Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

Psoralens activate and photosensitize Transient Receptor Potential channels Ankyrin type 1 (TRPA1) and Vanilloid type 1 (TRPV1).

BACKGROUND: PUVA (psoralen UVA) therapy is used to treat a variety of skin conditions, such as vitiligo psoriasis, eczema and mycosis fungoides, but it is frequently accompanied by phototoxicity leading to burning pain, itch and erythema. METHODS: We used a combination of calcium and reactive oxygen species (ROS) imaging, patch clamp and neuropeptide release measurement to investigate whether certain ion channels involved in pain and itch signalling could be responsible for these adverese effects of PUVA. RESULTS: Clinically used psoralen derivatives 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen at physiologically relevant concentrations were able to activate and photosensitize two recombinant thermoTRP (temperature-gated Transient Receptor Potential) ion channels, TRPA1 (Transient Receptor Potential Ankyrin type 1) and TRPV1 (Transient Receptor Potential Vanilloid type 1). 8-MOP enhanced ROS production by UVA light, and the effect of 8-MOP on TRPA1 could be abolished by the antioxidant N-acetyl cysteine and by removal of critical cysteine residues from the N-terminus domain of the channel. Natively expressed mouse TRPA1 and TRPV1 both contribute to photosensitization of cultured primary afferent neurons by 8-MOP, while direct neuronal activation by this psoralen-derivative is mainly dependent on TRPV1. Both TRPA1 and TRPV1 are to a large extent involved in controlling 8-MOP-induced neuropeptide release from mouse trachea. CONCLUSIONS: Taken together our results provide a better understanding of the phototoxicity reported by PUVA patients and indicate a possible therapeutic approach to alleviate the adverse effects associated with this therapy. SIGNIFICANCE: Our work provides evidence for the involvement of thermoTRP channels TRPA1 and TRPV1 in the activation and photosensitization of peripheral nociceptors during PUVA (Psoralen UVA) therapy.

The roles of TRPV1, TRPA1 and TRPM8 channels in chemical and thermal sensitivity of the mouse oral mucosa.

Spices in food and beverages and compounds in tobacco smoke interact with sensory irritant receptors of the transient receptor potential (TRP) cation channel family. TRPV1 (vanilloid type 1), TRPA1 (ankyrin 1) and TRPM8 (melastatin 8) not only elicit action potential signaling through trigeminal nerves, eventually evoking pungent or cooling sensations, but by their calcium conductance they also stimulate the release of calcitonin gene-related peptide (CGRP). This is measured as an index of neuronal activation to elucidate the chemo- and thermosensory transduction in the isolated mouse buccal mucosa of wild types and pertinent knockouts. We found that the lipophilic capsaicin, mustard oil and menthol effectively get access to the nerve endings below the multilayered squamous epithelium, while cigarette smoke and its gaseous phase were weakly effective releasing CGRP. The hydrophilic nicotine was ineffective unless applied unprotonated in alkaline (pH9) solution, activating TRPA1 and TRPV1. Also, mustard oil activated both these irritant receptors in millimolar but only TRPA1 in micromolar concentrations; in combination (1 mm) with heat (45 °C), it showed supraadditive, that is heat sensitizing, effects in TRPV1 and TRPA1 knockouts, suggesting action on an unknown heat-activated channel and mustard oil receptor. Menthol caused little CGRP release by itself, but in subliminal concentration (2 mm), it enabled a robust cold response that was absent in TRPM8-/- but retained in TRPA1-/- and strongly reduced by TRPM8 inhibitors. In conclusion, all three relevant irritant receptors are functionally expressed in the oral mucosa and play their specific roles in inducing neurogenic inflammation and sensitization to heat and cold.

Photosensitization of TRPA1 and TRPV1 by 7-dehydrocholesterol: implications for the Smith-Lemli-Opitz syndrome.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

Crotalphine desensitizes TRPA1 ion channels to alleviate inflammatory hyperalgesia.

Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.

Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity.

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

Photosensitization in Porphyrias and Photodynamic Therapy Involves TRPA1 and TRPV1.

UNLABELLED: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. SIGNIFICANCE STATEMENT: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.

Taurolidine and congeners activate hTRPA1 but not hTRPV1 channels and stimulate CGRP release from mouse tracheal sensory nerves.

