The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli

Abstract

Background & aims: The transient receptor potential (TRP) channel family includes transducers of mechanical and chemical stimuli for visceral sensory neurons. TRP ankyrin 1 (TRPA1) is implicated in inflammatory pain; it interacts with G-protein-coupled receptors, but little is known about its role in the gastrointestinal (GI) tract. Sensory information from the GI tract is conducted via 5 afferent subtypes along 3 pathways.

Methods: Nodose and dorsal root ganglia whose neurons innnervate 3 different regions of the GI tract were analyzed from wild-type and TRPA1(-/-) mice using quantitative reverse-transcription polymerase chain reaction, retrograde labeling, and in situ hybridization. Distal colon sections were analyzed by immunohistochemistry. In vitro electrophysiology and pharmacology studies were performed, and colorectal distension and visceromotor responses were measured. Colitis was induced by administration of trinitrobenzene sulphonic acid.

Results: TRPA1 is required for normal mechano- and chemosensory function in specific subsets of vagal, splanchnic, and pelvic afferents. The behavioral responses to noxious colonic distension were substantially reduced in TRPA1(-/-) mice. TRPA1 agonists caused mechanical hypersensitivity, which increased in mice with colitis. Colonic afferents were activated by bradykinin and capsaicin, which mimic effects of tissue damage; wild-type and TRPA1(-/-) mice had similar direct responses to these 2 stimuli. After activation by bradykinin, wild-type afferents had increased mechanosensitivity, whereas, after capsaicin exposure, mechanosensitivity was reduced: these changes were absent in TRPA1(-/-) mice. No interaction between protease-activated receptor-2 and TRPA1 was evident.

Conclusions: These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.

Figure 1. Expression of TRPA1 in visceral sensory pathways

A. Agarose gel electrophoresis of amplified RT-PCR products from TRPA1 wild-type (+/+) and null-mutant (−/−) mice using primers specific for TRPA1 and β-actin. Amplified TRPA1 transcripts were detected in nodose ganglia (NG), thoracolumbar (TL; T10-L1) and lumbosacral (LS; L6-S1) dorsal root ganglia (DRG) from TRPA1+/+ mice. TRPA1 transcripts were absent in ganglia from TRPA1−/− mice, whereas β-actin was present. (no = no RNA template added). B. Fluorescence in situ hybridization of TRPA1 mRNA expression (red) in TRPA1+/+ nodose ganglia (i), thoracolumbar (ii) and lumbosacral DRG (iii), combined with retrograde labeling of visceral sensory neurons from stomach (i), and distal colon (ii&iii) with CTB-FITC (green). Arrows indicate neurons showing retrograde labeling from the gut: blue arrows indicate those positive for TRPA1, and yellow arrows denote TRPA1-negative neurons. (Scale bar 25μm). C. Quantitative RT-PCR analysis indicated a similar level of TRPA1 transcript expression between whole NG and thoracolumbar and lumbosacral DRG. D. Neuronal counts of the proportion of neurons expressing TRPA1 in either whole ganglia or in retrogradely labeled neurons from viscera. Graphs indicate that a similar proportion of neurons in the general population (non-labeled neurons, open bars) express TRPA1 in the different ganglia. However, significantly more neurons innervating the stomach and colon (filled bars) express TRPA1. (P<0.001 retrogradely labeled gut neurons vs. general population, t-test). E. We have previously shown that ASIC1,2,3 and TRPV4 contribute in various ways to colonic mechanosensory function. To determine if compensatory changes in the expression of these transcripts occurs in TRPA1−/− mice which may explain the changes observed in TRPA1−/− mechanosensory function we performed quantitative RT-PCR analysis. This analysis revealed that there were no significant changes in ASIC1,2,3 or TRPV4 expression between whole TL DRG in TRPA1+/+ and −/− mice. Moreover, there were no significant changes in TRPV1, Bradykinin 2, TRPV2, TREK-1, or NaV1.8 receptor expression.

Figure 2. Expression of TRPA1 protein in peripheral fibers in mouse colon

A. Immunohistochemical analysis of a colonic section from a TRPA1+/+ mouse showing co-localization of the sensory marker TRPA1 exclusively with CGRP in a nerve fiber within the colonic mucosa. B. Wholemount of mesenteric blood vessels from a TRPA1+/+ mouse also showing co-localization of CGRP and TRPA1 in dense nerve fiber networks. C. Wholemount of colonic wall from a TRPA1+/+ mouse showing co-localization of CGRP and TRPA1 in a single colonic afferent with morphology and location consistent with serosal afferents. D. Wholemount of colonic wall from a TRPA1−/− mouse showing that the pattern of CGRP expression shown in C was also observed whereas TRPA1 expression was absent. E. Wholemount of colonic wall from a TRPA1+/+ mouse showing an example of colocalization of CGRP and TRPA1 in mesenteric blood vessels (BV), but not in endings within muscle.

