Vanilloid Agonists Research Articles

Positive Allosteric Modulation of TRPV1 as a Novel Analgesic Mechanism

BackgroundThe prevalence of long-term opiate use in treating chronic non-cancer pain is increasing, and prescription opioid abuse and dependence are a major public health concern. To explore alternatives to opioid-based analgesia, the present study investigates a novel allosteric pharmacological approach operating through the cation channel TRPV1. This channel is highly expressed in subpopulations of primary afferent unmyelinated C- and lightly-myelinated Aδ-fibers that detect low and high rates of noxious heating, respectively, and it is also activated by vanilloid agonists and low pH. Sufficient doses of exogenous vanilloid agonists, such as capsaicin or resiniferatoxin, can inactivate/deactivate primary afferent endings due to calcium overload, and we hypothesized that positive allosteric modulation of agonist-activated TRPV1 could produce a selective, temporary inactivation of nociceptive nerve terminals in vivo. We previously identified MRS1477, a 1,4-dihydropyridine that potentiates vanilloid and pH activation of TRPV1 in vitro, but displays no detectable intrinsic agonist activity of its own. To study the in vivo effects of MRS1477, we injected the hind paws of rats with a non-deactivating dose of capsaicin, MRS1477, or the combination. An infrared diode laser was used to stimulate TRPV1-expressing nerve terminals and the latency and intensity of paw withdrawal responses were recorded. qRT-PCR and immunohistochemistry were performed on dorsal root ganglia to examine changes in gene expression and the cellular specificity of such changes following treatment.ResultsWithdrawal responses of the capsaicin-only or MRS1477-only treated paws were not significantly different from the untreated, contralateral paws. However, rats treated with the combination of capsaicin and MRS1477 exhibited increased withdrawal latency and decreased response intensity consistent with agonist potentiation and inactivation or lesion of TRPV1-containing nerve terminals. The loss of nerve endings was manifested by an increase in levels of axotomy markers assessed by qRT-PCR and colocalization of ATF3 in TRPV1+ cells visualized via immunohistochemistry.ConclusionsThe present observations suggest a novel, non-narcotic, selective, long-lasting TRPV1-based approach for analgesia that may be effective in acute, persistent, or chronic pain disorders.

Vanilloid, purinergic, and CCK receptors activate glutamate release on single neurons of the nucleus tractus solitarius centralis

Baroreceptor inputs to nucleus of the tractus solitarius medialis (mNTS) neurons can be differentiated, among other features, by their response to vanilloid or purinergic agonists, active only on C- or A-fibers, respectively. A major aim of this study was to examine whether neurons of NTS centralis (cNTS), a subnucleus dominated by esophageal inputs, exhibit a similar dichotomy. Since it has been suggested that cholecystokinin (CCK), exerts its gastrointestinal (GI)-related effects via paracrine activation of vagal afferent C-fibers, we tested whether CCK-sensitive fibers impinging upon cNTS neurons are responsive to vanilloid but not purinergic agonists. Using whole cell patch-clamp recordings from cNTS, we recorded miniature excitatory postsynaptic currents (mEPSCs) to test the effects of the vanilloid agonist capsaicin, the purinergic agonist α,β-methylene-ATP (α,β-Met-ATP), and/or CCK-octapeptide (CCK-8s). α,β-Met-ATP, capsaicin; and CCK-8s increased EPSC frequency in 37, 71, and 46% of cNTS neurons, respectively. Approximately 30% of cNTS neurons were responsive to both CCK-8s and α,β-Met-ATP, to CCK-8s and capsaicin, or to α,β-Met-ATP and capsaicin, while 32% of neurons were responsive to all three agonists. All neurons responding to either α,β-Met-ATP or CCK-8s were also responsive to capsaicin. Perivagal capsaicin, which is supposed to induce a selective degeneration of C-fibers, decreased the number of cNTS neurons responding to capsaicin or CCK-8s but not those responding to α,β-Met-ATP. In summary, GI inputs to cNTS neurons cannot be distinguished on the basis of their selective responses to α,β-Met-ATP or capsaicin. Our data also indicate that CCK-8s increases glutamate release from purinergic and vanilloid responsive fibers impinging on cNTS neurons.

