Dr S. Vanner, Hotel Dieu Hospital, 166 Brock St., Kingston, Ontario, Canada K7L 5G2. Tel: 613- 544- 3310; fax: 613 544- 3114; e-mail: email@example.com
Abstract Gastrointestinal (GI) inflammation modulates the intrinsic properties of nociceptive dorsal root ganglia neurones, which innervate the GI tract and these changes are important in the genesis of abdominal pain and visceral hyperalgesia. neurones exhibit hyperexcitability characterized by a decreased threshold for activation and increased firing rate, and changes in voltage-gated Na+ and K+ channels play a major role in this plasticity. This review highlights emerging evidence that specific subsets of channels and signalling pathways are involved and their potential to provide novel selective therapeutic targets for the treatment of abdominal pain.
Abdominal pain and discomfort are amongst the most vexing symptoms for patients and present a major therapeutic challenge for physicians. This pain is a frequent manifestation of inflammatory diseases of the gastrointestinal (GI) tract and is a defining feature of functional GI conditions such as the irritable bowel syndrome, non-cardiac chest pain and functional dyspepsia. Treatments are often ineffective or fraught with undesirable side-effects, such as the altered motility, sedation and dependence seen with narcotic analgesics. Our current treatment of pain arising from the GI tract is limited by our knowledge of visceral sensory neurobiology. Visceral sensory nerves share many common properties with somatic sensory nerves, however, a number of important differences exist.1 For example, the local microenvironment confronting the epithelium of the GI tract, continuously exposes it to a variety of potentially noxious stimuli, such as acid in the stomach or billions of bacteria in the colon. Furthermore, the enteric nervous system lies within the wall of the GI tract, outnumbering extrinsic neurones by nearly 1000 : 1 and is capable of controlling basic functions such as absorption, secretion, motility and blood flow independent of input from the central nervous system (CNS). Another important factor is the GI tract immune system, the body's largest immune organ, with its rich supply of immune cells capable of secreting cytokines and trophic factors, which can impact on sensory nerve function. Finally, the visceral sensory nerve may also differ from somatic nerves in terms of myelination, neurochemical markers and receptors for growth factors. Because of the uniqueness of the enteric microenvironment and the potentially important role of inflammation in functional GI disorders, an important advance has been the combined approach of retrograde tracing from the gut with electrophysiological and immunohistochemical techniques. This approach has enabled nociceptive neurones, which innervate sites of inflammation in the GI tract to be identified and the impact of intestinal inflammation on their intrinsic properties to be determined.
Sensory afferent nerves are stimulated by mechanical, chemical or thermal stimuli which depolarize the nerve terminal, activating voltage-gated Na+ and K+ channels. This leads to action potential electrogenesis and the transmission of sensory information including pain to the CNS. Recent evidence suggests that inflammatory mediators may sensitize GI afferents, allowing them to encode non-noxious stimuli or develop enhanced firing in response to noxious stimuli.1 Thus, the ionic conductance which underlie the generation of action potentials and regulate the threshold and firing pattern of GI dorsal root ganglia (DRG) neurones are fundamentally important in the transduction of inflammatory pain from the GI tract to the CNS. This review will focus primarily on these ionic conductance, namely voltage-gated Na+ and K+ channels and how their properties are altered by GI inflammation. The effects of GI inflammation on voltage-gated ion channels in enteric sensory neurones (intrinsic primary afferent neurones, IPANs) have also been recently reviewed.2
Neuroanatomy and functional correlations