Taurolidine has long been in clinical use as an antimicrobial irrigation that does not impede wound healing. It can even be administered intravenously (30 g/day) to treat sepsis or to exert newly recognized antineoplastic actions. Only one irritant effect is reported, that is, to temporarily induce burning pain of unknown origin when applied to body cavities or peripheral veins. The structure of the molecule suggested the chemoreceptor channel TRPA1 as a potential target, which was verified measuring stimulated CGRP release from sensory nerves of the isolated mouse trachea and calcium influx in hTRPA1-transfected HEK293 cells. With both methods, the concentration-response relationship of taurolidine exceeded the threshold value below 500 µmol/L and 100 µmol/L, respectively, and reached saturation at 1 mmol/L. The clinical 2% taurolidine solution did not evoke greater or longer lasting responses. The reversible tracheal response was abolished in TRPA1(-/-) but retained in TRPV1(-/-) mice. Consistently, hTRPV1-HEK showed no calcium influx as a response, likewise native HEK293 cells and hTRPA1-HEK deprived of extracellular calcium did not respond to taurolidine 1 mmol/L. The metabolite taurultam and its oxathiazine derivative, expected to cause less burning pain, showed weak tracheal irritancy only at 10 mmol/L, acting also through hTRPA1 but not hTRPV1. In conclusion, taurolidine, its metabolite, and a novel derivative showed no unspecific cellular effects but selectively, concentration-dependently and reversibly activated the irritant receptor TRPA1 in CGRP-expressing, thus nociceptive, neurons. The clinical solution of 2% taurolidine (~70 mmol/L) can, thus, rightly be expected to cause transient burning pain and neurogenic inflammation.

Cigarette smoke has sensory effects through nicotinic and TRPA1 but not TRPV1 receptors on the isolated mouse trachea and larynx.

Cigarette smoke (CS) exposes chemosensory nerves in the airways to a multitude of chemicals, some acting through the irritant receptors TRPV1 and TRPA1 but potentially also through nicotinic acetylcholine receptors (nAChR). Our aim was to characterize the differences in sensory neuronal effects of CS, gas phase, and particulate matter as well as of typical constituents, such as nicotine and reactive carbonyls. Isolated mouse trachea and larynx were employed to measure release of calcitonin gene-related peptide (CGRP) as an index of sensory neuron activation evoked by CS, by filtered CS gas phase essentially free of nicotine, and by dilute total particulate matter (TPM) containing defined nicotine concentrations. With CS stimulation of the superfused trachea, TRPV1 null mutants showed about the same large responses as wild-type mice, whereas both TRPA1(-/-) and double knockouts exhibited 80% reduction; the retained 20% response was abolished by mecamylamine (10 µM), indicating a distinct contribution of nAChRs. These phenotypes were accentuated by using TPM to stimulate the immersed trachea; 50% of response was retained in TRPA1(-/-) and abolished by mecamylamine. In contrast, the gas phase acted like a sheer TRPA1 agonist, consistent with its composition, among other compounds, of volatile reactive carbonyls like formaldehyde and acrolein. In the trachea, the gas phase and CS were equally effective in releasing CGRP, whereas the larynx showed much larger CS than gas phase responses. Thus nicotinic receptors contribute to the sensory effects of cigarette smoke on the trachea, which are dominated by TRPA1. How this translates to human perception affords future research.

Irritant volatile anesthetics induce neurogenic inflammation through TRPA1 and TRPV1 channels in the isolated mouse trachea.

BACKGROUND: Irritating effects of volatile general anesthetics on tracheal nerve endings and resulting spastic reflexes in the airways are not completely understood with respect to molecular mechanisms. Neuropeptide release and neurogenic inflammation play an established role. METHODS: The basal and stimulated calcitonin gene-related peptide (CGRP) release from the isolated superfused mouse trachea was analyzed as an index of sensory neuron activation, applying irritant (desflurane and isoflurane) and nonirritant (sevoflurane) volatile anesthetics as stimuli. Various gas concentrations (0.5-, 1-, or 2-fold minimum alveolar concentration [MAC]) and different O2 atmospheres were used for tracheal stimulation at 38°C. Null mutants of the capsaicin receptor TRPV1 and of the chemoreceptor TRPA1, as well as double knockout mice, were used as tissue donors. RESULTS: Desflurane and, less so, isoflurane caused a concentration-dependent tracheal CGRP release, both saturating at 1 MAC (human), that is, 6% and 1.25%, respectively. With desflurane, the O2 concentration (25% or 94%) did not make a difference. Sevoflurane 1 MAC did not activate tracheal CGRP release. TRPV1 mice showed 75% reduced desflurane responses, and TRPA1 and double-null mutants showed no responses at all. CONCLUSIONS: Our results confirm the clinical experience that desflurane is more irritating than isoflurane at equal anesthetic gas concentration, whereas sevoflurane does not activate tracheobronchial sensory nerves to release neuropeptides and induce neurogenic inflammation. Both irritant receptor channels, TRPA1 more than TRPV1, are involved in mediating the adverse effects that may even extend to systemic proinflammatory sequelae.

