TRP Channels in the Viscera

Visceral (nociceptive) stimuli are sensed by a specialized set of neurons with their cell body in the dorsal root ganglion and free sensory nerve endings in the intestinal wall. These nerve terminals reside in a complex signaling environment where they are subjected to mechanical distortion during distension and a changing milieu of neuroactive signaling molecules that can be modulated by stress, immune cells, and the microbiome (20). The peripheral nerve endings in the gut are equipped with numerous receptors and ion channels that allow them to detect and respond to diverse chemical, mechanical, and thermal stimuli. These visceral signals are then transduced to interneurons in the dorsal horn of the spinal cord that transmit the signal to the brainstem and, if intensely enough, to the cortex for conscious perception. The best studied set of molecular sensors are the TRP channels, as they can bind many endogenous lipids and exogenous natural or synthetic compounds (17).

Somatic pain studies repeatedly show that direct activation of TRP channels in sensory nerves triggers protective mechanisms that lead to withdrawal from danger (pain), removal of irritants (itch and cough), and resolution of infection (neurogenic inflammation) (114). These physiologic processes are essential for survival, and are normally under tight control and cease when the initial trigger, for example, inflammation, is removed. However, in the diseased state, longer lasting and sometimes even persistent neuronal hypersensitivity is maintained by TRP channel sensitization (49, 114). This process is characterized by aberrant pain responses to noxious and nonnoxious stimuli, and is a major cause of chronic disorders such as asthma, psoriasis, and FGIDs. The exact mechanisms involved are not fully understood, but it seems that TRP channels act as targets for major downstream effectors of GPCR signaling. Stimulation of GPCR signaling by inflammatory mediators enhances the response to TRP agonists via sensitization, making them very attractive therapeutic targets in various disorders that are characterized by neuronal hypersensitivity.

To date there are 28 TRP genes described in mammals that are grouped into six TRP channel subfamilies: TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), TRPA (ankyrin), TRPML (mucolipin), and TRPP (or PKD, polycystin) (130). TRP channels nonselectively conduct cations and, when activated, lead to increased intracellular Na+ and Ca2+ concentrations, the initiation of neuronal excitation, and a plethora of cellular responses that are relevant to chemo-, thermo-, and/or mechanosensation.

In the gastrointestinal tract, multiple cells express a variety of TRP channels (TRPV1, TRPV3, TRPA1, TRPM2, TRPM5, and TRPM8) that are crucial in tasting seasoned food, thermoregulation of the gut, peristalsis, secretion, mucosal homeostasis, tissue protection, epithelial restitution, controlling of the membrane potential and excitability of neurons, epithelial cells, muscle cells and interstitial cells of Cajal, and visceral sensation (49). Emerging clinical evidence demonstrates aberrant TRP channel expression or function in FGIDs (4, 5, 128), while preclinical models using TRP agonists and transgenic mouse models lacking TRP channels confirm the crucial role of TRP channels in the development and maintenance of colonic afferent hypersensitivity (27, 31, 49, 112). In the following paragraphs, we present an overview of the function and mediators involved in sensitization of TRPV1, TRPV4, TRPA1, TRPM2, and TRPM8 channels in the pathophysiology of VHS as seen in FGIDs.