Sensory neurotransmission from the GI tract follows two pathways to the CNS through vagal and spinal afferents, also termed extrinsic primary afferent neurones (EPANs).3,4 Spinal afferents project in the splanchnic or pelvic nerves (Fig. 1). These neurones, whose cell bodies lie in the DRG, appear to mediate nociceptive sensations (i.e. noxious stimuli perceived as discomfort and pain)3 whereas vagal afferents may relay more graded innocuous sensations. However, there is ongoing controversy as to whether vagal afferents may also act as nociceptors. In electrophysiological studies,5–7 nociceptor properties are implied by the presence of toxin, tetrodotoxin-resistant (TTX-R) action potentials, capsaicin sensitivity and the small size of the neurones. Limitations of this approach are possible contamination by non-nociceptor neurones and that findings at the level of the cell body are inferred to be representative of changes occurring at the sight of sensory transduction on the nerve terminal. Recent studies also suggest a subpopulation of splanchnic DRG neurones innervate several visceral organs and may underlie recent reports8 of inflammation-induced cross-sensitization of visceral organs (Fig. 1).9
The ability to combine anatomical labelling and electrophysiological recording has significantly advanced our ability to correlate structure and function of nociceptive splanchnic and pelvic nerves. Using antegrade labelling techniques, terminal specializations unique to various populations of extrinsic sensory nerves have been identified.3,4 This approach can be combined with single-fibre recording and has the potential to correlate functional studies (e.g. capsaicin sensitivity) with subsequent immunohistochemical examination of the expression of receptors or ion channels on these identified fibres, although these latter techniques have proven technically challenging. Retrograde labelling with fast blue (Fig. 2) or DiI into the wall of the intestine has enabled the target organ of DRG neurones to be identified and correlated with their immunohistochemical and electrophysiological properties by studying the properties of labelled cell bodies in the DRG. To date, immunohistochemical studies suggest two general populations of nociceptive DRG neurones exist. One group expresses the peptidergic marker calcitonin gene related peptide (CGRP) and colocalize the nerve growth factor (NGF) receptor, but TrkA does not bind the plant lectin IB4. The other group does not express peptides such as CGRP but does express IB4 and P2X3 receptor immunoreactivity. Despite this evidence, not all studies support the suitability of IB4 in differentiating these groups.10,11 Nonetheless, in the mouse colon several studies have shown that ∼80% of fast blue-labelled small neurones are CGRP immunoreactive and only 20% bind IB4 (Fig. 2).12,13 Over 80% of these neurones also exhibited TrkA and TRPV1 immunoreactivity. Together, these findings suggest that CGRP, TrkA, immunoreactive neurones predominate in the GI tract, at least in the colon whereas in the somatic sensory system, IB4-positive appear to predominate.14,15 The functional implications of these differences remain to be fully elucidated but reports suggest that IB4 binding correlated with expression of Nav 1.9 TTX-R Na+ currents,16,17 provides an example to suggest they may be markers of important differences in the repertoire of voltage-gated ion channels found in populations of somatic DRGs compared with those innervating the GI tract.
The close proximity of sensory nerve terminals to the epithelial barrier and translocation of bacteria from the lumen is also emerging as an important issue. Recent studies have shown that stress can induce visceral hyperalgesia.18 Although central mechanisms are clearly involved,19,20 it seems likely that peripheral mechanisms involving DRG neurones also contribute. Acute stress has been shown to cause a break in the epithelial tight junctions with paracellular and transcellular translocation of bacteria.21 The implication is that this transient translocation of bacteria initiates cytokine signalling to sensory nerves, which could perpetuate peripheral sensitization and recent work in neonatally stressed rats supports the contention that peripheral (i.e. gut) factors such as permeability, mast cells and NGF levels do contribute to stress-induced visceral hypersensitivity.22 However, the hypothesis that this causes direct changes on gut DRG neurones remains to be tested.
