Targeting of Transient Receptor Potential Channels in Digestive Disease

Chapter 21


Targeting of Transient Receptor Potential Channels in Digestive Disease



Daniel P. Poole1,2,*; TinaMarie Lieu1; Nicholas A. Veldhuis1,3; Pradeep Rajasekhar1; Nigel W. Bunnett1,4    1 Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia
2 Department of Anatomy & Neuroscience, The University of Melbourne, Parkville, Victoria, Australia
3 Department of Genetics, The University of Melbourne, Parkville, Victoria, Australia
4 Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia
* Corresponding author: Daniel.Poole@monash.edu




Acknowledgments


We thank Professor John Furness and Dr. Hyun Jung Cho (The Department of Anatomy & Neuroscience, The University of Melbourne, Australia) for assistance with figure preparation. Research in the authors’ laboratories has been supported by the National Health & Medical Research Council (Australia), The Australian Research Council, and Monash University.


Overview


Transient receptor potential (TRP) channels are integral components of a network of receptors and ion channels that detect chemical and mechanical stimuli. They are widely expressed throughout the digestive tract, with important roles in taste, visceral sensation, gastrointestinal (GI) motility, as well as absorptive and secretory functions [1]. Dysregulation of these functions through increased expression or aberrant sensitivity also plays a major role in the etiology of digestive diseases. These changes may underlie both the initiation and maintenance of visceral hyperalgesia and inflammation associated with inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), pancreatitis, and functional disorders, including irritable bowel syndrome (IBS; see Figure 21.1). The GI tract is a major source and target of TRP channel activators, including protons, prostaglandin and arachidonic acid derivatives, G protein-coupled receptor (GPCR) agonists, and mechanical stimuli. A wide variety of pungent substances commonly associated with spices are potent TRP channel agonists and are often key active ingredients in traditional treatments for gut-related disorders [3]. This chapter provides an overview of the expression and function of TRP channels in the GI tract in health and disease and discusses the contributions of TRP channels to disease development and progression. Emphasis is placed on disorders related to GI motility, neurogenic inflammation, and sensation. The potential for TRP-targeted therapies in GI disease is discussed in detail where appropriate.


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Figure 21.1 Potential use of TRP channel targeted therapeutics for the treatment of GI diseases and disorders. TRP channels play integral roles in the development and maintenance of pain and neurogenic inflammation, conditions in which TRP channel inhibition is likely to be beneficial. TRP agonists have spasmolytic, prokinetic, and prosecretory effects that may alleviate constipation and symptoms of IBS. See text for details.

Expression of TRP Channels in the Gut and Their Role in GI Motility and Secretion


The GI tract is intrinsically innervated by the enteric nervous system (ENS), comprising two major ganglionated plexuses: the myenteric plexus, which controls motility, and the submucosal plexus, which regulates fluid exchange and blood flow [2]. The gut also receives extrinsic innervation by vagal and spinal nerves that also regulate GI functions, notably sensory signaling [4]. Studies using plant extracts or selective pharmacological tools suggest that TRP channels influence GI function and provide a possible explanation for the anecdotal effects of commonly ingested spices and foods on the gut. The presence of established TRP channel activators in traditional herbal remedies establishes a mechanism through which these may exert their proposed therapeutic actions. Certain plant-derived TRP channel activators alter GI motility and secretion in isolated preparations, including capsaicin [5], piperine [6], carvacrol/thymol [7], allyl isothiocyanate (AITC)/cinnamaldehyde [8], zingerone [9], and menthol [10]. However, the involvement of TRP channels and the molecular targets of many of these compounds in the gut are unresolved or under debate.


To determine the cellular sites of TRP channel-mediated effects, a thorough understanding of the distribution of TRP channels within the gut is required. Key cell types that influence motility include myenteric neurons, extrinsic nerves, smooth muscle of the muscularis externa, interstitial cells of Cajal (ICC), and enteroendocrine cells (see Figure 21.2). The expression of TRP channels by enteric neurons is an area of much debate. At present, there is evidence that TRPV2, TRPA1, and TRPV1 are expressed in the ENS of a number of species, including humans. However, in the case of TRPV1 in particular, there is relatively limited direct functional data to support these findings (see the section “TRPV1”). Moreover, the presence of TRP channel immunoreactivity is not always supported by pharmacological studies using selective agonists and antagonists, and key controls to confirm antibody specificity are often omitted. There is also no definitive evidence for expression of mRNA for TRP channels by enteric neurons.


