Activation Of Vanilloid Receptors Research Articles

Identification of a helix-turn-helix motif for high temperature dependence of vanilloid receptor TRPV2.

Pain and thermosensation rely on temperature-sensitive ion channels at peripheral nerve endings for transducing thermal cues into electrical signals. Members of the transient receptor potential (TRP) family are prominent candidates for temperature transducers in mammals. These thermal TRP channels possess an unprecedentedly steep temperature dependence, allowing them to discriminate small temperature variations. Thermodynamically, it is understood that the strong temperature sensitivity of the channel arises because opening of the channel undergoes reactions involving large enthalpy and entropy changes. However, the underlying molecular mechanisms have remained elusive. Here we investigated the molecular basis for heat activation of TRPV2, a thermal TRP channel in the vanilloid subfamily with the strongest temperature dependence among TRP channels. We unravel a minimum molecular region in the proximal N-terminus which dictates the slope temperature sensitivity of the channel. Structurally, the region comprises a helix-turn-helix motif and is positioned among the TRP helix from the C-terminus, the S2-S3 linker from the transmembrane domain and the ankyrin repeats from the distal N-terminus. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results support a pivotal role for the structural assembly around the TRP domain in the gating of thermal TRP channels by temperature. The findings also shed insight into how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors. KEY POINTS: The vanilloid receptor subtype 2 (TRPV2) is a heat-sensitive transient receptor potential (TRP) channel with the strongest temperature dependence among thermal TRP channels. The channel also has a high temperature activation threshold above 50°C which has rendered it difficult to study by conventional patch-clamp methods. Here we utilize fast laser temperature jumps to address the challenges of technical accessibility and explore the molecular basis underlying the high temperature dependence of the channel. We unravel a short helix-turn-helix motif in the proximal N-terminus, which controls the heat activation profile of the channel. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results provide insights on how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors.

11,12-Epoxyeicosatrienoic acid induces vasodilator response in the rat perfused mesenteric vasculature.

Summary Epoxyeicosatrienoic acids (EETs) are endogenous ligands that undergo hydrolysis by soluble epoxide hydrolase (sEH). The responses of 11, 12‐EET in comparison with other vasodilator agonists including carbachol and sodium nitroprusside (SNP) were investigated. The effect of 1‐cyclohexyl‐3‐dodecyl urea (CDU), a sEH, was tested on the vasodilator effect induced by 11, 12‐EET in the perfused mesenteric beds isolated from normo‐glycaemic and type‐1 STZ‐diabetic rats.In the perfused mesenteric beds of control and diabetic animals, 11, 12‐EET produced vasodilation in a dose‐dependent manner. The vasodilator response induced by 11, 12‐EET was significantly decreased in tissues obtained from diabetic animals, but this was significantly corrected through inhibition of sEH.The effects of nitric oxide synthase inhibitor, cyclo‐oxygenase inhibitor, specific potassium channel inhibitors, soluble guanylyl cyclase inhibitor and transient receptor potential channel V4 inhibitor, on vasodilator response to 11, 12‐EET were investigated.In tissues isolated from control animals, vasodilator responses to 11, 12‐EET were not inhibited by acute incubation with l‐NAME, l‐NAME with indomethacin, glibenclamide, iberiotoxin, charybdotoxin, apamin or ODQ.Incubation with the transient receptor potential channel V4 inhibitor ruthenium red caused significantly reduced vasodilator responses induced by 11, 12‐EET.In conclusion, results from this study indicate that 11, 12‐EET has a vasodilator effect in the perfused mesenteric bed, partly through activation of vanilloid receptor. A strategy to elevate the levels of EETs may have a significant impact in correcting microvascular abnormality associated with diabetes.