As Ritchie's original description of visceral hyperalgesia in patients with irritable bowel syndrome (IBS) in 1973, where he showed that patients experience pain in response to lower distending volumes than the healthy controls and subsequent similar observations were made in patients with other functional GI disorders, there has been an exponential increase in studies examining the underlying mechanisms. Broadly speaking, sensitization of nociceptive pathways can occur at the level of the DRG neurone (peripheral sensitization), or spinal cord and brain (central sensitization). There is ongoing debate as to the relative role of these two processes (see Woolf23 for review of central sensitization) but increasing evidence, such as persistent low-grade inflammation in the gut of IBS patients, suggests an important role for peripheral sensitization in the development of visceral hyperalgesia. In keeping with this notion, there is ample evidence in the inflamed gut, that neurotransmission in DRG neurones is increased. For example, experimental colonic inflammation results in sensitization of nociceptors, allowing them to respond to non-noxious stimuli or become active (so-called ‘silent nociceptors’).1 Inflammatory mediators such as bradykinin,24 5HT25, histamine26 and prostaglandins27–29 sensitize intestinal afferent nerves. In addition, the observation that IBS patients frequently report an antecedent history of enteritis30 and experimental colonic inflammation can cause long-term increases in visceral sensitivity31,32 even after the inflammation has resolved, suggests that inflammation may induce long-term changes (i.e. transcriptional) in visceral DRG nerves. This contention is further supported by studies of infectious or chemical intestinal inflammation, which show that following resolution of the inflammation, responses of DRG neurones evoked by either electrical stimulation of the DRG cell body or intestinal balloon distention remain enhanced.33–35
One of the first suggestions that long-term changes in DRG membrane excitability were induced by visceral inflammation originated from studies by Yoshimura and DeGroat using whole cell-patch clamp electrophysiological techniques on isolated DRG neurones labelled with fast blue from the bladder. Using an experimental model of cystitis, they demonstrated that these cells were hyperexcitable, exhibiting a lower current threshold and an increased number of action potentials during a prolonged depolarizing stimulus.7 Because these cell bodies lie outside the intestine and have been removed for hours from the animal, this strongly suggests that the neurones innervating the inflamed organ remained hyperexcitable, in the absence of inflammatory mediators. This implies there were long-term changes that occurred, e.g. changes in expression of ion channels. In the GI tract, Moore et al.5 and Stewart et al.6 showed similar findings in a trinitrobenzene sulphonic acid (TNBS) ileitis guinea-pig model (Fig. 3). These changes were limited to those cells innervating the inflamed organ, indicating that these changes were not due to circulating mediators. Thus it seems that inflammation at the level of the peripheral terminal can send signals, which alter, in the long term, the behaviour of the sensory neurone, likely through changes in multiple ion channels. These channels potentially include voltage-gated sodium, potassium, chloride inward rectifier and calcium channels but sodium and potassium channels appear to be of particular importance and have been the major focus of study to date.
Voltage-gated sodium channels in GI DRG neurones
Voltage-gated sodium currents (NaV) are responsible for the rapid upstroke of the action potential and their properties contribute to setting the firing threshold. In addition, a novel NaV with persistent kinetics has been recently described, which does not play a role in action potential electrogenesis, but can affect resting membrane potential and threshold.36 NaV can be grouped into two broad groups based on their sensitivity to nanomolar concentrations of the puffer fish (fugu sp.) TTX. The expression of TTX-R NaV is a relatively unique property of smaller diameter (unmyelinated) primary sensory neurones and a subpopulation of sensory neurones fire action potentials in the presence of TTX.5 TTX-S and TTX-R NaV differ in their biophysical properties as well.
There are at least three predominant types of NaV alpha subunits in sensory neurones. The first is NaV 1.7, a TTX-S channel, with rapid activation kinetics, low threshold for activation, hyperpolarized steady-state availability curve and relatively slow repriming kinetics. NaV 1.8 is a TTX-R channel, with a more depolarized threshold and availability curve, slower inactivation kinetics and rapid repriming kinetics.37–40 NaV 1.9 is responsible for a current with persistent inactivation kinetics, very low threshold and hyperpolarized availability curve.36 While the Nav subtypes underlying sodium currents in GI DRG neurones have yet to be conclusively identified, whole cell currents with properties corresponding to NaV 1.8 channels have been identified in labelled rat gastric,6,41–43 guinea-pig ileal6 and colonic12,28 DRG (mouse, rat) neurones in a variety of species. mRNA and immunoreactivity for NaV 1.8 has also been identified in labelled bladder afferents.44 In addition, Beyak et al.12 have demonstrated a persistent TTX-R NaV in a small proportion of colonic DRG sensory neurones of the mouse with similar kinetics to that reported for NaV 1.9. Although NaV 1.9 immunoreactivity has also been reported in bladder afferents, these data also suggest that its expression is less than in somatic afferents.16,44 While a number of other NaV subunits (e.g. 1.2, 1.3, 1.5, 1.6) have been identified in DRG neurones, their expression and function in visceral DRG neurones remains to be demonstrated.
