TRPV1 Agonist Capsaicin Research Articles

A novel role for TRPM8 in visceral afferent function

Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperatures and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of bowel hypersensitivity; however, the underlying mechanisms of action are unclear. Here we determined the role of TRPM8 in colonic sensory pathways. Laser capture microdissection, quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and retrograde tracing were used to localise TRPM8 to colonic primary afferent neurons. In vitro extracellular single-fibre afferent recordings were used to determine the effect of TRPM8 channel activation on the chemosensory and mechanosensory function of colonic high-threshold afferent fibres. TRPM8 mRNA was present in colonic DRG neurons, whereas TRPM8 protein was present on nerve fibres throughout the wall of the colon. A subpopulation (24%, n=58) of splanchnic serosal and mesenteric afferents tested responded directly to icilin (5μmol/L). Subsequently, icilin significantly desensitised afferents to mechanical stimulation (P<.0001; n=37). Of the splanchnic afferents responding to icilin, 21 (33%) also responded directly to the TRPV1 agonist capsaicin (3μmol/L), and icilin reduced the direct chemosensory response to capsaicin. Icilin also prevented mechanosensory desensitization and sensitization induced by capsaicin and the TRPA1 agonist AITC (40μmol/L), respectively. TRPM8 is present on a select population of colonic high threshold sensory neurons, which may also co-express TRPV1. TRPM8 couples to TRPV1 and TRPA1 to inhibit their downstream chemosensory and mechanosensory actions.TRPM8 was localised to high-threshold visceral afferent neurons. On visceral afferent peripheral endings, TRPM8 activation affected TRPV1 and TRPA1 downstream chemosensory and mechanosensory actions.

Serotonin enhances CGRP release from female human trigeminal nociceptors

Migraine, a pain disorder mediated by the trigeminal system, is often treatment resistant and more prevalent in women. The neurotransmitter serotonin (5HT) has been implicated in many trigeminal pain states and therapeutics targeting the 5HTsystem are successful in treatingmigraine. Trigeminal pain is often evoked by the transient receptor potential V1 channel (TRPV1), which is expressed by nociceptive trigeminal neurons and induces release of proinflammatory calcitonin gene-related peptide (CGRP). While 5HT attenuates central nociceptive processing, it is pronociceptive in the periphery. In our preclinical models, 5HT evokes thermal hyperalgesia and enhances CGRP release from trigeminal nociceptors. Whether this effect is similar in humans is unknown as no study has evaluated the pharmacology of 5HTon human nociceptors. As dental pulp is densely innervated by trigeminal nociceptors, routine tooth extractions offer a unique opportunity to examine whether 5HT enhances CGRP release from human nociceptors. Pulpal tissue was collected from extracted molar teeth from male and female patients between the ages of 14 and 35 visiting the UT Health Science Center. Following an in vitro superfusion protocol, tissue was washed in buffered saline and basal release samples were collected. Tissue was then treated with saline (n=35) or 100 mM 5HT (n=36) followed by the TRPV1 agonist capsaicin (1 mM) and CGRP release was measured by human CGRP enzyme immunoassay. Additional samples were collected to examine anatomical 5HT receptor expression. We report that 5HT induced a significant increase in CGRP release compared to vehicle controls. In addition,we found that 5HT-induced CGRP release was limited to female trigeminal nociceptors, while there was no significant effect of 5HT in males. These results indicate that 5HT enhances CGRP release from female but not male human trigeminal nociceptors and may provide a mechanistic basis for prevalence of trigeminal pain disorders, such as migraine, in women.

Coupling of Coronary Blood Flow to Cardiac Metabolism: Role of TRPV1 Channels and Local Changes in pH

TRPV1 channels, known to be activated by noxious stimuli such as pH changes, are thought to play a role in the regulation of the vascular tone. Accordingly, we hypothesized that TRPV1 activation by local pH changes during increased metabolic demand and/or ischemia would lead to modulation of myocardial blood flow (MBF). Further we will examine if TRPV1 channel dysfunction could contribute to the microvascular impairment in diabetic cardiomyopathy. To test our hypothesis, control (C57/B6), db/db and TRPV1 null mice (TRPV1−/−) were subjected to contrast stress echocardiography for quantification of MBF at baseline and during the infusion of the TRPV1 agonist capsaicin (CAP; 1–100 μg/kg). Arterial pressures (AP) and heart rate (HR) were simultaneously recordedfor double product (DP) measurement as a surrogate of myocardial oxygen consumption. In controls, CAP produced a dose‐dependent increase in MBF, which was significantly blunted in db/db mice, despite db/db having a higher DP. Low pH changes induced vasodilation in isolated mesenteric microvessels from controls, which was blunted in microvessels from db/db, TRPV1−/− and control after TRPV1 inhibition (SB366791), suggesting a portion of low pH induced vasodilation may be due to TRPV1 activation. We conclude that TRPV1 channels may be involved in the metabolic regulation of myocardial blood flow and this mechanism appears to be corrupted in diabetic mice.