The Capsaicin Paradox: Pain Relief by an Algesic Agent

Chemosensitive primary sensory neurones expressing the TRPV1 receptor, a molecular integrator of diverse noxious stimuli, play a fundamental role in the sensation of pain. Capsaicin, the archetypical ligand of the TRPV1 receptor, is one of the most painful chemical irritants, and its acute administration onto the skin and mucous membranes elicits severe pain. However, repeated or high-dose applications of capsaicin, and/or its administration through specific routes dramatically decreases the sensitivity of the innervated tissues to noxious chemical and heat stimuli. This review surveys the mechanisms of the antinociceptive, anti-inflammatory and anti-hyperalgesic effects of vanilloid agonists applied topically, or perineurally, or injected into the subarachnoid space in animal experiments and to put these data into a clinical perspective. The great body of available experimental evidence indicates that vanilloid agonists exert their antinociceptive actions through TRPV1 receptor-mediated selective neurotoxic/neurodegenerative effects directed against somatic and visceral C-fibre nociceptive primary afferent fibres. It is expected that vanilloid agonists will broaden the palette of analgesic drugs which do not cause addiction and tachyphylaxis. Keywords: Analgesia, capsaicin, neurogenic inflammation, pain management, primary sensory neurone, spinal cord, TRPV1, Sensory efferent, Vasodilation

The investigation into indomethacin‐induced potentiation of the contractile response to antigen in ovalbumin‐sensitized guinea‐pig tracheas

In the present study, we investigated the mediators involved in the potentiation of antigen-induced contractions by indomethacin in tracheas isolated from ovalbumin (OA)-sensitized guinea-pigs. Indomethacin-induced potentiation of OA contraction was mimicked by prostaglandin DP/EP(1) /EP(2) receptor antagonist, AH-6809 but not by phospholipase A(2) enzyme inhibitor mepacrine. The lipoxygenase inhibitor AA-861 did not affect the contraction response to OA but prevented its potentiation by indomethacin, while the leukotriene receptor antagonist cinalukast inhibited both the OA response and its potentiation. However, the antagonists of platelet-activating factor (PAF) (BN-52021), adenosine (CGS-15943), endothelin ET(A) and ET(B) receptors (BQ-123, BQ-788), and the neutral endopeptidase inhibitor phosphoramidon did not alter the OA-induced contraction and its potentiation by indomethacin. Furthermore, capsaicin and neuropeptide receptor NK1, NK2, and NK3 antagonists (L-732128, MEN-10376, and SB-218795, respectively) also did not affect the OA-induced contractions and its potentiation. On the other hand, the 'transient receptor potential vanilloid 1' (TRPV1) antagonist capsazepine inhibited the potentiation response, while it did not alter the OA contraction itself. In conclusion, the potentiation of OA-induced contraction by indomethacin is more likely due to the increase in lipoxygenase products by the shift of arachidonic acid towards lipoxygenase pathway. Because some of the lipoxygenase products are potent vanilloid agonists, the stimulation of TRPV1 receptors besides leukotriene receptors seems to participate in the potentiation of contraction response in sensitized guinea-pig tracheas. PAF, adenosine, endothelins, and the neuropeptides present in the afferent neurons do not contribute to the potentiation of OA-induced contraction by indomethacin.

Evaluation of Novel Vanilloid-Based Hemostatic Agents in Rats

: Hemostatic dressings containing clotting factors, biodegradable matrices, and recombinant proteins have been developed to control bleeding for battlefield trauma and trauma in clinical settings. Our present study evaluates the use of a vanilloid compound in biodegradable hemostatic dressings in a rat model of trauma. : Male Sprague-Dawley rats (n = 180) were randomly divided into treatment groups and control groups and subjected to a lethal groin injury at 30 degrees C and 37 degrees C. Treatment groups included hemostatic matrices consisting of Protosan and graded doses of 2.5%, 5%, 10%, 15%, and 20% of the vanilloid agonist CAP-305. Powder or bilayer patch formulations were applied to the injury site. The seal integrity was assessed by reperfusion of the animal to a minimum mean-arterial pressure (MAP) of 80 mm Hg and monitoring for 60 minutes postinjury. : Powder and patch formulations loaded with varying concentrations of CAP-305 were evaluated. Powders containing 2.5% to 20% drug by weight showed 40% to 80% seal rates at 37 degrees C (p < 0.0001), whereas no significant results were obtained at 30 degrees C compared with the control animals. Conversely, bilayer patches loaded with 5% to 20% drug exhibited a consistent 70% seal rate (p < 0.0001) at 37 degrees C and 70% to 90% seal rates (p < 0.0001) in hypothermic animals when compared with controls. : Our study demonstrates the efficacy of CAP-305 loaded hemostatic dressings in the rat model of lethal groin injury. This study provides relevant proof of concept for the development of vanilloid agonists as hemostatic agents.