Inflammation and chronic alterations in NaV function
A change in the expression pattern of NaV is now recognized to be a key component of the changes experienced by primary sensory neurones in a variety of experimental pain models. For example, in experimental axotomy, the TTX-R NaV is downregulated, while a rapidly repriming, low-threshold TTX-S current is increased (NaV 1.3), enabling repetitive firing.45,46 In contrast to axotomy, a number of models of somatic inflammatory hyperalgesia indicate that the inactivating TTX-R Ina are increased while TTX-S channels are affected to a lesser degree or not at all47–49 (however, see Black et al.Pain 2004; 108: 237). In the GI tract, recent work has examined the effects of inflammation on sodium currents in sensory neurones innervating the stomach, ileum and colon. Bielefeldt et al. have demonstrated alterations in gastric DRG NaV in two different models of gastric inflammation. In a model of severe gastric ulceration, the proportion of the total Na current that was TTX-R increased and the activation curve for the TTX-R NaV was shifted in a hyperpolarizing direction. In addition, the repriming kinetics of the TTX-S NaV were accelerated in DRG neurones.41 A more mild form of gastric injury induced by iodoacetamide did not have any effect on no-dose neurones, but significantly increased TTX-R NaV in gastric DRG neurones.42 In models of inflammatory bowel disease, ileitis (guinea-pig) also increased the TTX-R NaV in ileal DRGs, with no effect on the TTX-S6 (see Fig. 4) and TNBS colitis12 increased the TTX-R NaV and shifted its activation threshold in a hyperpolarizing direction. This latter study did not observe obvious changes in the persistent NaV 1.9, like currents in the small numbers of cells, which expressed them, however, a recent preliminary report suggests they could play a role in postinflammatory hyperexcitability.50 The consistently observed increase in the TTX-R NaV 1.8 currents can contribute to repetitive firing, by virtue of this channel's rapid repriming kinetics. A recent interesting finding suggests that the effects on NaV 1.8 may not just be limited to sensory neurones of the inflamed organ, but that sensory neurones of adjacent organs may undergo similar changes (cross-sensitization). A preliminary report by Akbarali et al. indicates that inflammation of the colon results in increases in NaV 1.8 in bladder DRGs and that these neurones co-localize retrograde labels from each organ.9 This suggests that some DRG neurones innervate several organs and may be one mechanism underlying this cross-sensitization. This raises interesting questions regarding the frequent coexistence of IBS and functional bladder syndromes such as interstitial cystitis.
Gene deletion models provide further evidence that TTX-R NaV are important in visceral hyperalgesia. NaV 1.8 knockout models demonstrate normal responses to noxious stimuli, but blunted sensitization of visceral stimuli by topical irritants or inflammation.51 In another ‘knockdown’ model of the NaV 1.8 gene using antisense oligodeoxynucleotides, bladder hyperreflexia induced by inflammation was significantly reduced following antisense treatment.52
K+ channels in GI DRG neurones
K+ (Kv) currents also play a fundamental role in regulating the excitability of neurones by modulating the threshold for activation and/or the firing rate or sustained discharge of action potentials.52,53 The repertoire of currents, which subserve this function in DRG neurones relates to the size and presumably function of the neurones.54 Small neurones, many of which are presumably nociceptive,6,52 are dominated by two voltage-gated Kv currents, a transient IA type current and a sustained delayed rectifier (IK) current. These currents may consist of several related currents, separated by their kinetics.55 Recent studies of small DRG neurones innervating the several organs in guinea-pig, rat and mouse GI tracts5,6,56,57 have confirmed similar IA and IK currents exist in these functionally identified neurones. Other K+ currents, particularly the Ca2+-activated K+ and IH current,58 do not appear to be as predominant in nociceptive neurones; however, these currents have yet to be studied in identified DRG neurones innervating the GI tract in detail and at least one preliminary study35 suggests Ca2+-activated K+ currents may play a role in inflammation-induced long-term excitability.
The voltage-gated potassium channel gene family is likely the most complex ion channel family known and the considerable molecular diversity of the α subunit, confounds the study of these channels. Heterologous expression systems have allowed correlations to be made between known channel subtypes and properties of the currents carried by these channels. In these systems Kv 1.4, 3.3, 3.4, 4.1, 4.2 and 4.3 give rise to currents, which resemble the IA current whereas Kv 1.1, 1.2, 1.5, 1.6, 2.1, 2.2 and 3.1 give rise to currents resembling the IK current.59 In DRG neurones, studies which compared cell size and the colocalization of TRPV1 and CGRP suggest that Kv 1.4 or 4.2 are prime candidates for the IA current in putative nociceptive neurones.60 Which of these candidate subunits are expressed in the nociceptive neurones innervating the GI tract remains to be determined.