Figure 3. TRPA1−/− mice display selective deficits in visceral afferent function

A Splanchnic i) Mesenteric and ii) Serosal colonic afferents showed dramatically reduced stimulus response functions to focal compression of mechanoreceptive fields with static von Frey hair (vfh) application (70mg–2000mg) in TRPA1−/− mice (P<0.0001, 2-way ANOVA). This was also significant at most individual stimulus intensities (*P<0.05, **P<0.01, ***P<0.001, Bonferroni post-hoc test). B. i) Pelvic serosal colonic afferents were hyposensitive TRPA1−/− mice. ii) Mucosal afferents also displayed reduced stimulus response functions in TRPA1−/− mice. iii) Muscular/mucosal afferents, which respond to both low intensity mucosal stroking and low intensity stretch, only displayed significant deficits in the mucosal component (P<0.001, 2-way ANOVA; *P<0.05, ***P<0.001, Bonferroni test). iv) Mechanosensory function of stretch sensitive muscular afferents was unaltered in TRPA1−/−. Changes in length of tissue in response to stretch (1–15g) were similar in both genotypes (not shown). C. Abdominal EMG responses of conscious mice to colorectal balloon distensions (5×80mmHg) were significantly reduced in TRPA1−/− mice (**N=5, P<0.01), implicating TRPA1 in the signaling of colonic pain. D. Two classes of gastroesophageal vagal afferents were recorded; mucosal and tension receptors. The deletion of TRPA1 caused modest but significant deficits in i) mucosal mechanosensory function (P<0.01, 2-way ANOVA).

Figure 4. TRPA1 agonists induce mechanical hypersensitivity

A. i) In untreated TRPA1+/+ splanchnic colonic afferents the TRPA1 agonists allyl-isothiocyanate (AITC; 40μM) or trans-cinnamaldehyde (TCA; 100μM) induced mechanical hypersensitivity of serosal fibers to a 2000mg vfh, typically by 25%. ii) Mechanical hypersensitivity was absent in TRPA1−/− serosal afferents. iii) The mechanical hypersensitivity evoked by both agonists is significantly enhanced in acute TNBS inflammation. iv) Change in mechanosensory response in inflammation compared with control (calculated from i) & iii). The selective TRPA1 antagonist HC-030031 (10μM for 10mins) had similar effects on mechanosensory responses to gene deletion (not shown) B. In pelvic colonic afferents 100μM TCA also induced significant mechanical hypersensitivity of TRPA1+/+ i) serosal and iii) mucosal colonic afferents which was lost in TRPA1−/− mice (ii & iv).

Figure 5. TRPA1 mediates the bradykinin-induced mechanical hypersensitivity of splanchnic colonic afferents

A. TRPA1+/+ splanchnic colonic serosal afferent responding to bradykinin (1μM; for 2mins). Note increased mechanosensory response to a 2000mg vfh after the chemosensory response to bradykinin. B. TRPA1−/− splanchnic serosal afferent responding to bradykinin (1μM; for 2mins). Note mechanosensory response is unchanged after bradykinin. C. i) TRPA1+/+ serosal fibers that responded to bradykinin subsequently displayed mechanical hypersensitivity (*P<0.05, paired t-test). This effect was not observed in the corresponding TRPA1−/− serosal afferents that responded to bradykinin. ii) Mechanical hypersensitivity after bradykinin application only occurred in TRPA1+/+ afferents that responded to bradykinin and was not observed in unresponsive fibers (NS, P>0.05, paired t-test). D. The chemosensory response bradykinin was similar in TRPA1+/+ and −/− fibers (NS, P>0.05, t-test). E. The proportion of serosal afferents responding to bradykinin was similar in TRPA1+/+ and −/− mice (NS, P>0.05, Fisher’s exact test).

Figure 6. TRPA1 mediates the capsaicin-induced mechanical desensitization of splanchnic colonic afferents

A. TRPA1+/+ splanchnic serosal afferent responding to capsaicin (3μM; 2mins). Note decreased mechanosensory response to 2000mg vfh after chemosensory response to capsaicin. B. TRPA1−/− splanchnic serosal afferent responding to capsaicin (3μM; 2mins). Note mechanosensory response is unchanged after capsaicin. C i) TRPA1+/+ serosal fibers responding to capsaicin subsequently displayed mechanical desensitization (*P<0.05; paired t-test). This effect was not observed in TRPA1−/− serosal afferents that responded to capsaicin (NS, P>0.05; paired t-test). ii) Mechanical desensitization after capsaicin application only occurred in TRPA1+/+ afferents that responded to capsaicin and was not observed in unresponsive fibers. D. Magnitude of chemosensory response to capsaicin was similar in TRPA1+/+ and −/− fibers (NS, P>0.05, paired t-test). E. The proportion of serosal afferents responding to capsaicin in either TRPA1+/+ or −/− mice was unchanged (NS, P>0.05, Fisher’s exact test).