blue represent muscular low threshold afferents (which terminate in muscle layers or around myenteric ganglia), and those in green represent mucosal afferents sensitive to tactile luminal stimuli.
Proteases (e.g. thrombin, trypsin and mast cell tryptase) are likely to be present on GI afferent nerve terminals when the intestine is ulcerated or when mast cells degranulate following inflammation. Until recently the role of mast cell proteases was considered to be restricted to the destruction of pathogens; however, somatic and pancreatic nociceptive afferents were shown to express specific protease-activated receptors. The ligand is tethered to the receptor and allowed to bind following proteolytic cleavage. These receptors have recently been demonstrated on small intestinal afferents [38], although whether vagal or spinal in origin remains to be established. PGs are produced in a wide range of cell types, and are important mediators of pain. PGs are often produced in response to other stimuli, and they in turn potentiate responses of GI afferents to other stimuli [39], constituting a positive feedback system. Blockade of PG synthesis is a common means to reduce pain and inflammation. Five classes of prostanoid receptors (DP, EP, FP, IP and TP receptors) have been identified and distinguished by rank order of agonist potency (e.g. PGE2 acts at EP receptors). EP1 receptors were recently shown to mediate rapid excitation of small intestinal afferents and EP2 receptors to cause longer term activation [10]. Because blockade of PG synthesis compromises other processes, such as mucosal defence, approaches to reducing visceral pain based on receptor antagonism may prove to be more selectively targeted.
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Vanilloids, such as the chilli extract capsaicin, are well-established excitants of somatic nociceptors, giving rise to a burning sensation mediated through the vanilloid receptor 1 (VR1 or TRPV1), which is also a natural sensor for heat and low pH [40•]. Capsaicin activates a proportion of all classes of vagal and spinal afferents throughout the GI tract, but the sensations evoked are not obvious. An early report found that the majority of GI afferents were activated by capsaicin [41]. Recent reports found that as few as 30% of vagal afferents [42], 42% of nodose ganglion cells [43] and 46% of colon dorsal root ganglion cells [44] were activated. There is no correlation between expression of VR1 and other properties of primary afferents, such as their peptide content, location or pathway; however, recent preliminary evidence points to a co-expression of VR1 with purinergic and bradykinin receptors on colon afferents in the pelvic pathway [45]. In addition, VR1 are expressed in bladder epithelial cells, suggesting a functional association of urothelial cells with afferent fibre nerve endings [46]. Endogenous activators of VR1 have recently been identified, suggesting contributions to physiological and/or pathophysiological processes [40•]. This endogenous pathway is yet to be investigated in the GI tract.
peripheral innervation of the gut a novel, κ opioid-like receptor that could be targeted to develop an analgesic for visceral discomfort and pain [49]. Recently, however, it was determined that these κ opioid agonists, which share structural similarity, significantly reduce voltage-activated sodium currents in colon sensory neurons [50]. This effect was shown not to be opioid receptor-mediated and to mimic the effects of other sodium channel blockers (e.g. effects were tonic and use-dependent). Metabotropic glutamate receptors (mGluR) may be divided into eight molecular subtypes, which currently fall into three pharmacological and functional groups: group I (mGlu 1 and 5 receptors) are excitatory, whereas group II (mGlu 2 and 3 receptors) and group III (mGlu 4, 6, 7 and 8 receptors) are inhibitory to neuronal function. Preliminary data from in vitro studies in ferret and mouse indicates that group I mGluR do not affect vagal afferents, but both group II and III inhibit mechanical sensitivity in a similar manner to GABAB receptors [51]. mRNA and protein for all of the mGluR subtypes are found in the nodose ganglia [52]. Pharmacological tools for mGluR are currently limited in selectivity but, as this hopefully improves, the role of individual subtypes may become more apparent.