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Figure 21.2 Potential therapeutic targeting of TRP channels for treatment of GI motility and secretion disorders. TRP channels are expressed by major cell types that regulate motility and secretion, as outlined. TRP agonists acting on these cells have spasmolytic, prokinetic, and prosecretory effects that may alleviate constipation and symptoms of IBS. (1) Enteric neurons: TRPA1, TRPV2, TRPC. (2) Enterochromaffin cells: TRPA1. (3) Smooth muscle: TRPC1, 3, 4, 6; TRPM4. (4) Interstitial cells of Cajal: TRPC1, TRPC2, TRPC4, TRPM7, TRPC4. (5) Epithelial cells: TRPC1; TRPV1, TRPV3, TRPV4, TRPV5, TRPV6; TRPM6, TRPM7. (6) Extrinsic primary afferent neurons: TRPV1, TRPV2, TRPV4; TRPA1, TRPM8; TRPC4. Details are provided in the text. Figure is modified from Ref. [11].

Although the expression of TRP channels in the GI tract and the effects of TRP channel activators and inhibitors on gut function have been widely examined, there is a lack of consistency across these studies. Many of these differences may be attributable to species- or region-specific effects and to differences in methods and selectivity of reagents. The involvement of TRP channels is often implied, but not specifically examined. Moreover, there are a number of examples in which promising results obtained using laboratory species have not yielded equivalent results in clinical trials. Examples include the use of TRPV1 antagonists for alleviation of symptoms of esophageal reflux disease [12,13] and the use of traditional medicines in constipation [14]. An examination of the current literature also highlights the lack of a thorough understanding of the distribution of TRP channels in the human GI tract.


Regulating GI function using TRP activators holds some therapeutic promise, given the fact that many of these compounds are regularly ingested as part of the diet. Most of the current preclinical and clinical studies have focused on the therapeutic application of TRP channel modulators for treatment of constipation-related disorders, functional dyspepsia, and GERD. Notably, much of the current work relates to the validation of traditional remedies and natural products, which may target TRPA1, TRPV1, and TRPM8. These compounds have functional effects in animal and human tissues, in addition to experimental models of digestive disease. However, these studies have not necessarily translated to effectiveness in clinical trials. One remedy that has shown promise is peppermint oil for the treatment of IBS, with a number of clinical studies suggesting efficacy [15]. It should be noted that there is no clear identification of molecular targets underlying these effects of menthol or peppermint oil, although it is likely to be through TRPM8 or TRPA1 and involve a neurogenic mechanism (see the section “TRPM8”). The use of peppermint oil in clinical trials without definitive knowledge of where it acts demonstrates a key difference between studies involving novel synthetic drugs and those of established, commonly used traditional remedies.


TRPA1


TRPA1 is expressed by cell types involved in controlling GI motility including enterochromaffin (EC) cells [8,16] and enteric neurons [17,18]. Importantly, these studies also identify equivalent expression across species, including humans. Studies in isolated intestine have identified two different mechanisms through which AITC may exert its effects, namely, release of 5-hydroxytryptamine (5-HT) from EC cells [8,19] and activation of enteric reflexes [18,20]. Non-TRPA1-dependent effects of AITC have been identified, although the precise molecular targets involved remain undefined [21,22]. The relative role of 5-HT release in stimulating propulsive motility is debatable given that AITC contracts the mucosa-free ileum [22] and recent reexamination of the role of mucosally derived 5-HT in propulsive motility [23].