TRPV1. The best characterized and most studied nociceptor in VHS is TRPV1. TRPV1 is a voltage-gated outwardly rectifying cation channel activated by noxious heat, acidosis (pH < 6) (110), exogenous irritants such as capsaicin (the active component of hot peppers) (25), allyl isothiocyanate (AITC, i.e., mustard oil) (34), and a variety of endogenous lipid compounds, including anandamide (140) and some lipoxygenase metabolites of arachidonic acid (53). In the gastrointestinal tract, TRPV1 is highly expressed by extrinsic sensory neurons and by intrinsic enteric neurons (7, 8, 121). An overview of clinical and preclinical studies providing evidence for the role of TRPV1 in VHS is presented in Table 1. For example, Akbar et al. (4, 5) showed that, in comparison with healthy individuals, quiescent inflammatory bowel disease (IBD) patients with IBS-like symptoms (4) and IBS patients (5) showed increased numbers of TRPV1-positive nerve fibers that correlate with abdominal pain scores. Others provided rather functional evidence for TRPV1 deregulation, as ingestion of capsaicin capsules caused increased pain responses in patients with diarrhea-predominant IBS and FD patients compared with healthy individuals (41, 46, 68). These findings were corroborated by our group; visceral hypersensitive IBS patients, identified by colorectal balloon distention, experienced more pain during rectal application of capsaicin compared with normosensitive patients and healthy individuals (113). Even though hypersensitive patients reported more pain to rectal capsaicin application, rectal TRPV1 mRNA and protein expression was similar between IBS patients and healthy individuals, suggesting that TRPV1 is sensitized rather than upregulated (113). In a follow-up study, TRPV1 responses to capsaicin were indeed potentiated in rectal submucosal neurons of IBS patients but not in healthy subjects (128). Additionally, murine primary sensory DRG neurons revealed an increased capsaicin-induced intracellular Ca2+ response after overnight incubation with rectal biopsy supernatants of IBS patients but not of healthy subjects, indicating that submucosal biopsies of IBS patients release mediators that can sensitize TRPV1 (128). Table 1. Implications of TRPV1 in the pathophysiology of visceral hypersensitivity in FGIDs Tissue (Disease) Species Tissue or Cell Type Technique Result Reference Expression profiles Colon (IBD) Human Rectosigmoid biopsies Immunohistochemistry Increased TRPV1+ nerve fibers 4 Colon (IBS) Human Rectosigmoid biopsies Immunohistochemistry and symptom questionnaires Increased TRPV1+ nerve fibers correlating with abdominal pain 5 Colon (IBS) Human Rectal biopsies Immunohistochemistry, RT-qPCR No upregulation of TRPV1 113 Colon (DSS colitis) Mice HEK-293 cells and dorsal root ganglia Immunohistochemistry, RT-qPCR, Western blot Upregulation TRPV1 by substance P 66 Colon (TNBS colitis) Rats Dorsal root ganglia Immunohistochemistry Increased TRPV1 immunoreactivity 78 Functional data Colon (IBS-D) Human Symptom questionnaires Increased pain sensation to capsaicin capsules 41 Stomach and small intestine (FD) Human Symptom questionnaires Increased pain sensation to capsaicin capsules 46, 68 Colon (IBS) Human Symptom questionnaires Rectal capsaicin application induced increased pain perception. 113 Rectum and colon (IBS) Human and mice Submucosal neurons (human) and dorsal root ganglia (mice) Calcium imaging and symptom questionnaires Increased TRPV1 sensitivity in IBS mediated by histamine and Hrh1. Symptom reduction after treatment with Hrh1 antagonist 128 Colon (DSS colitis) Mice Colorectal distention TRPV1 deficiency prevents postinflammatory VHS 6 Colon Mice Serosal afferent nerves Colorectal distention and afferent nerve recording Inflammatory mediators sensitize TRPV1 resulting in VHS, an effect lacking in TRPV1 knockout mice 56 Colon Mice Dorsal root ganglia Patch-clamp TRPV1 sensitization by 5-HT 106 Colon Rat Maternal separation: colorectal distention VHS after maternal separation reversed by TRPV1 antagonist 112 Colon Rat Dorsal root ganglia Colorectal distention and patch-clamp Depletion 5-HT decreases capsaicin response and VHS 92 The involvement of TRPV1 in VHS has also been demonstrated in various preclinical models of visceral hypersensitivity. For example, increased TRPV1 immunoreactivity was detected in mouse DRG neurons of a post-2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model and was linked to chemical (capsaicin) and mechanical (colonic distention) VHS (78). Moreover, mice deficient in TRPV1 failed to develop postinflammatory VHS following acute colitis induced by dextran sulfate sodium (DSS) (66). Finally, in a rat stress model of maternal separation, visceral hypersensitivity in adult rats was reversed by a TRPV1 antagonist (112), further underscoring the role of TRPV1 in VHS.