Inhibitory receptors
γ-Amino butyric acid (GABA) activates ionotropic (GABAA and GABAC) receptors and metabotropic (GABAB) receptors on neurons of the central and enteric nervous systems. In these systems GABA functions as a direct inhibitor of neuron excitability and an inhibitory modulator of synaptic transmission. The role of endogenous GABA in modulation of vagal and spinal primary afferents is considered to be minor, but exogenous GABAB receptor agonists (but not GABAA agonists) are potent inhibitors of mechanosensitivity in a subpopulation of vagal afferents. This has stimulated clinical interest in the use of these drugs to reduce signalling of gastric distension to the brainstem. This in turn reduces triggering of motor patterns that lead to gastroesophageal acid reflux [47•]. The potential for GABAB receptors agonists in reducing signalling through spinal pathways is yet to be explored. κ Opioids Interest in κ opioids was stimulated by initial reports that a compound (fedotozine) with κ opioid efficacy was effective in reducing visceral pain in humans [48]. Subsequent electrophysiological studies of pelvic afferent responses to colon distension and vagal afferent responses to gastric distension confirmed dose-dependent attenuation of responses by κ, but not either µ or δ opioid agonists [37•], suggesting a potentially useful strategy to modulate visceral pain; however, several features of the κ opioid effects were unusual. The effects were not fully reversed or prevented by opioid receptor antagonists, including κ opioid receptor selective antagonists. In addition, the effective concentrations of different κ opioids for reduction of pelvic afferent responses to colon distension were similar and overlapping, despite wide divergence of potencies in other models. Initially it was considered that there existed in the
Somatostatin Several clinical studies concluded that the somatostatin (SST) analogue, octreotide, inhibited visceral afferent fibres and by so doing reduced discomfort and pain produced by balloon distension of the colon in normal and IBS patients [53,54]; however, two recent tests of this hypothesis produced divergent outcomes, leaving unclear the site of action of octreotide. In one study, octreotide was ineffective in the rat in reducing responses of pelvic afferents to distension in either normal or acetic acid inflamed colon, but did reduce behavioural responses when given intrathecally, suggesting a central site of action of octreotide as a visceral analgesic [55]. Conversely, in a study of mesenteric afferents innervating the rat jejunum, octreotide and a selective SST2 receptor agonist (BIM 23027) both attenuated baseline discharge and responses to ramp distension, suggesting that SST2 receptors inhibit populations of mesenteric afferents likely to be involved in nociceptive transmission [56]. Neurotrophins
There are five main endogenous neurotrophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT) 3 and 4, and glial cell linederived NF (GDNF). Others besides these may prove important in the GI tract as the field grows rapidly. mRNAs for NGF, BDNF, GDNF, NT3 and NT4, and protein content for NGF are significantly increased after acute and chronic cyclophosphamide-induced cystitis [57]. NGF is induced within 3 hours of chemical inflammation of the urinary bladder, and leads to lowering of pelvic bladder afferent mechanical thresholds [19]. These changes may underlie persistent symptoms of cystitis. NGF is also required for development and survival of many populations
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of spinal afferents. Therefore a balance must be struck to maintain normal sensory function. An imbalance in NGF secretion by the gut may be a mechanism for clinical hypersensitivity (following inflammation or not). Other neurotrophins are required for development and survival of other, non-nociceptive afferents, including vagal afferents sensitive primarily to BDNF [58].
cell bodies of colonic afferents in the DRG [64], but little information is available about the potentially important roles of voltage-gated K+ (Kv) channels in visceral sensation. A recent report suggests that reduction in the expression of Kv channel subunits in small diameter DRG cells after peripheral nerve injury leads to increased excitability and contributes to alterations in pain sensitivity [65].