TRESK Channel Contribution to Nociceptive Sensory Neurons Excitability: Modulation by Nerve Injury

BackgroundNeuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K+ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K2P channels after peripheral axotomy in mammals.ResultsHere we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.ConclusionsIn summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.

Endocannabinoid-Dependent LTD in a Nociceptive Synapse Requires Activation of a Presynaptic TRPV-Like Receptor

Recent studies have found that some forms of endocannabinoid-dependent synaptic plasticity in the hippocampus are mediated through activation of transient potential receptor vanilloid (TRPV) receptors instead of cannabinoid receptors CB1 or CB2. The potential role for synaptic localization of TRPV receptors during endocannabinoid modulation of nociceptive synapses was examined in the leech CNS where it is possible to record from the same pair of neurons from one preparation to the next. Long-term depression (LTD) in the monosynaptic connection between the nociceptive (N) sensory neuron and the longitudinal (L) motor neuron was found to be endocannabinoid-dependent given that this depression was blocked by RHC-80267, an inhibitor of DAG lipase that is required for 2-arachidonoyl glycerol (2AG) synthesis. Intracellular injection of a second DAG lipase inhibitor, tetrahyrdolipstatin (THL) was also able to block this endocannabinoid-dependent LTD (ecLTD) when injected postsynaptically but not presynaptically. N-to-L ecLTD was also inhibited by the TRPV1 antagonists capsazepine and SB 366791. Bath application of 2AG or the TRPV1 agonists capsaicin and resiniferatoxin mimicked LTD and both capsaicin- and 2AG-induced depression were blocked by capsazepine. In addition, pretreatment with 2AG or capsaicin occluded subsequent expression of LTD induced by repetitive activity. Presynaptic, but not postsynaptic, intracellular injection of capsazepine blocked both activity- and 2AG-induced ecLTD, suggesting that a presynaptic TRPV-like receptor in the leech mediated this form of synaptic plasticity. These findings potentially extend the role ecLTD to nociceptive synapses and suggest that invertebrate synapses, which are thought to lack CB1/CB2 receptor orthologues, utilize a TRPV-like protein as an endocannabinoid receptor.

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Intranasal TRPV1 agonist capsaicin challenge and its effect on c-fos expression in the guinea pig brainstem

Central neuronal interaction seems to play a role in pathogenesis of upper airway cough syndrome. In the guinea pig model we used the method c-fos expression to identify neurons involved in processing of nociceptive nasal stimuli and their contribution to enhancement of cough. 21 spontaneously breathing, urethane anaesthetized animals were used. The controls received intranasal saline, stimulation group received capsaicin (15 microl, 50 microM), and not-treated group was free of nasal challenge. After 2 h animals were deeply anaesthetized, exsanguinated and transcardially perfused with saline and paraformaldehyde. The brainstems were removed, post-fixed, and slices were processed immunohistochemically for c-fos. In capsaicin group the FLI was detected in the nTs 0.5 mm caudal, 1.5 mm lateral to the obex, the area postrema, LRN and VRG. Intensive FLI was identified in trigeminal nuclear complex. Mean number of FOS positive neurons per section was significantly higher in capsaicin group than that in no-treatment controls or saline controls at the level of obex (p<0.01). Neurons of nTs and VRG clearly activated after nasal provocation may participate in enhancement of cough.

‘TRPing’ Synaptic Ribbon Function in the Rat Pineal Gland: Neuroendocrine Regulation Involves the Capsaicin Receptor TRPV1

Synaptic ribbons (SRs) are presynaptic structures thought to regulate and facilitate multivesicular release. In the pineal gland, they display a circadian rhythm with higher levels at night paralleling melatonin synthesis. To gain more insight into the processes involved and the possible functions of these structures, a series of experiments were conducted in rodents. We studied the regional distribution of a molecular marker of pineal SRs, the kinesin motor KIF3A in the gland. Respective immunoreactivity was abundant in central regions of the gland where sympathetic fibers were less dense, and vice versa, revealing that intercellular communication between adjacent pinealocytes is enhanced under low sympathetic influence. KIF3A was found to be colocalized to the transient receptor potential channel of the vanilloid receptor family, subtype 1 (TRPV1). The TRPV1 agonist capsaicin increased melatonin secretion from perifused pineals in a dose-dependent manner that was blocked by the competitive TRPV1 antagonist capsazepine. No change in free intracellular calcium was observed in response to TRPV1 ligands applied to pinealocytes responding to norepinephrine, bradykinin and/or depolarization. These data clearly indicate that TRPV1 actively regulates pineal gland function.

