Transient Receptor Potential Vanilloid Subtype Research Articles

Characterization of acid signaling in rat vagal pulmonary sensory neurons

Local tissue acidosis frequently occurs in airway inflammatory and ischemic conditions. The effect of physiological/pathophysiological-relevant low pH (7.0-5.5) on isolated rat vagal pulmonary sensory neurons was investigated using whole cell perforated patch-clamp recordings. In voltage-clamp recordings, vagal pulmonary sensory neurons exhibited distinct pH sensitivities and different phenotypes of inward current in responding to acidic challenge. The current evoked by lowering the pH of extracellular solution to 7.0 consisted of only a transient, rapidly inactivating component with small amplitude. The amplitude of this transient current increased when the proton concentration was elevated. In addition, a slow, sustained inward current began to emerge when pH was reduced to <6.5. The current-voltage curve indicated that the transient component of acid-evoked current was carried predominantly by Na+. This transient component was dose-dependently inhibited by amiloride, a common blocker of acid-sensing ion channels (ASICs), whereas the sustained component was significantly attenuated by capsazepine, a selective antagonist of transient receptor potential vanilloid receptor subtype-1 (TRPV1). The two components of acid-evoked current also displayed distinct recovery kinetics from desensitization. Furthermore, in current-clamp recordings, transient extracellular acidification depolarized the membrane potential and generated action potentials in these isolated neurons. In summary, our results have demonstrated that low pH can stimulate rat vagal pulmonary sensory neurons through the activation of both ASICs and TRPV1. The relative roles of these two current species depend on the range of pH and vary between neurons.

Gastro-protective action of lafutidine mediated by capsaicin-sensitive afferent neurons without interaction with TRPV1 and involvement of endogenous prostaglandins

Lafutidine, a histamine H2 receptor antagonist, exhibits gastro-protective action mediated by capsaicin-sensitive afferent neurons (CSN). We compared the effect between lafutidine and capsaicin, with respect to the interaction with endogenous prostaglandins (PG), nitric oxide (NO) and the afferent neurons, including transient receptor potential vanilloid subtype 1 (TRPV1). Male SD rats and C57BL/6 mice, both wild-type and prostacyclin IP receptor knockout animals, were used after 18 h of fasting. Gastric lesions were induced by the po administration of HCl/ethanol (60% in 150 mmol/L HCl) in a volume of 1 mL for rats or 0.3 mL for mice. Both lafutidine and capsaicin (1-10 mg/kg, po) afforded dose-dependent protection against HCl/ethanol in rats and mice. The effects were attenuated by both the ablation of CSN and pretreatment with N(G)-nitro-L-arginine methyl ester, yet only the effect of capsaicin was mitigated by prior administration of capsazepine, the TRPV1 antagonist, as well as indomethacin. Lafutidine protected the stomach against HCl/ethanol in IP receptor knockout mice, similar to wild-type animals, while capsaicin failed to afford protection in the animals lacking IP receptors. Neither of these agents affected the mucosal PGE2 or 6-keto PGF(1alpha) contents in rat stomachs. Capsaicin evoked an increase in [Ca2+]i in rat TRPV1-transfected HEK293 cells while lafutidine did not. These results suggest that although both lafutidine and capsaicin exhibit gastro-protective action mediated by CSN, the mode of their effects differs regarding the dependency on endogenous PGs/IP receptors and TRPV1. It is assumed that lafutidine interacts with CSN at yet unidentified sites other than TRPV1.

No Pain, No Gain

Myocardial ischemia, the major cause of mortality and morbidity in the United States, accounts for nearly 20% of all deaths.1 The prognosis of patients with acute myocardial infarction is strongly related to the amount of tissue destroyed during ischemia/reperfusion; hence, limiting this injury is of paramount importance for reducing the morbidity and mortality associated with ischemic heart disease.2,3 Over the past 40 years, several hundred pharmacological or nonpharmacological therapies have been reported to alleviate ischemia/reperfusion injury in experimental animal models; unfortunately, few of these results have been reproducible, and, with the exception of early reperfusion, none have been translated into clinical therapies.4 These sobering facts provide a cogent rationale for a reassessment of the models and methodologies that have been used heretofore to test the efficacy of putative cardioprotective interventions. The obvious translational failure in this field calls for a new paradigm in which preclinical studies must be done as rigorously as their clinical counterparts, that is, using clinically relevant animal models, appropriate statistical methods, and a multicenter, randomized, blinded design using centralized core laboratories for data analysis.4 This paradigm would also improve our understanding of the mechanism of cardiomyocyte death during ischemia/reperfusion. Article p 3617 One facet of ischemic biology that has received relatively little attention is the role of cardiac sensory nerves. Myocardial ischemia/reperfusion is known to cause the release of bradykinin and protons, both of which activate the vanilloid receptor 1 (VR1, also known as transient receptor potential vanilloid type 1 [TRPV1]) on the capsaicin-sensitive cardiac sensory nerves (unmyelinated [type C] or thinly myelinated [A-δ] fibers).5 The VR1 is a nonselective cation channel that plays an important role in the polymodal detection of noxious stimuli such as low pH and heat.6 It can also be activated by chemicals such as capsaicin …