Ion channels
Effects of inflammation
This is an area studied extensively in cell bodies of sensory neurons generally, but hitherto inaccessible in GI afferent endings (where transduction occurs). It addresses the fundamentals of sensory transduction. In addition to being themselves therapeutic targets, ion channels also constitute the final pathway underlying many drug actions on sensory endings. The gut is endowed with afferents showing different rates of adaptation to mechanical deformation that are almost certain to depend on expression of different channels. The coupling of G protein receptors is often direct to cation channels (e.g. [59]). In sensory neurons these are assumed to be N-type calcium channels because of studies of cell somata and central transmitter release, but this is questionable in peripheral endings where potassium channels may be more important.
GI infections or inflammatory diseases are not necessarily associated with visceral hypersensitivity; however, in subgroups of IBS and functional dyspepsia patients, symptoms do not occur until after an episode of GI infection or inflammation. The sensitisation of somatic afferents, principally if not exclusively nociceptors, by inflammatory and immune mediators is well documented and many of the established principles apply to GI afferents. Release of many of the excitatory substances listed above is increased in inflammatory conditions, and their effects on GI afferents are therefore relevant. That virtually all mechanosensitive spinal afferents that innervate the GI tract exhibit the ability to sensitise suggests that, unlike afferents in the somatic realm, all such GI afferents can contribute to discomfort and pain in circumstances where the chemical milieu at the receptive ending changes receptor excitability. Accordingly, GI afferents that encode into the noxious range generally show increased responses to mechanical stimuli following inflammation; the effect of inflammation on chemoreceptive function is relatively unexplored experimentally.
Sodium channels of the Deg/ENaC family, which include acid-sensing ion channels, have very recently been shown to be involved in visceral and cutaneous mechanosensitivity [60•]. This has been demonstrated using pharmacological blockade with the lanthanide ion gadolinium and with amiloride and analogues, and most recently by gene targeting. In addition, voltage-gated Na+ (NaV) channels, which are principally responsible for the rising phase of neuron action potentials, have become a target of interest. NaV1.3 is a tetrodotoxin (TTX)-sensitive sodium channel present principally in the CNS in adults; however, when peripheral nerves are injured, NaV1.3 is expressed in peripheral sensory neurons where it is considered to contribute to increased excitability. Two TTX-resistant channels, NaV1.8 and NaV1.9, are present principally in a subset of small dorsal root ganglion (DRG) neurons, those commonly considered to be the cell bodies of nociceptors. Recent reports show that NaV1.8 is upregulated in sacral DRG after urinary bladder inflammation [61] and that the increase in peak sodium current recorded in nodose and thoracic DRG cells after gastric insult is contributed to principally by the TTX-resistant component of the current [62,63]. Because the sodium current is important to generation of action potentials, these outcomes suggest that increased excitability of visceral sensory neurons contributes to visceral hypersensitivity. It is important to recognise that cell bodies of vagal afferents also exhibited changes in excitability, suggesting that in circumstances of organ insult vagal afferents could contribute to altered sensations, including discomfort and pain that arise from the gut. Mechanosensitive potassium channels may be important in regulating activity of GI afferents so that intense mechanical activation does not lead to toxic effects of cation imbalance. Their role as mechanosensors has been demonstrated at the
Conclusions Primary afferent neurons are among promising targets for future pharmacotherapy of visceral pain, as they are the direct link from the GI tract to the CNS, before processing and convergence have made the targets more distant from the circulation and more alike pharmacologically. We have outlined how GI afferent neurons express a variety of molecular targets, many of which are present on only a subpopulation of neurons, providing an opportunity for selective modulation of particular afferent modalities, and possibly therefore reducing particular sensations. Superimposed on the choice of which afferents to target is the choice of excitatory and inhibitory mechanisms. Thus, we may antagonise the excitatory action of an endogenous mediator that excites afferent endings, or we may activate inhibitory receptors whose endogenous ligand is rarely encountered. There are further choices provided by developing those drugs that may act directly on ion channels. These channels may be those that normally transduce the signal from membrane receptors, those that are regulatory for cellular excitability or those that transduce mechanical stimuli directly into membrane depolarisation.
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