Anti-nociceptive mechanism of baicalin involved in intervention of TRPV1 in DRG neurons in vitro

Ethnopharmacological relevance Scutellaria baicalensis Georgi (Lamiaceae) is often included as an ingredient in traditional Chinese compound prescriptions for the treatment of fever-related or inflammatory conditions. Aim of the study The present work was to further uncover the analgesic mechanisms of baicalin (a known principal constituent of Scutellaria baicalensis) by investigating its effects on the expression of TRPV1 mRNA as well as on its functions as mediators of calcium entrance into the cytoplasm of dorsal root ganglion (DRG) neurons in vitro. Materials and methods By using CPT as an agent to eliminate the non-neuronal cells and using serum-free neurobasal as culture medium, primary cultures of rat DRG neurons with high purity and viability were established. On this basis, effects of baicalin on both the expression of TRPV1 mRNA and on the function of TRPV1 in vitro under two various temperature conditions were studied. The TRPV1 mRNA expression levels were examined by using qRT-PCR and analyzed by the method of 2 −ΔΔCT. The elevation amplitudes of intracellular [Ca 2+]i evoked by TRPV1 agonist capsaicin in DRG neurons were examined by the calcium fluorescence imaging method under confocal microscopy. Results Baicalin was shown to down-regulate the mRNA expression levels of TRPV1 at both 37 and 39 °C, and under the latter temperature, the intracellular fluorescent intensity evoked by capsaicin was significantly decreased following incubation with baicalin in vitro. We also demonstrated that the actions of baicalin to TRPV1 were not achieved through pathways of TRPA1 or TRPV subfamily members. Conclusions Collectively, these results provide compelling evidence that the down-regulated actions of baicalin to TRPV1 in DRG neurons might account for part of the anti-nociceptive mechanism of baicalin.

The TRPV1 ion channel antagonist capsazepine inhibits osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo

The vanilloid type 1 ion channel (TRPV1) is known to play an important role in the regulation of pain and inflammation. Pharmacological ligands of TRPV1 regulate human osteoclast formation in vitro, but the effects of these agents on osteoblast function have not been studied and their effects on bone loss in vivo are unknown. Here we examined the effects of the TRPV1 ion channel antagonist capsazepine on mouse osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo. Capsazepine inhibited osteoclast formation and bone resorption in a dose dependent manner in bone marrow–osteoblast co-cultures and RANKL generated osteoclast cultures, whereas the TRPV1 agonist capsaicin enhanced RANKL and M-CSF stimulated osteoclast formation. Capsazepine also suppressed RANKL induced IκB and ERK1/2 phosphorylation and caused apoptosis of mature osteoclasts and also inhibited alkaline phosphatase activity and bone nodule formation in calvarial osteoblast cultures. Studies in vivo showed that capsazepine (1 mg/kg/day) inhibited ovariectomy induced bone loss in mice and histomorphometric analysis showed inhibitory effects on indices of bone resorption and bone formation. We conclude that pharmacological blockade of TRPV1 ion channels by capsazepine inhibits osteoclastic bone resorption and protects against ovariectomy induced bone loss in mice, but also inhibits osteoblast activity and bone formation.

Functional Expression of TRPV4 Cation Channels in Human Mast Cell Line (HMC-1).

Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23~25℃ to a moderately high temperature (MHT, 37~39℃ to activate TRPV3/4, a high temperature (HT, 44~46℃ to activate TRPV1, or a very high temperature (VHT, 53~55℃ to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4α-phorbol 12,13-didecanoate (4αPDD, 1µM) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca(2+)](c) of HMC-1 cells was also increased by MHT or by 4αPDD. In summary, our present study indicates that HMC-1 cells express Ca(2+)-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.