TRPV4. The fourth member of the vanilloid subfamily of TRP channels, TRPV4, is a Ca2+-permeable cation channel that has been detected in both sensory and nonsensory cells. In the gastrointestinal tract, TRPV4 has been reported to be primarily expressed on extrinsic afferent nerve fibers and a variety of nonneuronal cells such as epithelial and endothelial cells. Although TRPV4 was originally identified as a channel activated by hypo-osmotic swelling (69, 105, 127), recent evidence indicates that the channel can be activated by diverse stimuli, including shear stress (38), nonnoxious warm temperatures (44, 124), acidity (108), phorbol esters (both protein kinase C-activating and nonactivating phorbol esters) (38, 122, 131), and downstream metabolites of arachidonic acid (epoxyeicosatrienoic acids) (29, 119, 123). Accumulating evidence indicates that TRPV4 activation triggers VHS (overview in Table 2). For example, Cenac et al. (29) elegantly demonstrated that the levels of the TRPV4 agonist 5,6-EET, but not of TRPV1 or TRPA1 agonists, were increased in IBS biopsies compared with controls, and that these increased levels correlated with abdominal pain and bloating scores. Intracolonic infusion of supernatants from IBS biopsies, but not from controls, induced VHS in mice, while knockdown of TRPV4 in mouse primary afferent neurons by siRNA inhibited the hypersensitivity caused by supernatants from IBS biopsies (29). Moreover, polyunsaturated fatty acid metabolites extracted from IBS biopsies or colons of mice with VHS activated mouse sensory neurons in vitro, an effect that was mediated by TRPV4 activation. Intriguingly, the supernatants of IBS biopsies itself did not contain 5,6-EET, but triggered the production of 5,6-EET by mouse sensory neurons via a mechanism that involved the proteinase-activated receptor-2 (PAR-2) and cytochrome epoxygenase (29), indicating that sensory neurons themselves produce TRPV4 agonists upon activation by proteases. Moreover, recently it was shown that human serosal nociceptor mechanosensitivity was attenuated by application of the TRPV4 antagonist HC067047, further underscoring the potential role of TRPV4 in VHS (74). Using live imaging of rectal biopsies, we recently found increased Ca2+ responses to TRPV4 agonist GSK1016790A in submucosal neurons of IBS patients compared with healthy controls, an effect that could be mimicked by histamine in submucosal neurons of healthy subjects. As no increased TRPV4 messenger RNA (mRNA) was found, we hypothesize that TRPV4 is rather sensitized than upregulated (11). Also, in patients suffering from acute IBD, TRPV4 mRNA is highly enriched in colonic sensory neurons (21) and in colonic biopsies obtained from patients with Crohn’s disease and ulcerative colitis compared with healthy subjects (36). Data on TRPV4 expression in IBD patients in remission and suffering from VHS are lacking so far. Table 2. Implications of TRPV4 in the pathophysiology of visceral hypersensitivity in FGIDs Tissue (disease) Species Tissue or cell type Technique Result Reference Expression profiles Colon (IBD) Human Surgical resections and colonic biopsies Immunohistochemistry and RT-qPCR Upregulation TRPV4 in sensory neurons, serosal blood vessels and colonic biopsies 21, 36 Functional data Ilium, colon, and rectum Human Serosal afferent nerves Afferent nerve recording Application of TRPV4 antagonist HC067047 attenuated serosal nociceptor mechanosensitivity 74 Rectum and colon (IBS) Human and mice Submucosal neurons (human) and dorsal root ganglia (mice) Calcium imaging Increased TRPV4 sensitivity in IBS mediated by histamine and Hrh1. 11 Colon Human and mice Colonic biopsies (human) and dorsal root ganglia (mice) Calcium imaging Knockdown of TRPV4 inhibited hypersensitivity caused by supernatants from IBS biopsies 29 Colon (TNBS colitis) Mice Evaluation of pain-related behavior TRPV4 antagonists alleviates colitis and inflammatory pain 36 Colon Mice Colorectal distention Intracolonic administration of TRPV4 agonists induces VHS which was inhibited by TRPV4 siRNA treatment 27 Colon Mice Serosal and mesenteric afferent nerves Colorectal distention and afferent nerve recording TRPV4 knockout mice or treatment with TRPV4 siRNA decreases visceromotor response. Serosal and mesenteric afferent nerves response to TRPV4 agonist 5,6-EET 21 Colon Mice Dorsal root ganglia Colorectal distention and calcium imaging Intracolonic administration of histamine