Inflammation and chronic changes in Kv function
In models of chronic inflammation in the GI tract, including peptic ulcer and ileitis,6,56 inflammation suppressed the transient IA or, in the latter study, IA and the sustained, delayed rectifier IK currents in small DRG neurones. Similar effects on IA have been observed in a model of cystitis7 but not in a model of mild gastritis,42 possibly reflecting differences in the degree of inflammation. At least two different types of IA currents are expressed in sensory neurones, a slow and fast inactivating current.7 Most studies suggest the slowly inactivating IA currents predominate on the nociceptive neurones including those innervating the viscera.6,7,56 Models of visceral inflammation6,7,56 have also shown that the reduction in peak IA current was associated with a hyperpolarizing shift in the inactivation curve which results in fewer IA channels available at or near the resting membrane potential.
Inflammatory mediators, signalling pathways and post-transcriptional and transcriptional modulation of the voltage-gated Na+ and K+ currents
The signalling pathways which modulate these K+ and Na+ voltage-gated channels have yet to be elucidated in the GI tract in detail, but findings from studies of DRG neurones targeting sites outside the GI tract suggest changes in the properties of these channels can result from acute post-transcriptional and chronic transcriptional events and that multiple signalling pathways are involved. For example, in the acute setting (i.e. within minutes) the application of inflammatory mediators such as PGE2, adenosine, serotonin and NGF5,28,58,61–65 increase the TTX-R Na+ currents channels and/or suppress IK currents signalling through PKA and/or PKC pathways. Other pathways also exist, for example, in the case of NGF, the sphingomyelin pathway activates the intracellular messenger ceramide.66 Two studies have examined in detail the effects of potential inflammatory mediators in GI neurones. Surprisingly the first67 failed to observe any effect of 5-HT, PGE2 or adenosine on colonic Na currents. In contrast, Gold et al. demonstrated that PGE2 markedly increases the magnitude of TTX-R currents.28 In addition, further studies indicated that there may be important differences between cutaneous and visceral DRG neurones. For example, the proportion of cutaneous vs colonic DRG neurones responding to PGE2 differ significantly, with nearly all colonic DRG neurones being excited by PGE2, while less than half cutaneous DRG neurones were sensitized by PGE2.27 Thus the acute presence of inflammatory mediators, seems to be a potent and at least in the case of PGE2, somewhat selective stimulus for GI afferent neurones, with the effects seeming to, in part at least, being mediated through the TTX-R NaV.
There is also growing evidence that inflammation leads to other post-translational events (e.g. increase insertion of TTX-R Na+ channels into the membrane) or transcriptional events which might underlie the development of chronic pain.23,38–40 Of particular interest are recent studies highlighting additional complexities underlying plasticity, such as the suggestion inflammation results in ‘hyperalgesic priming’.68,69 In these studies, following resolution of the inflammation rechallenge with inflammatory mediators such as PGE2 switches from a sole dependence on a PKA-dependent pathway signalling to TTX-R Na+ channels to one also depending on PKC and ERK1/2 activation coupled to cytoskeleton scaffold. Moreover, disruption of the cytoskeleton now inhibits the PGE2-mediated sensitization leading to the suggestion that a possible treatment of chronic pain may involve maintaining the cytoskeleton in a ‘quiescent’ rather than a ‘primed’ state.69 It is intriguing to speculate that similar mechanisms could be involved in the development of pain in conditions such as postinfectious IBS where acute bacterial enterocolitis may prime the neurones to subsequent persisting low levels of inflammation.