Suppression of pain‐related behavior in two distinct rodent models of peripheral neuropathy by a homopolyarginine‐conjugated CRMP2 peptide

The N-type voltage-gated calcium channel (CaV2.2) is a clinically endorsed target in chronic pain treatments. As directly targeting the channel can lead to multiple adverse side effects, targeting modulators of CaV2.2 may prove better. We previously identified ST1-104, a short peptide from the collapsin response mediator protein 2 (CRMP2), which disrupted the CaV2.2-CRMP2 interaction and suppressed a model of HIV-related neuropathy induced by anti-retroviral therapy but not traumatic neuropathy. Here, we report ST2-104 -a peptide wherein the cell-penetrating TAT motif has been supplanted with a homopolyarginine motif, which dose-dependently inhibits the CaV2.2-CRMP2 interaction and inhibits depolarization-evoked Ca(2+) influx in sensory neurons. Ca(2+) influx via activation of vanilloid receptors is not affected by either peptide. Systemic administration of ST2-104 does not affect thermal or tactile nociceptive behavioral changes. Importantly, ST2-104 transiently reduces persistent mechanical hypersensitivity induced by systemic administration of the anti-retroviral drug 2',3'-dideoxycytidine (ddC) and following tibial nerve injury (TNI). Possible mechanistic explanations for the broader efficacy of ST2-104 are discussed.

Activation of vanilloid receptor upregulates expression of tight junction protein zonula occludens-1, claudin-3 and claudin-11 in rabbit submandibular gland

Background and Objectives: Tight junction is a structural and functional basis for material transport through paracellular pathway. Our previous studies showed that vanilloid receptor subtype 1 (VR1) was expressed in rabbit submandibular gland (SMG). Activation of VR1 by capsaicin increases saliva secretion of SMG, but its underlying mechanism is unknown. The aim of this study was to explore the effect of VR1 activation on the tight junction protein in rabbit SMG.

Possible Role of Spleen Derived Factors, Vanilloid Receptors and Calcitonin Gene-related Peptide in Diabetes Induced Hyperalgesia in Mice

The present study was designed to investigate a possible role of vanilloid receptors, CGRP and spleen in the induction of diabetes induced hyperalgesia in mice. Tail flick latency, an index of hyperalgesia, was assessed using analgesiometer. Serum nitrite levels and an index of nitric oxide were analyzed using Griess reaction. Mice were rendered diabetic with streptozotocin (200 mg/kg(-1), i.p.) and kept for 30 days for development of diabetic pain. To explore the involvement of spleen in diabetic pain, spleen homogenate supernatant (SHS) was prepared from spleen of 30th day diabetic mice and administered in normal mice for 14 days. Both in diabetic and SHS treated mice, significant degree of hyperalgesia was developed, suggesting the possible role of spleen derived factor in induction of diabetic pain. Moreover, the levels of nitric oxide were also elevated in 30 day diabetic mice and SHS treated mice. Administration of ruthenium red (1 mg/kg(-1), i.p.), vanilloid receptor antagonist, and sumatriptan (50 mg/kg(-1), i.p.), a CGRP release inhibitor, attenuated diabetes and SHS induced decrease in nociceptive threshold and increase in serum nitrite oxide levels. These results suggest that spleen derived factor induced activation of vanilloid receptors and CGRP release may be contributing in the development of hyperalgesia in diabetic mice.

Anandamide inhibits IL-12p40 production by acting on the promoter repressor element GA-12: possible involvement of the COX-2 metabolite prostamide E2