Emodin Down-Regulates Expression of TRPV1 mRNA and Its Function in DRG Neurons in Vitro

Emodin is a principle ingredient isolated from rhubarb rhizome, which is commonly used for constipation or pain-related diseases in traditional Chinese medicine (TCM) practice. The transient receptor potential vanilloid 1 ion channel proteins (TRPV1) are abundantly expressed in the peripheral sensory neurons and are assumed to act as a kind of nociceptor involved in the perception of pain and development of hyperalgesia. The aim of this study was to further unravel the analgesic mechanisms of rhubarb through investigating the effects of its main constitutive ingredient emodin on the expression of TRPV1 mRNA as well as on its calcium- mediating functions in vitro. The primary DRG neurons with a high purity and viability were obtained, and the TRPV1 mRNA expression levels were examined by using real-time RT-PCR and the elevated amplitudes of intracellular [Ca(2+)]i in the DRG neurons evoked by TRPV1 agonist capsaicin were examined by confocal microscopy. The results showed that emodin could significantly down-regulate both the mRNA expression of TRPV1 and the capsaicin-evoked intracellular fluorescent intensity in the DRG neurons under both 37 degrees C and 39 degrees C in vitro. Concomitantly, all of the changes induced by emodin could not be blocked by pretreatment of the primary neurons with capsazepine, an antagonist of TRPV1. In conclusion, we established that the mRNA expression level of TRPV1 and its calcium-mediating function in naive DRG neurons could be down-regulated by emodin through perhaps the non-TRPV1 channel pathways, and this might be the molecular mechanisms for rhubarb to inhibit hyperalgesia induced by inflammatory stimuli.

Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells

Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

Optical tracking of phenotypically diverse individual synapses on solitary tract nucleus neurons

The solitary tract nucleus (NTS) is the termination site for cranial visceral afferents–peripheral primary afferent neurons which differ by phenotype (e.g. myelinated and unmyelinated). These afferents have very uniform glutamate release properties calculated by variance mean analysis. In the present study, we optical measured the inter-terminal release properties across individual boutons by assessing vesicle membrane turnover with the dye FM1–43. Single neurons were mechanically micro-harvested from medial NTS without enzyme treatment. The TRPV1 agonist capsaicin (CAP, 100 nM) was used to identify afferent, CAP-sensitive terminals arising from unmyelinated afferents. Isolated NTS neurons retained both glutamatergic and inhibitory terminals that generated EPSCs and IPSCs, respectively. Visible puncta on the neurons were stained positively with monoclonal antibody for synaptophysin, a presynaptic marker. Elevating extracellular K + concentration to 10 mM increased synaptic release measured at individual terminals by FM1–43. Within single neurons, CAP destained some but not other individual terminals. FM1–43 positive terminals that were resistant to CAP could be destained with K + solution. Individual terminals responded to depolarization with similar vesicle turnover kinetics. Thus, vesicular release was relatively homogenous across individual release sites. Surprisingly, conventionally high K + concentrations (> 50 mM) produced erratic synaptic responses and at 90 mM K + overt neuron swelling—results that suggest precautions about assuming consistent K + responses in all neurons. The present work demonstrates remarkably uniform glutamate release between individual unmyelinated terminals and suggests that the homogeneous EPSC release properties of solitary tract afferents result from highly uniform release properties across multiple contacts on NTS neurons.

Calcitonin Gene-Related Peptide Selectively Relaxes Contractile Responses to Endothelin-1 in Rat Mesenteric Resistance Arteries

We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wire-myography, and pharmacological tools. ET(A)- and ET(B)-receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET(B)-receptors, NO-synthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET(A)-antagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 microM) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET(A)-mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K(+) channels.

Functional TRPV4 channels and an absence of capsaicin-evoked currents in freshly-isolated, guinea-pig urothelial cells

Previously we have shown that the transient receptor potential vanilloid 4 (TRPV4) channel regulates urinary bladder function, and that TRPV4 is expressed in both smooth muscle and urothelial cell types within the bladder wall (Thorneloe et al. 2008). Urothelial cells have also been suggested to express TRPV1 channels (Birder et al., 2001). Therefore, we enzymatically isolated guinea-pig urothelial cells in an attempt to record TRPV4 and TRPV1-mediated currents. The identity of the isolated cells was confirmed by quantitative PCR for the urothelial marker uroplakin 1A. Whole-cell patch-clamp recordings with the TRPV4 agonist, GSK1016790A, activated urothelial currents with an EC50 of 11 nM that were completely inhibited by the TRPV4 inhibitor ruthenium red (5 µM). Urothelial currents were also activated by challenge with hypotonic extracellular solution (220 mOsm) known to activate TRPV4 channels. However, the TRPV1 agonist capsaicin, which activated TRPV1 currents in HEK cells expressing TRPV1, was unable to evoke current in these freshly-isolated guinea-pig urothelial cells. We demonstrate that TRPV4 channels are functionally expressed at the plasma membrane of freshly-isolated, guinea-pig urothelial cells, further supporting the important role of TRPV4 in urinary bladder physiology.