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO-TRPA1-CGRP pathway. We propose that this neuroendocrine HNO-TRPA1-CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons.

High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (-)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, - five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy.

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

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.

TRPV1 controls acid- and heat-induced calcitonin gene-related peptide release and sensitization by bradykinin in the isolated mouse trachea.

Chronic cough derives from inflammatory hypersensitivity of tracheobronchial nerve endings, most of which express the polymodal capsaicin receptor-channel transient receptor potential vanilloid (TRPV) type 1 and the secretory neuropeptide calcitonin gene-related peptide (CGRP). An isolated mouse trachea preparation was established to measure chemically and thermally stimulated CGRP release as an index for sensory transduction of potential cough-inducing stimuli. TRPV1 knockout mice were employed to assess the TRPV1 contribution to tracheal responsiveness and sensitization. Graded heat-induced CGRP release depended entirely on extracellular calcium and partly on TRPV1; knockout mice showed 60% less CGRP release at 45 degrees C (for 5 min) than wild-types. This heat response was facilitated by the TRPV1 agonist ethanol and the TRPV1-3 agonist 2-aminoethoxydiphenyl borate, effects that were reduced or absent in TRPV1(-/-), respectively. The TRPV1 antagonists ruthenium red and N-(4-t-butylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide were ineffective on the basal heat response. A step increase of temperature from 22 to 40 degrees C caused a TRPV1-independent CGRP release that was doubled by bradykinin in wild-types but not TRPV1(-/-). Proton stimulation resulted in a bell-shaped concentration-response curve with threshold at pH 6.7 and a maximum at pH 5.7; responses were greatly reduced but not abolished in TRPV1(-/-). Coadministration of amiloride (30 microm), the blocker of acid-sensing ion channels, was ineffective in both TRPV1 genotypes. The data suggest that tracheal acid sensing mainly involves TRPV1 but not acid-sensing ion channels, whereas noxious heat responsiveness partly depends and (inflammatory) sensitization to heat largely depends on the capsaicin receptor in tracheal nerve endings. Lowering of their heat threshold to near body temperature may sustain hypersensitivity and neurogenic inflammation of the upper airways.

Why cooling is beneficial: non-linear temperature-dependency of stimulated iCGRP release from isolated rat skin.

The capsaicin receptor in nociceptive neurons is a target for the sensitizing actions of algogenic inflammatory mediators. Capsaicin and potential endogenous ligands are thought not to gate this heat-activated ion channel but to sensitize it so profoundly that even room temperature can open it. We investigated the temperature dependency of capsaicin-induced CGRP release from nociceptive nerve fibers in isolated rat skin over a range of ambient temperatures using different agonist concentrations (10(-7)-10(-5)M) and KCl (60 mM) for control. Ambient temperature (4-40 degrees C) showed no significant influence on the basal iCGRP outflow. The supramaximal capsaicin concentration of 10(-6)M as a stimulus evoked a response that was not significantly diminished by temperatures decreasing from 40 to 24 degrees C but lost 65% of its amplitude between 24 and 14 degrees C (Q(10) approximately 6.7). Such a collapse of the response occurred between 40 and 32 degrees C at lower capsaicin concentration (10(-7)M). The concentration-response curves showed a rightward shift upon cooling from 40 to 24 degrees C and a major loss of slope and maximum effect at 14 degrees C which formally describes a noncompetitive antagonism. KCl-induced iCGRP release showed a much more linear temperature dependency (Q(10) approximately 2.4 between 24 and 14 degrees C). Significant capsaicin responses even at 8 degrees C suggest a contribution of noxious-cold sensitive neurons known to coexpress CGRP and the capsaicin receptor. The heat-activated ion channels (TRPV1-4) are thought to play a significant role in inflammatory pain which is effectively relieved by cooling. The present results contribute to understanding this phenomenon.