The eCB [endoCB (cannabinoid)] system is being considered as a novel therapeutic target for immune disorders. Cytokines of the IL-12 (interleukin-12) family have essential functions in cell-mediated immunity. In the present study, we have addressed the mechanisms of action of the eCB AEA (anandamide) on the regulation of IL-12p40 in activated microglia/macrophages. We demonstrated that AEA can inhibit the expression of p35, p19 and p40 subunits, which form the biologically-active cytokines IL-12 and IL-23 in microglia stimulated with LPS (lipopolysaccharide)/IFNgamma (interferon gamma). Additionally, we have provided evidence that AEA reduces the transcriptional activity of the IL-12p40 gene in LPS- and IFNgamma-co-activated cells, and this is independent of CB or vanilloid receptor activation. Site-directed mutageneis of the different elements of the p40 promoter showed that AEA regulates IL-12p40 expression by acting on the repressor site GA-12 (GATA sequence in IL-12 promoter). Prostamide E(2) (prostaglandin E(2) ethanolamide), a product considered to be a putative metabolite of AEA by COX-2 (cyclo-oxygenase 2) oxygenation, was also able to inhibit the activity of the IL-12p40 promoter by acting at the repressor site. The effects of AEA and prostamide E(2) on p40 transcription were partially reversed by an antagonist of EP(2) (prostanoid receptor-type 2), suggesting the possibility that prostamide E(2) may contribute to the effects of AEA on IL-12p40 gene regulation. Accordingly, the inhibition of COX-2 by NS-398 partially reversed the inhibitory effects of AEA on IL-12 p40. Overall, our findings provide new mechanistic insights into the activities of AEA in immune-related disorders, which may be relevant for the clinical management of such diseases.

Calcium-dependent desensitization of vanilloid receptor TRPV1: a mechanism possibly involved in analgesia induced by topical application of capsaicin

The rationale for the topical application of capsaicin and other vanilloids in the treatment of pain is that such compounds selectively excite and subsequently desensitize nociceptive neurons. This desensitization is triggered by the activation of vanilloid receptors (TRPV1), which leads to an elevation in intracellular free Ca2+ levels. Depending on the vanilloid concentration and duration of exposure, the Ca2+ influx via TRPV1 desensitizes the channels themselves, which may represent not only a feedback mechanism protecting the cell from toxic Ca2+ overload, but also likely contributes to the analgesic effects of capsaicin. This review summarizes the current state of knowledge concerning the mechanisms that underlie the acute capsaicin-induced Ca2+-dependent desensitization of TRPV1 channels and explores to what extent they may contribute to capsaicin-induced analgesia. In view of the polymodal nature of TRPV1, we illustrate how the channels behave in their desensitized state when activated by other stimuli such as noxious heat or depolarizing voltages. We also show that the desensitized channel can be strongly reactivated by capsaicin at concentrations higher than those previously used to desensitize it. We provide a possible explanation for a high incidence of adverse effects of topical capsaicin and point to a need for more accurate clinical criteria for employing it as a reliable remedy.

Capsaicin-induced death of cultured neurons of the rat hippocampus

We studied the effects of an agonist of vanilloid receptors (VRs), capsaicin, and of an antagonist of VRs of type 1, capsazepin, on cultured neurons of the rat hippocampus. In cultures incubated for 1 day in a medium containing 10 µM capsaicin, the numbers of cytologically normal cells and those with manifestations of necrosis and apoptosis were, on average, 46.4 ± 3.3, 30.7 ± 2.4, and 22.9 ± 5.4%. The latter two values were more than three times greater than the respective indices under control conditions (P < 0.05). Coincubation of the cells with 10 µM capsaicin and 25 µM capsazepin decreased the normalized number of apoptotic units by about one-third, while the number of cells with necrotic changes showed nearly no changes. Using confocal microscopy and staining the cells with a fluorescent dye, JC-1, we found that incubation with capsaicin resulted in a dramatic drop in the mitochondrial potential in the great majority of cultured cells, while capsazepin somewhat smoothed this effect. Thus, our data show that the cytotoxic effect of capsaicin is related to changes in the mitochondrial potential and is at least partially mediated by activation of type-1 VRs.

Depression of Presynaptic Excitation by the Activation of Vanilloid Receptor 1 in the Rat Spinal Dorsal Horn Revealed by Optical Imaging