A Transient Receptor Potential-Like Channel Mediates Synaptic Transmission in Rod Bipolar Cells

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists L-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1(-/-) mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Activation of Transient Receptor Potential Vanilloid‐1 (TRPV1) Channels in the Rostral Ventrolateral Medulla (RVLM) Produces Renal Sympathoinhibition

The RVLM plays an important role in the regulation of sympathetic nerve activity (SNA) and arterial blood pressure (ABP). The TRPV1 channel is sensitive to temperature, chemical and mechanical stimuli, but recent evidence suggests that TRPV1 channels also modulate neurotransmission. The present study examined whether activation of TRPV1 channels in the RVLM altered SNA and ABP. In urethane‐chloralose anesthetized rats, unilateral injection (60nL) of the TRPV1 agonist capsaicin into the RVLM dose‐dependently decreased renal SNA (0.5nmol: ‐9±1%; 1.0nmol: ‐44±6%; n&gt;4) and mean ABP (0.5nmol: ‐4±1; 1 nmol: ‐15±2 mmHg; n&gt;4). The sympathoinhibitory response to 1nmol capsaicin was eliminated by pretreatment with 600pmol of the TRPV1 antagonist capsazepine (renal SNA: ‐1±1%; mean ABP: 0±0 mmHg, n=6). Blockade of GABA receptors by pretreatment with bicuculline (60pmol) and CGP35348 (400pmol) did not prevent, but rather enhanced the sympathoinhibitory response to 1nmol capsaicin (renal SNA: ‐58±1%; mean ABP: ‐51±3; n=3). Blockade of ionotropic glutamate receptors by injection of kynurenic acid (3nmol) did not alter the capsaicin‐induced decrease in renal SNA (‐42±9%) or mean ABP (‐13±3mmHg, n=4). The present findings suggest that activation of TRPV1 channels in the RVLM inhibit renal SNA and ABP independently of GABAergic or glutamatergic neurotransmission. Supported by NIH HL090826, AHA 0630202N &amp; 0625260B

Cross-inhibition between native and recombinant TRPV1 and P2X 3 receptors

Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X 3 receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X 3 receptors. At a negative holding potential, the P2X 3 receptor agonist α,β-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba 2+-containing superfusion medium, or when the buffering of intrapipette Ca 2+ was altered. The C-terminal truncation at Glu362 of P2X 3 receptors abolished the TRPV1/P2X 3 cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X 3 showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X 3 interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X 3 receptor distal to Glu362.

Nitrooleic Acid, an Endogenous Product of Nitrative Stress, Activates Nociceptive Sensory Nerves via the Direct Activation of TRPA1

Transient Receptor Potential A1 (TRPA1) is a nonselective cation channel, preferentially expressed on a subset of nociceptive sensory neurons, that is activated by a variety of reactive irritants via the covalent modification of cysteine residues. Excessive nitric oxide during inflammation (nitrative stress), leads to the nitration of phospholipids, resulting in the formation of highly reactive cysteine modifying agents, such as nitrooleic acid (9-OA-NO(2)). Using calcium imaging and electrophysiology, we have shown that 9-OA-NO(2) activates human TRPA1 channels (EC(50), 1 microM), whereas oleic acid had no effect on TRPA1. 9-OA-NO(2) failed to activate TRPA1 in which the cysteines at positions 619, 639, and 663 and the lysine at 708 had been mutated. TRPA1 activation by 9-OA-NO(2) was not inhibited by the NO scavenger carboxy-PTIO. 9-OA-NO(2) had no effect on another nociceptive-specific ion channel, TRPV1. 9-OA-NO(2) activated a subset of mouse vagal and trigeminal sensory neurons, which also responded to the TRPA1 agonist allyl isothiocyanate and the TRPV1 agonist capsaicin. 9-OA-NO(2) failed to activate neurons derived from TRPA1(-/-) mice. The action of 9-OA-NO(2) at nociceptive nerve terminals was investigated using an ex vivo extracellular recording preparation of individual bronchopulmonary C fibers in the mouse. 9-OA-NO(2) evoked robust action potential discharge from capsaicin-sensitive fibers with slow conduction velocities (0.4-0.7 m/s), which was inhibited by the TRPA1 antagonist AP-18. These data demonstrate that nitrooleic acid, a product of nitrative stress, can induce substantial nociceptive nerve activation through the selective and direct activation of TRPA1 channels.