TRPV1


Most studies conclude that functional TRPV1 expression in the gut is restricted to extrinsic primary afferent neurons of spinal or vagal origin, and that TRPV1 is not expressed by enteric neurons [2426]. The pharmacological effects of capsaicin on intestinal contractility are generally tetrodotoxin resistant and are blocked by neurokinin receptor antagonists, suggesting that they are mediated through substance P (SP) release from extrinsic nerve terminals [5]. Capsaicin simulates secretion in the human colon through the same mechanism [27]. Capsaicin can also inhibit GI contractions through release of nitric oxide (NO), 5-HT, opioids, or calcitonin gene-related peptide (CGRP) from these nerves [28]. Some effects of established TRPV1 activators on intestinal motility may be TRPV1-independent [29], highlighting potential issues with translation from basic science to the clinic, and the importance of studies using human tissues.


TRPV2


TRPV2 is expressed by gastric and intestinal enteric neurons [3032]. TRPV2 activation relaxes the stomach and intestine and promotes GI transit [31]. These studies are limited by agonist specificity, although selective antagonists have been used to confirm TRPV2-dependence. The mechanosensitivity of TRPV2 is probably of most relevance to the GI tract, given that viscera are unlikely to be exposed to noxious heat. TRPV2 has been proposed as a therapeutic target for the treatment of functional dyspepsia associated with impaired gastric adaptive relaxation [32]. However, there are no equivalent preliminary studies to determine the expression and role of TRPV2 in human tissues.


TRPV3


TRPV3 is expressed by colonic epithelial cells, but expression by smooth muscle and the ENS has not been examined [33]. The TRPV3 activators thymol and eugenol stimulate 5-HT release from EC cells [34], with implications for colonic motility and secretion [8]. Active compounds from oregano, thyme, and cloves (carvacrol, thymol, and eugenol) have spasmolytic effects on the GI tract through both direct actions on smooth muscle and via the ENS [35]. Although activation of TRPA1 and TRPV3 by these compounds has since been determined [36], subsequent studies have not been performed to determine the respective role of these channels.


TRPM8


Both a TRPM8GFP reporter mouse and immunofluorescence have been used to define TRPM8 expression in the mouse colon, where it was localized to the epithelium and extrinsic nerve fibers, with little convincing evidence for expression by the ENS [37,38]. Although there is a consistent inhibitory effect of menthol on contractile activity, the molecular target of menthol in the GI tract remains undetermined [10].


TRP Channel Expression in Smooth Muscle and ICC


ICCs, smooth muscle cells, and EC cells also regulate GI motility, and consideration of possible effects on these cells must be given with regard to therapeutic targeting of TRP channels. These cells do not express the major neuronal TRP channels outlined earlier. ICCs are the major interface between the ENS and smooth muscle contraction, and their activity is required for slow wave generation. TRPs act as nonselective cation channels in both ICCs and smooth muscle, where their activity is coupled to GPCRs. TRP expression by different subsets of ICC has been characterized, with TRPC1, 2, and 4, and TRPM4 and TRPM7 identified at the mRNA level [39,40]. Functional expression of TRPC4 [41] and TRPM7 [42] have been confirmed and may play a role in slow wave generation. Smooth muscle cells mainly express TRPC channels [43], which are involved in receptor- and store-operated Ca2 + entry and release. TRPC4 and TRPC6 couple to muscarinic receptor activation; their deletion attenuates muscarinic receptor signaling and delays GI transit [44].


TRP Channels as Targets for Prokinetic Drugs


Constipation may be due to a combination of two factors: reduced or nonpropulsive motility and increased relative absorptive activity. Delayed transit is correlated with an increased time for water absorption, and the two factors are therefore interrelated. Chronic constipation, including constipation-predominant IBS and chronic idiopathic constipation, is poorly managed by current bulk-forming, osmotic, and secretory laxatives [45]. Severe constipation is also a significant limiting side effect of opiate use and affects the majority of patients taking this class of drugs. Regulating GI function using TRP activators certainly holds some promise. However, a more rigorous experimental examination of the proposed actions of these agents and the specific involvement of TRP channels is required before the effectiveness of TRP channel activators in modifying GI motility can be proven.