In this study, we show that capsaicin (CAP) depresses primary afferent fiber terminal excitability by acting on vanilloid receptor 1 (TRPV1 channels) of primary afferent fibers in adenosine 5'-triphosphate (ATP)- and temperature-dependent manner using two optical imaging methods. First, transverse slices of spinal cord were stained with a voltage-sensitive dye and the net excitation in the spinal dorsal horn was recorded. Prolonged treatment (>20 min) with the TRPV1 channel agonist, CAP, resulted in a long-lasting inhibition of the net excitation evoked by single-pulse stimulation of C fiber-activating strength. A shorter application of CAP inhibited the excitation in a concentration-dependent manner and the inhibition was reversed within several minutes. This inhibition was Ca++-dependent, was antagonized by the TRPV1 channel antagonist, capsazepine (CPZ), and the P2X and P2Y antagonist, suramin, and was facilitated by the P2Y agonist, uridine 5'-triphosphate (UTP). The inhibition of excitation was unaffected by bicuculline and strychnine, antagonists of GABAA and glycine receptors, respectively. Raising the perfusate temperature to 39°C from 27°C inhibited the excitation (-3%/°C). This depressant effect was antagonized by CPZ and suramin, but not by the P2X antagonist, 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Second, in order to record the presynaptic excitation exclusively, we stained the primary afferent fibers anterogradely from the dorsal root. CAP application and a temperature increase from 27°C to 33°C depressed the presynaptic excitation, and CPZ antagonized these effects. Thus, this study showed that presynaptic excitability is modulated by CAP, temperature, and ATP under physiological conditions, and explains the reported central actions of CAP. These results may have clinical importance, especially for the control of pain.

Mechanisms of HIV-1 Inhibition by the Lipid Mediator N-Arachidonoyldopamine

Several linear fatty acid dopamides (N-acyldopamines) have been identified recently in the brain. Among them, N-arachidonoyldopamine (NADA) is an endogenous lipid mediator sharing endocannabinoid and endovanilloid biological activities. We have reported previously that NADA exerts some of its biological activities through inhibition of the NF-kappaB pathway and, because this transcription factor plays a key role in HIV-1-long terminal repeat (LTR) trans activation, we have evaluated the anti-HIV-1 activity of NADA. In this study, we show that NADA inhibits vesicular stomatitis virus-pseudotyped HIV-1 infection in the human leukemia T cell line Jurkat, in primary T cells, and in the human astrocytic cell line U373-MG. Other endocannabinoids such as anandamide, 2-arachidonoylglycerol, and noladin ether did not show inhibitory activity in the HIV-1 replication assays. The anti-HIV-1 activity of NADA was independent of known cannabinoid and vanilloid receptor activation. In addition, NADA did not affect reverse transcription and integration steps of the viral cycle, and its inhibitory effect was additive with that of the reverse transcriptase inhibitor azidothymidine. NADA inhibited both TNF-alpha and HIV-1 trans activator protein-induced HIV-1-LTR activation. We also show that NADA counteracts the TNF-alpha-mediated trans activation capacity of the p65 NF-kappaB subunit without affecting its physical association to the HIV-1-LTR promoter. Moreover, NADA inhibited the p65 transcriptional activity by specifically targeting the phosphorylation of this NF-kappaB subunit at Ser(536). These findings provide new mechanistic insights into the biological activities of NADA, and highlight the potential of lipid mediators for the management of AIDS.

The cardiovascular actions of anandamide: more targets?

British Journal of Pharmacology (2005) 145, 565–566. doi:10.1038/sj.bjp.0706234

Evaluation of the anti-emetic potential of anti-migraine drugs to prevent resiniferatoxin-induced emesis in Suncus murinus (house musk shrew)