and serotonin potentiated TRPV4-induced VHS, absent mice treated with TRPV4 siRNA. Histamine and serotonin potentiate the TRPV4 response on mouse dorsal root ganglia 28 Colon Mice Dorsal root ganglia Colorectal distention and calcium imaging Intracolonic administration of PAR-2 agonists induces VHS, absent in TRPV4 knockout mice. PAR-2 agonists potentiate the TRPV4 response in mouse dorsal root ganglia 27, 100 Colon Mice Dorsal root ganglia Colorectal distention and calcium imaging Intracolonic PAR-4 agonist inhibits PAR-2 agonist and TRPV4 agonist-induced VHS. PAR-4 agonist inhibited calcium response to PAR-2 and TRPV4 agonist in mouse dorsal root ganglia 10 In addition to the clinical data indicating a potential role for TRPV4 in VHS, various preclinical models already provide functional evidence. Activation of TRPV4 by the TRPV4 agonist 4α-phorbol 12,13-didecanoate (4α-PDD) in colonic projections of DRG neurons induced mechanical VHS in a dose-dependent manner (27). Moreover, mechanosensory responses of colonic serosal and mesenteric fibers were enhanced by the TRPV4 agonist 5,6-EET, and significantly reduced by targeted deletion of TRPV4 or by the TRPV4 antagonist ruthenium red (21). Others showed that intervertebral pretreatment of mice with TRPV4 directed small interfering RNA (siRNA) reduced basal visceral nociception, as well as 4α-PDD agonist-induced hypersensitivity (27). Furthermore, selective blockade of TRPV4 in the TNBS colitis mouse model alleviated colitis and pain associated with acute intestinal inflammation (36). On the basis of these data, TRPV4 seems an important colonic nociceptor that mediates both mechanical and chemical hyperalgesia. Despite these findings, more clinical studies investigating the role of TRPV4 in VHS in IBS, FD, and IBD patients in remission are warranted.

TRPA1. In mammals, TRPA1 is the sole member of the TRPA gene subfamily. TRPA1 is a cold- and mechanosensitive TRP channel activated by cooling to the noxious cold range of temperatures (<17°C) (104). TRPA1 is best known as an irritant sensor and is activated by a wide variety of pungent compounds, such as cinnamaldehyde (12), AITC (57), allicin (15), menthol (59), inflammatory fatty acids, prostaglandin metabolites, and hydrogen peroxide (9, 72). In addition, TRPA1 acts as a sensor of bacterial lipopolysaccharides (77, 102). In the gastrointestinal tract of mammals, TRPA1 has been shown to be expressed on extrinsic primary afferent nerves as well as in intrinsic enteric neurons (84, 104). Besides neuronal cells, TRPA1 is also highly expressed in nonneuronal 5-hydroxytryptamine-releasing enterochromaffin cells (80), cholecystokinin-releasing endocrine cells (91), and intestinal epithelial cells (63). Recent reports identified TRPA1 as a target for the noxious and inflammatory irritant AITC in peripheral sensory neurons, implicating a functional role in pain and neurogenic inflammation (overview in Table 3). Although the majority of the literature on TRPA1 in VHS is based on preclinical studies, a recent study reported upregulation of TRPA1 mRNA expression in biopsies of active IBD patients but not in quiescent IBD patients (65). This effect on acute pain perception was already described by Meseguer et al. (77) who found that TRPA1 channels mediate acute neurogenic inflammation and pain produced by LPS. Also, in mice, intracolonic administration of a TRPA1 agonist increased the visceromotor response, an effect that was absent in TRPA1 deficient mice (19, 26). Others showed upregulation of TRPA1 expression in colonic DRGs of mice suffering from acute TNBS-induced colitis that led to an enhanced visceromotor response to colorectal distention, an effect that was prevented by intrathecal pretreatment with a TRPA1 antisense oligodeoxynucleotide (134) and TRPA1 blockade (115). In addition, the TRPA1 agonist AITC induced colonic hypersensitivity in a mild DSS colitis model that was prevented by treatment with a TRPA1 antagonist (79). Table 3. Implications of TRPA1 in the pathophysiology of visceral hypersensitivity in FGIDs Tissue (disease) Species Tissue or Cell Type Technique Result Reference Expression profiles Colon (IBD) Human Colonic biopsies Immunohistochemistry and RT-qPCR Upregulation TRPA1 65 Colon (mustard oil colitis) Mice Colonic tissue RT-qPCR Upregulation TRPA1 on colonic afferent nerves 61 Colon Mice Dorsal root ganglia Immunohistochemistry Treatment mice pups with TRPA1 agonist increases TRPA1 expression 31 Colon (TNBS) Mice Dorsal root ganglia Upregulation TRPA1 115, 134 Colon Rats Dorsal root ganglia Western blot TRPA1 upregulation in stress-induced VHS 135 Functional data Rectum and colon (IBS) Human and mice Submucosal neurons (human) and dorsal root ganglia (mice) Calcium imaging Increased TRPA1 sensitivity in IBS mediated by histamine and Hrh1. 