TRPA1 Agonists


Anecdotal and experimental evidence suggest that TRPA1 agonists are prokinetic and reverse constipation, although specific TRPA1 involvement is unproven [46,47]. Oral administration of AITC restores transit in mouse models of atonic and spastic constipation [47], and activation of TRPA1 on colonic epithelial cells stimulates anion secretion [7,48]. Thus, TRPA1 is an ideal therapeutic target for the treatment of nonobstructive constipation associated with delayed transit or dry stool. Whether TRPA1 activation in the colon is desirable given the proinflammatory and proalgesic effects of AITC remains to be determined.


Dai Kenchu To


There has been renewed interest in the prokinetic actions of the traditional Japanese medicine Dai Kenchu To and its defined commercial formulation TU-100. TU-100 contains established TRPA1 and TRPV1 channel activators, including (6)-shogaol and hydroxyl-β-sanshool [49]. TU-100 has been proposed as a treatment for postoperative ileus, severe constipation, and symptoms of IBS and Crohn’s disease. TU-100 reverses opioid-induced constipation [50,51] and enhances colonic contractions [52]. However, there is no evidence for TRP channel involvement in these effects. Clinical trials have examined the therapeutic potential of TU-100 with limited success, with no significant effect on gastric emptying or on colonic motility [14,53]. The apparent inability to translate data from rodents to humans highlights potential limitations to this experimental approach.


Peppermint Oil


Peppermint oil has been traditionally used to aid digestion, and one of its biologically active constituents is the TRPM8 activator menthol. Despite the prevalent use of peppermint oil to alleviate IBS symptoms and use as a spasmolytic, there have been very few studies of the effects of peppermint oil, menthol, or icillin on GI motility. Several clinical studies have reported effectiveness of peppermint oil as a spasmolytic [15]. Although menthol consistently inhibits GI contractions, the specific involvement of TRP channels has not been examined [10].


Roles of TRP Channels in the Development of IBD


TRP Channels, Neurogenic Inflammation, and Visceral Hypersensitivity


Activation of TRP channels expressed by primary spinal afferents promotes neurogenic inflammation and visceral pain through neuropeptide release. Both TRP function and expression are upregulated in IBD, leading to the enhancement and prolongation of tissue damage and pain. Moreover, there is an increase in the density of TRPV1-positive innervation of the colon in both IBD and IBS. Thus, pharmacological targeting of key TRP channels may attenuate disease-associated proinflammatory and nociceptive signaling. For the sake of brevity, this chapter will focus on TRP channel expression by colonic afferents and expression and functional changes associated with IBD and IBS.


TRPV1


TRPV1 plays a role in both the initiation and maintenance of colitis. Conversely, there is also evidence of a protective role for TRPV1 in experimental colitis. Genetic deletion of TRPV1, TRPV1 inhibition, and afferent desensitization by capsaicin all exacerbate the early, acute phases of colitis [54]. In particular, the TRP channel-dependent release of SP from these terminals is of primary significance [55]. Capsazepine is protective against dextran sulfate sodium (DSS) colitis [56], as is treatment with capsaicin, the latter presumably acting through desensitization of TRPV1 signaling [57]. Long-term inhibition of TRPV1 or TRPA1 attenuates development of chronic colitis [58,59]. Whether these observations translate to human disease remains to be determined.


TRPV4


TRPV4 is expressed by visceral afferents and the colonic epithelium [6063]. TRPV4 is upregulated in colonic epithelial cells in experimental and clinical IBD [6365]. Altered expression of TRPV4 in nerve fibers in disease and increased functional TRPV4 activity in IBD were not specifically examined.


TRPA1


TRPA1 is an attractive target for potential anti-inflammatory therapeutics. Intracolonic administration of mustard oil induces experimental colitis [66], TRPA1 mRNA is upregulated in colitis [66], and endogenous TRPA1 activators are generated in inflamed tissues [67]. TRPA1 promotes, inhibits, or has no effect on experimental colitis [6870].


TRPM8


TRPM8 is expressed by colonic afferents and exposure to the TRPM8 agonist icillin inhibits chemo- and mechanosensory signaling by these neurons [37]. Icillin inhibits capsaicin-stimulated release of CGRP and reduces colitis severity [38]. Moreover, menthol and eucalyptol reduce acute visceral pain primarily through actions at TRPM8, possibly through opioid release [71].