Activation of vanilloid receptors has commonly been used to facilitate neurogenic inflammation and plasma exudation to model components of the pathogenesis of migraine; however, these studies have been performed mainly in species lacking the emetic reflex. In the present studies, therefore, we used Suncus murinus, a species of insectivore capable of emesis, to investigate if the vanilloid receptor agonist resiniferatoxin is capable of modeling the emesis associated with migraine. Resiniferatoxin (100 nmol/kg, s.c.) induced an emetic response that was antagonized significantly ( P<0.05) by ruthenium red (1–3 μmol), (2 R- trans)-4-[1-[3,5-bis(trifluromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]- N-(2,6-dimethylphenyl)-1-acetamide ( S)-hydroxybutanedioate (R116301; 10–100 μmol/kg), and scopolamine (1 μmol/kg), but not by dihydroergotamine (0.3–3 μmol/kg), sumatriptan (1–10 μmol/kg), methysergide (1–10 μmol/kg), tropanyl 3,5-dichlorobenzoate (MDL72222; 3–30 μmol/kg), ondansetron (0.3–3 μmol/kg), metoclopramide (3–30 μmol/kg), domperidone (3–30 μmol/kg), diphenhydramine (1–10 μmol/kg), or indomethacin (3–30 μmol/kg). The failure of a wide range of representative anti-migraine drugs to reduce retching and vomiting limits the use of this model to identify/investigate novel treatments for the emesis (and nausea) associated with migraine attacks in humans. However, the results provide further evidence for the involvement of a novel vanilloid receptor in resiniferatoxin-induced emesis and implicate both tachykinins and acetylcholine in the pathway(s) activated by resiniferatoxin in S. murinus.

Protective Effects of Evodiamine on Myocardial Ischemia-Reperfusion Injury in Rats

Previous investigations have indicated that the pharmacological activities of evodiamine, a major alkaloidal component of the dried, unripe fruit of Evodia rutaecarpa Bentham (Rutaceae), are associated with the stimulation of calcitonin gene-related peptide (CGRP) release and that CGRP protects the myocardium against ischemia-reperfusion injury. In the present study, we have examined whether evodiamine protects against myocardial ischemia-reperfusion injury in rats and whether the protective effects of evodiamine are related to the activation of capsaicin-sensitive sensory nerves. Rats were pretreated with evodiamine 10 min before the experiment, and then the left main coronary artery of rat hearts was subjected to 60 min occlusion followed by 180 min reperfusion. Infarct size, the activity of serum creatine kinase, serum concentrations of TNF-alpha and plasma concentrations of CGRP were measured. Pretreatment with evodiamine (30 or 60 microg/kg, i.v.) markedly increased the content of CGRP in plasma concomitantly with a significant reduction in infarct size, the activity of serum creatine kinase, and TNF-alpha level, and the effects of evodiamine were completely abolished by capsazepine (5.0 mg/kg, s.c.), a competitive vanilloid receptor antagonist. These results suggest that evodiamine exerts a protection against myocardial ischemia-reperfusion injury in rats and that the protective effects of evodiamine are related to stimulation of CGRP release via activation of vanilloid receptors.

R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression. To address this issue, we tested the influence of methyl-β-cyclodextrin (MCD), a membrane cholesterol depletor, on COX-2 expression by the endocannabinoid analogue R(+)-methanandamide (R(+)-MA). Incubation of H4 cells with MCD was associated with a loss of lipid raft integrity and a substantial inhibition of R(+)-MA-induced COX-2 expression and subsequent formation of prostaglandin E2. Moreover, MCD was shown to suppress signal transduction steps upstream to COX-2 induction by R(+)-MA. Accordingly, the cholesterol depletor suppressed R(+)-MA-induced formation of ceramide as well as phosphorylation of p38 and p42/44 MAPKs. Together, our results suggest that R(+)-MA induces COX-2 expression in human neuroglioma cells via a pathway linked to lipid raft microdomains.

The Vanilloid Receptor as a Putative Target of Diverse Chemicals in Multiple Chemical Sensitivity

The vanilloid receptor (TRPV1 or VR1), widely distributed in the central and peripheral nervous system, is activated by a broad range of chemicals similar to those implicated in Multiple Chemical Sensitivity (MCS) Syndrome. The vanilloid receptor is reportedly hyperresponsive in MCS and can increase nitric oxide levels and stimulate N-methyl-D-aspartate (NMDA) receptor activity, both of which are important features in the previously proposed central role of nitric oxide and NMDA receptors in MCS. Vanilloid receptor activity is markedly altered by multiple mechanisms, possibly providing an explanation for the increased activity in MCS and symptom masking by previous chemical exposure. Activation of this receptor by certain mycotoxins may account for some cases of sick building syndrome, a frequent precursor of MCS. Twelve types of evidence implicate the vanilloid receptor as the major target of chemicals, including volatile organic solvents (but not pesticides) in MCS.