The list of inflammatory mediators and growth factors which are released during inflammation in the GI tract and modulate voltage-gated ion channels in nerve terminals of DRG neurones continues to grow and another important challenge is to clarify the relative roles and interaction of these agents. For example, enthusiasm for the role of neurotrophins, particularly NGF, has increased recently with reports suggesting a central role in modulating visceral hypersensitivity in the GI tract and parallel effects on voltage-gated ion channels in DRG neurones.43,70,71 In whole animal models, visceral hyperalgesia induced by TNBS colitis can be blocked by antibodies to NGF and exogenous NGF can mimic the effects of TNBS. Similar findings have been reported for other viscera outside the GI tract.72 In parallel studies, most nociceptive DRG neurones express TrkA12 and the application of NGF to gastric DRG neurones in culture was shown to mimic the increase in TTX-R NaV induced by gastric ulcers, an effect that was also blocked by neutralizing antibody.43 Although not yet studied in the GI tract, it is highly probable that neutrophins signal to other voltage-gated channels, particularly K+ channels given the findings of others.73,74 Despite the evidence that NGF plays a central role in visceral hyperalgesia other mediators, such as proteases acting through PARs,75 have also been suggested to be important in inflammatory models and it is probable that neurotrophins also signal to other ligand gated receptors. No doubt future studies will clarify the relative role of these various mediators and the complexity of the interactions of downstream signalling following their activation of the neurones. One important variable may be the nature of inflammation (e.g. cell mediated vs mast cell mediated.), as recently suggested for enteric sensory neurones.2
Ligand gated channels
The Na+ and K+ voltage-gated ion channels on the nerve terminals of DRG neurones are intricately linked to a variety of ligand-gated ion channels. Among these in the GI tract include 5HT3,25 P2X,76,77 ASIC,78 NMDA79 and TRPV180,81 channels. The influx of cations through these receptor–channel complexes acts to depolarize the membrane, in turn activating voltage-gated Na+ and K+ currents. A number of emerging findings are likely to be important in GI inflammatory states. Firstly, many agonists of these receptors are increased in inflammation (e.g. 5-HT, ATP) and these have been implicated in somatic pain models.82,83 Secondly, known inflammatory mediators, acting through cellular second messenger systems have the capability of modulating the function of these channels. An example of this is the recent finding that PAR-2 receptor activation enhances currents carried by the TRPV1 receptor.84,85 In addition, TRPV1 activation is enhanced by other inflammatory substances such as bradykinin86 and lipoxygenase87,88 metabolites. Thirdly, inflammation may upregulate the expression or augments the function of these channels. For example, in somatic models, inflammation increases P2X,89 5HT-390 and TRPV1 expression91,92 while in human inflammatory bowel disease, there is evidence for increased immunoreactivity for TRPV1 and ASIC3 receptors.93,94 Furthermore, Page et al. have shown that P2X receptor activation sensitizes mechanoreceptors in the inflamed, but not in the non-inflamed esophagus.95 The mechanisms underlying increases in ligand-gated ion channel function are unknown, however, there is evidence that exogenous NGF augments both the expression and function of TRPV1 channels,96 suggesting that some of the inflammatory signalling pathways that influence voltage-gated channels, may similarly upregulate these channels.
In summary, inflammation in the GI tract causes hyperexcitability of DRG nociceptive neurones innervating the site of inflammation and as a result, for a given stimulus, results in increased nociceptive trafficking to the CNS. These changes can persist after removal of the inflammatory stimulus and involve alterations in the ionic determinants of neuronal firing, namely voltage-gated Na+ and K+ channels. Of the sodium channels, the TTX-R NaV 1.8 appears to be a particularly attractive target for novel analgesics, given its selective expression in small sensory neurones. These inflammatory changes are significantly different from those resulting from other forms of pain, such as neuropathic pain and while many of these inflammatory changes are similar to those described in inflammatory models outside the GI tract, such as the somatic nervous system, important differences may exist, which might be exploited to further enhance the selectivity of therapeutic agents. The field of GI nociception is rapidly expanding by making use of integrative strategies such as behavioural and electrophysiological techniques combined with modern molecular and transgenic approaches. Using these approaches, there is likely to be a rapid expansion of our knowledge in the near future which should clarify which inflammatory mediators are important in modulating voltage-gated ion channels under specific inflammatory conditions, the relative role of these changes in peripheral sensitization and which downstream pathways are the key signalling events underlying the changes in these channels.
SV is supported by CIHR. MB is supported by a Canadian Association of Gastroenterology/Astra Zeneca/CIHR Fellowship.