11 Colon Mice Colorectal distention Intracolonic administration of TRPA1 agonists induces VHS, absent in TRPA1 knockout mice 19, 26 Colon Mice Colorectal distention Treatment mice pups with TRPA1 agonist results in adult VHS 31 Colon Mice Colorectal distention Intracolonic PAR-2 agonist administration induces VHS, absent in TRPA1 knockout mice 26 Colon Mice Serosal and mesenteric afferent nerves Colorectal distention and afferent nerve recording Bradykinin increases mechanosensitivity in afferent nerves and VHS, absent in TRPA1 knockout mice 19 Colon Mice Serosal and mesenteric afferent nerves Afferent nerve recording No interaction of PAR-2 and TRPA1 in splanchnic afferents 19 Colon (TNBS and DSS colitis) Rats and mice Colorectal distention VHS absent in TRPA1 knockout mice and by TRPA1 blockade 26, 79, 115, 134 Besides its role in acute pain perception, preclinical models showed that intracolonic treatment of newborn mouse pups with the TRPA1 agonist AITC triggers a permanent increase in the percentage of TRPA1-positive DRG neurons and results in adult VHS (31). Increased responses of mechanosenstive colonic afferent neurons by TRPA1 agonists has been suggested to result from upregulation of TRPA1 mRNA in a model of mustard oil-induced colitis in mice (61). Moreover, in a model of chronic exposure to water avoidance stress, the increased visceromotor response to colorectal distention correlated with a significant protein upregulation of TRPA1 and TRPV1 in DRG neurons (135), indicative of a crucial role for TRPA1 and TRPV1 in VHS. Indeed, in sensory neurons, TRPA1 has been shown to act in concert with TRPV1 (see details below). Finally, we recently demonstrated an increased TRPA1 agonist-induced Ca2+ response in rectal submucosal neurons of IBS patients compared with those of healthy controls (11). Furthermore histamine was able to potentiate TRPA1 responses in submucosal neurons of healthy subjects. Again, TRPA1 mRNA expression was not upregulated in rectal biopsies of IBS patients compared with healthy individuals, suggesting that also TRPA1 is sensitized in IBS (11). Even though these studies are promising, more clinical studies are required to better understand the role of TRPA1 in VHS in FGIDs.

TRPM2. TRPM2 is a heat-sensitive TRP channel that belongs to the melastatin subgroup of the TRP channel superfamily. It can be activated by intracellular ADP ribose and extracellular stimuli such as reactive oxygen species (47, 85, 125). TRPM2 channels are expressed by intrinsic and spinal primary afferent neurons innervating the distal colon in rat (73). Besides neuronal cells, TRPM2 is also expressed in mucosal macrophages and mast cells and contributes to the progression of experimental colitis and food allergy in mice (81, 133). Several reports show that TRPM2 deficiency has anti-allodynic effects in a wide variety of inflammatory and neuropathic pain mouse models (101), suggesting that TRPM2 may be a new therapeutic target for controlling chronic pain. Furthermore, a recent study found evidence for a role of TRPM2 in visceral nociception and hypersensitivity (73) (overview in Table 4). TRPM2 expression was increased in distal colon of a TNBS colitis rat model, and oral administration of TRPM2 antagonist or TRPM2 deficiency reduced the visceromotor response to noxious colorectal distention in rats. These data suggest that TRPM2 is involved in VHS and may present a novel therapeutic target for VHS triggered by intestinal inflammation. To date clinical studies investigating the role of TRPM2 in visceral pain sensation in FGIDs are completely lacking, but are definitely warranted to establish preclinical evidence. Table 4. Implications of TRPM2 in the pathophysiology of visceral hypersensitivity in FGIDs Tissue (Disease) Species Tissue or Cell Type Technique Result Reference Expression profiles Colon (TNBS- colitis) Rat Distal colon Immunohistochemistry Increased TRPM2 expression 73 Functional data Colon (TNBS- colitis) Rat Distal colon Colorectal distention Treatment with TRPM2 antagonist restores VHS 73