Regulated Exocytosis Contributes to Protein Kinase C Potentiation of Vanilloid Receptor Activity

The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.

Purinergic and vanilloid receptor activation releases glutamate from separate cranial afferent terminals in nucleus tractus solitarius.

Vanilloid (VR1) and purinergic (P2X) receptors are found in cranial afferent neurons in nodose ganglia and their central terminations within the solitary tract nucleus (NTS), but little is known about their function. We mechanically dissociated dorsomedial NTS neurons to preserve attached native synapses and tested for VR1 and P2X function primarily in spindle-shaped neurons resembling intact second-order neurons. All neurons (n = 95) exhibited spontaneous glutamate (EPSCs) and GABA (IPSCs)-mediated synaptic currents. VR1 agonist capsaicin (CAP; 100 nm) reversibly increased EPSC frequency, effects blocked by capsazepine. ATP (100 microm) increased EPSC frequency, actions blocked by P2X antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS; 20 microm). In all CAP-resistant neurons, P2X agonist alphabeta-methylene-ATP (alphabeta-m-ATP) increased EPSC frequency. Neither CAP nor alphabeta-m-ATP altered EPSC amplitudes, kinetics, or holding currents. Thus, activation of VR1 and P2X receptors selectively facilitated presynaptic glutamate release on different NTS neurons. PPADS and 2',3'-O-(2,4,6-trinitrophenyl)-ATP blocked alphabeta-m-ATP responses, but P2X1-selective antagonist NF023 (8,8'-[carbonylbis (imino-3,1-phenylene carbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid) did not. The pharmacological profile and transient kinetics of ATP responses are consistent with P2X3 homomeric receptors. TTX and Cd(2+) did not eliminate agonist-evoked EPSC frequency increases, suggesting that voltage-gated sodium and calcium channels are not required. In nodose ganglia, CAP but not alphabeta-m-ATP evoked inward currents in slow conducting neurons and the converse pattern in myelinated, rapidly conducting neurons (n = 14). Together, results are consistent with segregation of glutamatergic terminals into either P2X sensitive or VR1 sensitive that correspondingly identify myelinated and unmyelinated afferent pathways at the NTS.

The complexities of the cardiovascular actions of cannabinoids.

The cardiovascular actions of cannbinoids are complex. In general they cause vasorelaxation in isolated blood vessels, while in anaesthetised animals they cause multiphasic responses which involve an early bradycardia and long-lasting hypotension. However, in conscious animals, the picture is one of bradycardia followed by pressor responses. Clearly, the responses to cannabinoids are dependent on the experimental conditions and synthetic cannabinoids and endocannabinoids exhibit different pharmacologies. In terms of mechanisms involved in the vascular responses to cannabinoids, the following have been implicated: the involvement of 'classical' cannabinoid receptors, the involvement of a novel endothelial cannabinoid receptor, the release of nitric oxide, the release of endothelium-derived hyperpolarising factor (EDHF), the activation of vanilloid receptors, metabolism of endocannabinoids to vasoactive molecules, and both peripheral inhibition and central excitation of the sympathetic nervous system.

Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons.

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt-uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration-dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 micromoles) for 10 min followed by a 2-min wash did not produce significant change in the capsaicin-induced labelling. Coapplication of insulin (10 micromoles) with capsaicin, however, potentiated the 50 and 100 nm capsaicin-evoked staining. Insulin itself also produced cobalt labelling in a concentration-dependent manner. The size-frequency distributions of neurons showing capsaicin- or insulin-induced cobalt accumulation were similar. The insulin-induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one-third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation- and/or phosphatidylinositol(4,5)biphosphate hydrolysis-evoked activation of the vanilloid receptor 1. Although the insulin-induced activation of vanilloid receptor 1 seems to be a short-lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.

Resiniferatoxin-Induced Loss of Plasma Membrane in Vanilloid Receptor Expressing Cells

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca 2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca 2+ accumulation, but not on cell phenotype.