TRPA1 and substance P mediate colitis in mice.

BACKGROUND & AIMS: The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS: We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS: TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS: Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Inhibitory CB1 and activating/desensitizing TRPV1-mediated cannabinoid actions on CGRP release in rodent skin.

Cannabinoid-induced antinociception relies on activation of inhibitory cannabinoid receptors (CB1) in the peripheral and central nervous system. However, most cannabinoids at higher concentration also activate excitatory ionotropic transient receptor potential (TRP) channels coexpressed with CB1 in primary nociceptive neurons that contain and release calcitonin gene-related peptide (CGRP) upon activation. Over a wide concentration range (0.01-100µM) we investigated the molecular action principles of the endocannabinoid anandamide and of the plant-derived Δ(9)-THC that can be prescribed for analgesia. Isolated rat and mouse skin preparations were used to measure CGRP release induced by noxious heat (47°C) and capsaicin (0.5µM), stimuli known to activate the capsaicin receptor TRPV1. At low concentration (0.1µM) both cannabinoids inhibited stimulated CGRP release by 34-65%, which effects were absent under CB1 block by AM 251 and in global CB1 but not TRPV1 knockout mice. At high concentration (100µM) both cannabinoids evoked CGRP release by themselves and desensitized subsequent heat responses, which effects were absent under TRPV1 block by BCTC and in global TRPV1 but not CB1 knockouts. A lower (0.01µM) and the intermediate concentrations (1 and 10µM) of cannabinoids were ineffective. Excitatory and desensitizing effects were not more expressed (disinhibited) in CB1(-/-), inhibitory effects not stronger in TRPV1(-/-). CGRP release induced by unspecific depolarization (KCl) was not modulated by cannabinoids. An incidental finding was that global CB1(-/-) showed reduced heat sensitivity, almost as low as TRPV1(-/-) and in accord with their behavioral phenotype. In conclusion, the antinociceptive potency of peripherally acting CB1 agonists is not restrained by opposing irritant effects through TRPV1 but by their own limited efficacy and narrow concentration-response relationship.

Prostaglandin E(2) and I(2) facilitate noxious heat-induced spike discharge but not iCGRP release from rat cutaneous nociceptors.

The bradykinin-induced sensitization of cutaneous nociceptors to heat was previously shown to be abolished by cyclooxygenase blockade suggesting that endogenous prostaglandins exerted a heat-sensitizing action. The present study aimed at investigating the effects of exogenous prostaglandin E(2) (PGE(2)) and I(2) (PGI(2)) on noxious heat-evoked responses of rat cutaneous nociceptors. As neuropeptides including calcitonin gene-related peptide (CGRP) can be released from the peptidergic subset of heat-sensitive nociceptors, both the spike-generating (afferent) and CGRP-releasing (efferent) responses to heat stimulation were assessed by recording action potentials from single cutaneous C-fibers and measuring immunoreactive CGRP (iCGRP) release from isolated skin flaps, respectively. A combination of PGE(2) and PGI(2) (100 microM for both) unlike 10 microM PGE(2) or PGI(2) increased the number of spikes discharged during a noxious heat stimulus whereas the heat threshold remained unchanged. In contrast, 100 microM PGE(2) plus PGI(2) failed to increase the iCGRP release induced by noxious heat (47 degrees C) from the isolated rat skin. PGE(2) (100 microM), however, augmented the iCGRP-releasing effect of protons (pH 5.7). The adenylyl cyclase activator forskolin and the protein kinase C activator phorbol ester (PMA, 10 microM for both) facilitated heat-induced iCGRP release whereas increasing the intracellular Ca(2+) concentration by 10 microM ionomycin produced a desensitization of the response. In conclusion, PGE(2) plus PGI(2) can sensitize the afferent function of nociceptors in the rat skin, by increasing heat-induced spike discharge, but not the heat-induced efferent response i.e. iCGRP release. This discrepancy might reflect the differences between mechanisms of Na(+) channel-dependent spike generation and Ca(2+)-dependent neuropeptide release.

Bradykinin-induced nociceptor sensitisation to heat depends on cox-1 and cox-2 in isolated rat skin.

Bradykinin is an important inflammatory mediator that can either activate and/or sensitise nociceptors to heat stimuli applied to the skin. Several studies have suggested that prostaglandins and thus the cyclooxygenase (cox) enzymes are important in the sensitisation process but little is known about the relative involvement of the two cox isoforms, cox-1 and cox-2. Extracellular recordings were made from C-mechanoheat-sensitive fibres in isolated rat skin-saphenous nerve preparations. Bradykinin-mediated sensitisation of heat responses in these afferents was significantly attenuated by the selective cox-1 inhibitor, SC-560, and by the selective cox-2 inhibitor, NS-398. In the same experiments, bradykinin-mediated induction of ongoing activity was reduced by SC-560 but not NS-398. In a second series of experiments, bradykinin-stimulated synthesis and release of prostaglandin E2 (PGE2) was measured in isolated skin-nerve preparations. Although the basal release of PGE2 appeared unaffected by either drug, bradykinin-stimulated PGE2 release from the skin was inhibited by both SC-560 and NS-398. Immunocytochemical evaluation revealed cox-1 immunostaining was present in large cutaneous nerve branches, small intradermal nerve bundles as well as nerve endings within the skin. Cox-1 labelling was also present in non-neuronal cell types such as mast cells. Cox-2 immunoreactivity was weak but where present was located in small nerve bundles, smaller intradermal nerve bundles and nerve endings. This study shows that both cox isoforms are present in skin and that they have an important role in mediating bradykinin-evoked heat sensitisation of C-MH sensitive fibres through cox-1 and cox-2 dependent prostaglandin synthesis.

Effects of TRPV1 receptor antagonists on stimulated iCGRP release from isolated skin of rats and TRPV1 mutant mice.

Capsaicin antagonists including ruthenium red, capsazepine and iodo-resiniferatoxin (I-RTX) have recently been shown to inhibit the activation by noxious heat of the capsaicin receptor (TRPV1) expressed in non-neuronal host cells, and natively, in cultured dorsal root ganglion cells. Noxious heat has been shown to release immunoreactive calcitonin gene-related peptide (iCGRP) from the isolated rat skin. In this model, ruthenium red, I-RTX as well as capsazepine 10 microM caused no alteration in iCGRP release at 32 degrees C by themselves whereas capsazepine 100 microM doubled it reversibly. In wild-type mice 100 microM capsazepine also stimulated iCGRP release while it was without effect in TRPV1 knockout littermates. In the rat skin, both ruthenium red and capsazepine (10/100 microM) reduced and abolished, respectively, capsaicin-induced iCGRP release while I-RTX (1/10 microM) was ineffective. Only ruthenium red 100 microM showed an unspecific effect inhibiting iCGRP release induced by KCl. Ruthenium red and capsazepine (10/100 microM) caused no significant alteration of iCGRP release induced by heat stimulation at 47 degrees C. Employing 45 degrees C stimulation intensity, capsazepine and I-RTX (in the higher concentrations) showed a significant facilitatory effect on the heat response suggesting a partial agonistic action of the compounds. It is concluded that noxious heat-induced iCGRP release in the isolated rat skin occurs through a mechanism that is not inhibited by TRPV1 antagonism reflecting a different pharmacological profile of noxious heat transduction in terminals of sensory neurons compared to that in cultured cell bodies and TRPV1-transfected host cells.