Opposing effects of cannabinoids and vanilloids on evoked quantal release at the frog neuromuscular junction

Cannabinoids and vanilloids are two distinct groups of substances that share some pharmacological targets. Here we report that two cannabinoid type 1 receptor (CB1) agonists, WIN 55212-2 (WIN) and arachidonyl-2′-chloroethylamide (ACEA) have opposing effects on evoked quantal acetylcholine release – WIN decreased quantal content while ACEA increased quantal content. The decrease in quantal content by WIN was blocked by the CB1 antagonist AM 251. The increase in quantal content by ACEA was not blocked by AM 251, indicating it acts through a receptor other than CB1. As ACEA is also an agonist for the vanilloid receptor (TRPV1) we tested the effect of vanilloids on quantal content. Similar to ACEA, the vanilloid agonist capsaicin increased quantal content, and this effect was blocked by capsazepine, a TRPV1 antagonist. Capsazepine also blocked the increase in quantal content by ACEA. Together these data show an inhibitory effect of CB1 activation on evoked acetylcholine release and the first evidence for the presence of a vanilloid receptor at the neuromuscular junction.

W2040 Responses of Nucleus Tractus Solitarius Neurons to CCK-8s, Vanilloid and Purinergic Agonists

W2040 Responses of Nucleus Tractus Solitarius Neurons to CCK-8s, Vanilloid and Purinergic Agonists

Resiniferatoxin Mediated Ablation of TRPV1+ Neurons Removes TRPA1 as Well

Resiniferatoxin, the most potent agonist of inflammatory pain/vanilloid receptor/cation channel (TRPV1) can be used for neuron subtype specific ablation of pain generating cells at the level of the peripheral nervous system by Ca(2+)-excytotoxicity. Molecular neurosurgery is an emerging technology either to alleviate severe pain in cancer or treat/prevent different local neuropathies. Our aim was determining sensory modalities that may be lost after resiniferatoxin treatment. Newborn or adult mice were treated with resiniferatoxin, then changes in chemical and heat sensitivity were correlated with alterations of the cell composition of sensory ganglions. Only mice treated at adult age became less sensitive to heat stimuli, while both treatment groups lost sensitivity to specific vanilloid agonists of TRPV1 and, interestingly, to allyl-isothiocyanate, a selective agonist of TRPA1. Our in vivo and post mortem analytical results confirmed that TRPV1 and TRPA1 function together and resiniferatoxin-mediated neurosurgery removes both sensor molecules. In adult mice resiniferatoxin causes: i) desensitization to heat and ii) sensitization to cold. Cold hyperalgesia, an imbalance in thermosensation, might be conferred by a prominent cold receptor that is expressed in surviving resiniferatoxin-resistant sensory neurons and compensates for pain signals lost with TRPA1 and TRPV1 double positive cells in the peripheral nervous system.

Contribution of Transient Receptor Potential Vanilloid Subtype 1 to the Analgesic and Antihyperalgesic Activity of Nefopam in Rodents

In order to further elucidate the mechanism(s) of action of analgesic and antihyperalgesic nefopam, its interactions with the transient receptor potential vanilloid subtype 1 (TRPV1) were investigated. In sensory neurons of rat embryos, dorsal root ganglion (DRG) in culture, nefopam (3–30 μmol/l) and capsazepine (TRPV1 antagonist, 10 μmol/l) prevented intracellular calcium elevation and calcitonin gene-related peptide release induced by vanilloid agonist capsaicin. Unlike nefopam, capsazepine failed to inhibit these same responses induced by KCl excess. In vivo, nefopam (0.5 and 2 mg/kg, i.v.) and capsazepine (40 mg/kg, i.p.) reduced the licking response due to intraplantar injection of capsaicin in mice. These findings suggest that nefopam exerts its analgesic and antihyperalgesic effects through multiple mechanisms including blockade of TRPV1 in addition to voltage-dependent calcium channels in the DRG.