Other TRP Channels


TRPM2 is expressed by monocytes, where it is involved in the release of CXCL8 (Interleukin-8). TRPM2 deletion reduces neutrophil infiltration and associated ulceration in DSS colitis [72]. TRPM2 also plays a role in inflammatory pain signaling, including mechanical and thermal pain [73]. The involvement of TRPM2 in visceral pain signaling is unexplored but is potentially very interesting given the role of TRPM2 in the development of colitis and the importance of mechanosensory input in visceral pain.


Another possible therapeutic target is TRPC4. TRPC4 deletion reduces visceral pain associated with mustard oil-induced colitis [74], although expression of TRPC4 by colonic afferents and a role in neurogenic inflammation has yet to be demonstrated.


TRP Channels and Clinical IBD


The expression and association of TRP channels with clinical IBD and IBS is best characterized for TRPV1, with almost no information on the other TRP channels. TRPV1-immunoreactive nerve fibers and mRNA are increased in IBS, active or quiescent IBD, and rectal hypersensitivity [7579]. The density of TRPV1-specific innervation has been positively correlated with abdominal pain scores [77,78] and with thermal and mechanical sensory thresholds [76]. Increased TRPV1-positive innervation of intestinal blood vessels occurs in Crohn’s disease [80], suggesting a role in IBD-related vascular leak and altered blood flow.


IBS is often associated with hypersensitivity to colorectal distension [81]. Increased innervation density and expression of TRPV1-positive nerve fibers within colon biopsies has been correlated with pain scores [77,78,82]. However, the roles and expression of other TRP channels in the etiology of IBS and IBD in humans remains largely unknown. This will no doubt change, given the emerging and established literature from animal models that have defined a clear role for TRP channels in intestinal inflammation. Whether hyperalgesia and increased sensitivity associated with clinical IBD is simply due to an increase in the extent to which the colon is innervated or also involves TRP sensitization has yet to be established.


Complementary Medicines and IBD: Role for TRP Channels


Complementary and alternative medicines, including herbal medications, are commonly used by IBD patients, primarily stemming from a desire to discontinue steroid treatment [83]. Herbal formulations used to treat IBD commonly contain TRP agonists. Examples include peppermint oil, thyme and oregano oil, and Japanese Kampo. At present only limited studies into their effectiveness and the specific involvement of TRP channels in their effects have been conducted. Many of these compounds can activate multiple TRP channels and other targets, thus caution must be taken when ascribing either a TRP-dependent or TRP-specific mechanism of action. Oregano and thyme oils reduce the severity of 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis in rats and mice. Carvacrol attenuates TNBS colitis in mice through TRPA1-dependent suppression of NO production by macrophages and TRPA1 desensitization [69]. DKT/TU-100 suppresses TNBS colitis through a TRPA1 and adrenomedullin-dependent vasodilatatory action [84,85]. Eugenol protects against gastric ulceration through increased mucus production [86]. An equivalent role in experimental colitis has not been examined in detail. Curcumin, present in turmeric, has protective effects in TNBS colitis that are blocked by capsazepine [87]. However, curcumin had no effect on TRPV1 currents in Xenopus oocytes, and critical experiments to examine curcumin-dependent regulation of TRPV1 activity were not conducted. Thus, at present there is little evidence that the protective effects of curcumin are indeed TRPV1-mediated.


TRP Channel Sensitization in IBS- and IBD-Related Pain


The etiology of IBS is unclear and is confounded by the diverse array of symptoms with which patients may present. Current theories suggest that IBS represents a postinfectious state [88,89] or a result of other stressors [90]. Abdominal pain associated with IBS and IBD is likely to involve hyperexcitability of visceral afferents [81,91]. The contribution of TRPs has recently been reviewed [1,92] and includes TRP sensitization, increased expression by afferents, and enhanced neuropeptide release.