Structure–activity relationships of 1,4-dihydropyridines that act as enhancers of the vanilloid receptor 1 (TRPV1)

Vanilloid agonists such as capsaicin activate ion flux through the TRPV1 channel, a heat- and ligand-gated cation channel that transduces painful chemical or thermal stimuli applied to peripheral nerve endings in skin or deep tissues. We have probed the SAR of a variety of 1,4-dihydropyridine (DHP) derivatives as novel ‘enhancers’ of TRPV1 activity by examining changes in capsaicin-induced elevations in 45Ca 2+-uptake in either cells ectopically expressing TRPV1 or in cultured dorsal root ganglion (DRG) neurons. The enhancers increased the maximal capsaicin effect on 45Ca 2+-uptake by typically 2- to 3-fold without producing an action when used alone. The DHP enhancers contained 6-aryl substitution and small alkyl groups at the 1 and 4 positions, and a 3-phenylalkylthioester was tolerated. Levels of free intracellular Ca 2+, as measured by calcium imaging, were also increased in DRG neurons when exposed to the combination of capsaicin and the most efficacious enhancer 23 compared to capsaicin alone. Thus, DHPs can modulate TRPV1 channels in a positive fashion.

Vanilloid-Induced Conduction Analgesia: Selective, Dose-Dependent, Long-Lasting, with a Low Level of Potential Neurotoxicity

Vanilloid agonists (capsaicin, resiniferatoxin, [RTX]) applied to the peripheral nerves provide conduction blockade. In contrast to the analgesic component of conduction anesthesia produced by local anesthetics, vanilloid agonists provide conduction analgesia not associated with suppression of motor or sensory functions not related to pain. Vanilloid agonists provide conduction analgesia selectively because their effect on the nerve trunks is limited to C- and ADelta-fibers. RTX is much more potent than capsaicin and has a wider therapeutic window. In rat experiments, perineural RTX produced a long-lasting thermal and mechanical hypoalgesia with a very wide separation between effective concentrations (from 0.00003% to 0.001%) providing an effect lasting from several hours to several weeks. A nerve block with RTX prevented the development of thermal and mechanical hyperalgesia as well as pain behavior in a model of incisional pain. RTX-induced conduction blockade has an inherent drawback of TRPV1 agonists, the initial excitation (pain); therefore, a local anesthetic should be injected to prevent it. When RTX was applied to the rat's sciatic nerve in doses necessary to provide conduction analgesia, the frequency of unmyelinated fiber degeneration was more than an order of magnitude lower than that with the therapeutic concentration of lidocaine. These promising results should be confirmed by experiments in species other than rodents (pigs, sheep). Taken together, the data indicate possible clinical applicability of vanilloid-induced conduction analgesia.

Peripheral inflammation increases Scya2 expression in sensory ganglia and cytokine and endothelial related gene expression in inflamed tissue

The sensation of pain (nociception) is a critical factor in host defense during tissue injury and inflammation and is initiated at the site of injury by activation of primary afferent C-fiber and A- partial differential nerve endings. Inflammation induces tissue alterations that sensitize these nociceptive nerve terminals, contributing to persistent pain. To understand this 'algesic tissue environment' and peripheral nervous signaling to the CNS and immune system, we examined cytokine and endothelial-related gene expression profiles in inflamed rat tissues and corresponding dorsal root ganglia (DRG) by microarray and RT-PCR following hind paw injection of carrageenan. In inflamed tissue, forty-two cytokine and endothelial-related genes exhibited elevated expression. In contrast, in DRG, only Scya2 (chemokine C-C motif ligand 2) mRNA was up-regulated, leading to an increase in its gene product monocyte chemoattractant protein-1. Scya2 mRNA was localized by in situ hybridization-immunocytochemical double-labeling to a subpopulation of vanilloid receptor-1 (transient receptor potential vanilloid subtype 1) containing neurons, and its expression was increased by direct transient receptor potential vanilloid subtype 1 stimulation with the vanilloid agonist resiniferatoxin, indicating sensitivity to nociceptive afferent activity. Our results are consistent with the idea that monocyte chemoattractant protein-1 at the site of peripheral injury and/or in DRG is involved in inflammatory hyperalgesia.