TRP sensitization is characterized by a reduction in activation threshold or augmented responsiveness to established activators or to mechanical distension [1]. TRPs expressed by colonic afferents are sensitized in disease models and following exposure to receptor agonists, including bradykinin [93], 5-HT [9496], histamine [94,95], prostaglandins [95], proteases [62,9799], growth factors [100], and cytokines [101]. In contrast, activation of protease-activated receptor-4 (PAR4) by cathepsin G is analgesic [102,103], suggesting that the balance of inflammatory proalgesic and analgesic agonists determines the extent to which TRP-dependent visceral hypersensitivity develops. Mechanisms underlying TRP channel sensitization include TRP phosphorylation [104], Phosphatidylinositol 4,5-bisphosphate (PIP2) depletion [105], and TRP recruitment to the cell surface [106], leading to rapid, transit upregulation of channel activity.


Hyperexcitability is maintained long after the resolution of inflammation and presumably involves other mechanisms such as TRP channel upregulation or increased nerve fiber sprouting. Elevated expression of TRPV1 and TRPA1 by colonic afferents occurs in experimental colitis [68,100,107109] and IBS/IBD [7578].


Therapeutic Targeting of TRP Channels for Sensory Disorders


Inhibition of TRP channels expressed by afferent terminals may represent a viable therapeutic target and may prove efficacious [12,110]. Alternatively, activation of TRPM8 suppresses colitis and visceral hyperalgesia [38,71]. However, peripheral expression of TRP channels, particularly TRPA1 and TRPV4, is not restricted to afferent terminals (see Figure 21.2), and global inhibition of these channels may have undesirable on-target effects on gut function. Desensitization or functional denervation of visceral afferents (e.g., by capsaicin) is also possible [57] but will effectively eliminate normal sensory signaling from the bowel.


Delivery of RNAi to visceral afferents theoretically enables long-term block of TRP channel expression, eliminating the requirement for regular drug administration. Viral vectors are the most effective means of delivery and could enable selective targeting of afferent neurons. However, selective delivery to these neurons remains a significant challenge [111].


Although intracellular signaling molecules, including protein kinase C (PKC), are key mediators of TRP sensitization [104], therapeutic targeting of kinases is limited by their ubiquitous expression, functional redundancy, and the lack of specificity of inhibitors [112]. Isoform-selective inhibition of PKC is possible and may prove to be more effective [113115] but is limited by drug delivery and duration of action [111]. As our basic understanding of GPCR-TRP channel interactions develops, it is likely that novel kinase targets with more discrete cellular distributions will be identified.


A novel target is the TRP domain, and TRPducin-like peptides derived from this sequence block TRPV1 activation and neuropeptide release, but not responses to mechanical stimuli [116]. The utility of these inhibitors in suppressing visceral pain is untested.


Augmented TRP channel activity and pore dilation may be harnessed to specifically target visceral afferents. The coadministration of both capsaicin and the membrane impermeant voltage-dependent sodium channel inhibitor QX-314 blocks action potential firing exclusively in TRPV1 expressing neurons [117]. However, other TRPs that mediate visceral pain, including TRPA1, are ineffective at delivering QX-314 [118]. To our knowledge the potential for treating chronic visceral pain has yet to be examined.


GPCR agonists that suppress TRP channel activity may be another therapeutic option. PAR4 agonists have protective roles in neurogenic inflammation and pain [103]. Similarly, there is potential for somatostatin receptor-dependent modulation of these channels, particularly TRPV1 [119,120].


Summary


Considerable evidence points to a major role of TRP channels in the regulation of GI functions in health and disease states. TRP channels are expressed by important regulatory cell types, including intrinsic and extrinsic neurons, enteroendocrine cells, myocytes, and ICCs, and activation and inhibition of these channels affects motility, secretion, inflammation, and visceral sensitivity (Table 21.1). TRP channel activity modifiers are potential therapeutics in the GI tract and may account for the beneficial actions of certain traditional and complementary medications. However, although TRP channels are major homeostatic regulators of GI function, contribute to the etiology of digestive disease, and are potential therapeutic targets, many unresolved questions remain. There is a distinct need to translate observations from laboratory species to human subjects. Many reports have failed to use sufficiently selective agonists and antagonists, which has severely limited the utility of these findings. This is particularly the case in those involving herbal remedies, which may contain a number of different TRP channel activators.


Nov 18, 2017 | Posted by in PHARMACY | Comments Off on Targeting of Transient Receptor Potential Channels in Digestive Disease
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