TRPV1 is a novel target for omega‐3 polyunsaturated fatty acids

Omega-3 (n-3) fatty acids are essential for proper neuronal function, and they possess prominent analgesic properties, yet their underlying signalling mechanisms are unclear. Here we show that n-3 fatty acids interact directly with TRPV1, an ion channel expressed in nociceptive neurones and brain. These fatty acids activate TRPV1 in a phosphorylation-dependent manner, enhance responses to extracellular protons, and displace binding of the ultrapotent TRPV1 ligand [3H]resiniferatoxin. In contrast to their agonistic properties, n-3 fatty acids competitively inhibit the responses of vanilloid agonists. These actions occur in mammalian cells in the physiological concentration range of 1-10 mum. Significantly, docosahexaenoic acid exhibits the greatest efficacy as an agonist, whereas eicosapentaenoic acid and linolenic acid are markedly more effective inhibitors. Similarly, eicosapentaenoic acid but not docosahexaenoic acid profoundly reduces capsaicin-evoked pain-related behaviour in mice. These effects are independent of alterations in membrane elasticity because the micelle-forming detergent Triton X-100 only minimally affects TRPV1 properties. Thus, n-3 fatty acids differentially regulate TRPV1 and this form of signalling may contribute to their biological effects. Further, these results suggest that dietary supplementation with selective n-3 fatty acids would be most beneficial for the treatment of pain.

Modulation of human TRPV1 receptor activity by extracellular protons and host cell expression system

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand-gated cation channel that can be activated by capsaicin, heat, protons and cytosolic lipids. We compared activation of recombinant human TRPV1 receptors stably expressed in human 293 cells, derived from kidney embryonic cells, and in human 1321N1 cells, derived from brain astrocytes. Cellular influx of calcium was measured in response to acid, endovanilloids ( N-arachidonoyl-dopamine, N-oleoyl-dopamine and anandamide), capsaicin and other traditional vanilloid agonists under normal (pH 7.4) and acidic (pH 6.7 and 6.0) assay conditions. The host cell expression system altered the agonist profile of endogenous TRPV1 receptor agonists without affecting the pharmacological profile of either exogenous TRPV1 receptor agonists or antagonists. Our data signify that the host cell expression system plays a modulatory role in TRPV1 receptor activity, and suggests that activation of native human TRPV1 receptors in vivo will be dependent on cell-specific regulatory factors/pathways.

Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83–90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca 2+] i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca 2+] i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE ≫ NADA), low pH and noxious heat (>42 °C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.

Enhancement of Spontaneous and Heat-Evoked Activity in Spinal Nociceptive Neurons by the Endovanilloid/Endocannabinoid N-Arachidonoyldopamine (NADA)

N-arachidonoyldopamine (NADA) is an endogenous molecule found in the nervous system that is capable of acting as a vanilloid agonist via the TRPV1 receptor and as a cannabinoid agonist via the CB1 receptor. Using anesthetized rats, we investigated the neural correlates of behavioral thermal hyperalgesia produced by NADA. Extracellular single cell electrophysiology was conducted to assess the effects of peripheral administration of NADA (i.pl.) on nociceptive neurons in the dorsal horn of the spinal cord. Injection of NADA in the hindpaw caused increased spontaneous discharge of spinal nociceptive neurons compared with injection of vehicle. The neurons also displayed magnified responses to application of thermal stimuli ranging from 34 to 52 degrees C. NADA-induced neural hypersensitivity was dose dependent (EC50 = 1.55 microg) and TRPV1 dependent, as the effect was abolished by co-administration of the TRPV1 antagonist 5'-iodoresiniferatoxin (I-RTX). In contrast, co-administration of the CB1 antagonist SR 141716A did not attenuate this effect. These results suggest that the enhanced responses of spinal nociceptive neurons likely underlie the behavioral thermal hyperalgesia and implicate a possible pain-sensitizing role of endogenous NADA mediated by TRPV1 in the periphery.

The Effects of Intraarticular Resiniferatoxin in Experimental Knee-Joint Arthritis

In this study we sought to determine whether an intraarticular administration of a vanilloid agonist resiniferatoxin (RTX) produces an analgesic effect in experimental arthritis. Knee joint inflammation was induced in rats by intraarticular carrageenan (2%, 30 microL). Pain score and left/right hind leg weight distribution ratio were used to assess pain behavior. Changes in knee dimensions were evaluated by measuring external circumference and intraarticular area (ultrasound scanning). The intraarticular administration of RTX (0.0003% or 0.003%, 30 microL) provided a significant analgesic effect. Twenty-four hours after RTX administration, the pain score was reduced from 15.1 +/- 4.7 to 6.9 +/- 4.4 (P < 0.01) with 0.0003% and was abolished (P < 0.0001) with 0.003%. The improvement in weight distribution ratio lasted for several days after the RTX administration. Reduction in knee circumference demonstrated that intraarticular RTX suppressed the carrageenan-induced edema by at least one third. Ultrasound scanning revealed no RTX-induced decrease of the intraarticular area. The experiments demonstrated that intraarticular RTX inhibits pain behavior in knee-joint arthritis and that this effect is dose-dependent. These results suggest a new direction for peripheral analgesia.

Pharmacological interactions between calcium/calmodulin-dependent kinase II α and TRPV1 receptors in rat trigeminal sensory neurons

Multiple lines of evidence suggest that calcium/calmodulin-dependent kinase II α (CaMKIIα) plays an important role in the spinal dorsal horn in nociceptive models of chemical, inflammatory and nerve injury. Moreover, CaMKIIα phosphorylates the vanilloid receptor type 1 (TRPV1), thereby regulating vanilloid agonist binding to the receptor. Herein, we have explored a possible interaction of CaMKIIα activity with the TRPV1 receptor in rat trigeminal ganglion (TG) neurons in vitro. Inhibition of CaMKIIα with KN-93 (5 μM) inhibited capsaicin (CAP)- and n-arachidonoyl-dopamine (NADA)-evoked calcitonin gene-related peptide (CGRP) release effectively decreasing the E max for both compounds. This effect was not mimicked by the inactive compound KN-92 (5 μM), indicating that the effect was mediated by CaMKIIα inhibition. CAP also stimulated a significant ∼50% increase in autophosphorylation of CaMKIIα at Thr 286/287. Immunocytochemistry for phospho-CaMKIIα indicated that this effect specifically occurred in TRPV1-positive TG neurons. These findings indicate that phopho-CaMKIIα is likely to play a role in presynaptic primary afferents in animal models of nociceptive hypersensitivity and provide support for CaMKIIα modulation of TRPV1 activity in sensory neurons.

Regulation of TRPV1 by a novel renally expressed rat TRPV1 splice variant

The capsaicin receptor and transient receptor potential channel TRPV1 senses heat, protons, and vanilloid agonists in peripheral sensory ganglia. Abundant data have suggested the presence of potentially novel splice variants in the kidney. We report a novel rat TRPV1 splice variant, TRPV1(VAR), cloned from kidney papilla. TRPV1(VAR) cDNA was identified in multiple kidney tissues. Its sequence was fully compatible with potential splice donor and acceptor sites in the rat TRPV1 gene. TRPV1(VAR) is predicted to encode a truncated form of TRPV1 consisting of the NH2-terminal 248 residues of TRPV1 (all within the NH2-terminal intracellular domain) followed by five nonconsensus amino acids (Arg-Glu-Ala-Met-Trp) and a stop codon. The variant utilizes the same consensus Kozak sequence as canonical TRPV1. A band of the appropriate molecular mass was identified in rat kidney papillary (but not medullary) lysates immunoblotted with an antibody directed against the NH2 terminus of TRPV1, whereas an antibody recognizing the TRPV1 COOH terminus failed to detect it. Upon heterologous expression in HEK 293 cells, TRPV1(VAR) potentiated the ability of cotransfected TRPV1 to confer calcium influx in response to resiniferatoxin. TRPV1(VAR) did not influence expression or cell surface localization of cotransfected TRPV1. TRPV1(VAR) protein product associated with the NH2 terminus of canonical TRPV1. Interestingly, when expressed in the COS-7 epithelial cell line, TRPV1(VAR) functioned in a dominant-negative acting capacity, partially blocking TRPV1-dependent resiniferatoxin responsiveness. We conclude that TRPV1(VAR) is one of perhaps several TRPV1 splice variants expressed in rat kidney and that it may serve to modulate TRPV1 responsiveness in some tissues.

The Taming of Capsaicin. Reversal of the Vanilloid Activity of N-Acylvanillamines by Aromatic Iodination

Aromatic iodination ortho to the phenolic hydroxyl reverts the activity of the ultrapotent vanilloid agonist resiniferatoxin (RTX, 1a), generating the ultrapotent antagonist 5'-iodoRTX (1b). To better understand the role of iodine in this remarkable switch of activity, a systematic investigation on the halogenation of vanillamides, a class of compounds structurally simpler than resiniferonoids, was carried out. The results showed that (a) the antagonistic activity depends on the site of halogenation and is maximal at C-6', (b) iodine is more efficient than chlorine and bromine at reverting the agonistic activity, and (c) iodine-carbon exchange decreases antagonist activity. Iodine-induced reversal of vanilloid activity was also observed in vanillamides more powerful than capsaicin, but a poor correlation was found between agonistic and antagonistic potencies, suggesting that differences exist in the way vanillamides and their 6'-iodo derivatives bind to TRPV1.' '