Camphor Activates and Strongly Desensitizes the Transient Receptor Potential Vanilloid Subtype 1 Channel in a Vanilloid-Independent Mechanism

Camphor is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite its long history of common medical use, the underlying molecular mechanism of camphor action is not understood. Capsaicin and menthol, two other topically applied agents widely used for similar purposes, are known to excite and desensitize sensory nerves by acting on two members of transient receptor potential (TRP) channel superfamily: heat-sensitive TRP vanilloid subtype 1 (TRPV1) and cold-sensitive TRP channel M8, respectively. Camphor has recently been shown to activate TRPV3, and here we show that camphor also activates heterologously expressed TRPV1, requiring higher concentrations than capsaicin. Activation was enhanced by phospholipase C-coupled receptor stimulation mimicking inflamed conditions. Similar camphor-activated TRPV1-like currents were observed in isolated rat DRG neurons and were strongly potentiated after activation of protein kinase C with phorbol-12-myristate-13-acetate. Camphor activation of rat TRPV1 was mediated by distinct channel regions from capsaicin, as indicated by camphor activation in the presence of the competitive inhibitor capsazepine and in a capsaicin-insensitive point mutant. Camphor did not activate the capsaicin-insensitive chicken TRPV1. TRPV1 desensitization is believed to contribute to the analgesic actions of capsaicin. We found that, although camphor activates TRPV1 less effectively, camphor application desensitized TRPV1 more rapidly and completely than capsaicin. Conversely, TRPV3 current sensitized after repeated camphor applications, which is inconsistent with the analgesic role of camphor. We also found that camphor inhibited several other related TRP channels, including ankyrin-repeat TRP 1 (TRPA1). The camphor-induced desensitization of TRPV1 and block of TRPA1 may underlie the analgesic effects of camphor.

Extracellular Cations Sensitize and Gate Capsaicin Receptor TRPV1 Modulating Pain Signaling

Transient receptor potential (TRP) channels detect diverse sensory stimuli, including alterations in osmolarity. However, a molecular detector of noxious hypertonic stimuli has not yet been identified. We show here that acute pain-related behavior evoked by elevated ionic strength is abolished in TRP vanilloid subtype 1 (TRPV1)-null mice and inhibited by iodoresiniferatoxin, a potent TRPV1 antagonist. Electrophysiological recordings demonstrate a novel form of ion channel modulation by which extracellular Na+, Mg2+, and Ca2+ ions sensitize and activate the capsaicin receptor, TRPV1. At room temperature, increasing extracellular Mg2+ (from 1 to 5 mM) or Na+ (+50 mM) increased ligand-activated currents up to fourfold, and 10 mM Mg2+ reduced the EC50 for activation by capsaicin from 890 to 450 nM. Moreover, concentrations of divalent cations >10 mM directly gate the receptor. These effects occur via electrostatic interactions with two glutamates (E600 and E648) formerly identified as proton-binding residues. Furthermore, phospholipase C-mediated signaling enhances the effects of cations, and physiological concentrations of cations contribute to the bradykinin-evoked activation of TRPV1 and the sensitization of the receptor to heat. Thus, the modulation of TRPV1 by cationic strength may contribute to inflammatory pain signaling.

Selective Blockade of the Capsaicin Receptor TRPV1 Attenuates Bone Cancer Pain

Cancer colonization of bone leads to the activation of osteoclasts, thereby producing local tissue acidosis and bone resorption. This process may contribute to the generation of both ongoing and movement-evoked pain, resulting from the activation of sensory neurons that detect noxious stimuli (nociceptors). The capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1) is a cation channel expressed by nociceptors that detects multiple pain-producing stimuli, including noxious heat and extracellular protons, raising the possibility that it is an important mediator of bone cancer pain via its capacity to detect osteoclast- and tumor-mediated tissue acidosis. Here, we show that TRPV1 is present on sensory neuron fibers that innervate the mouse femur and that, in an in vivo model of bone cancer pain, acute or chronic administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in a significant attenuation of both ongoing and movement-evoked nocifensive behaviors. Administration of the antagonist had similar efficacy in reducing early, moderate, and severe pain-related responses, suggesting that TRPV1 may be a novel target for pharmacological treatment of chronic pain states associated with bone cancer metastasis.

Acid-Sensing Properties in Rat Gastric Sensory Neurons from Normal and Ulcerated Stomach

Gastric acid contributes to dyspeptic symptoms, including abdominal pain, in patients with disorders of the proximal gastrointestinal tract. To examine the molecular sensor(s) of gastric acid chemonociception, we characterized acid-elicited currents in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons that innervate the stomach and examined their modulation after induction of gastric ulcers. A fluorescent dye (DiI) was injected into the stomach wall to retrogradely label gastric sensory neurons. After 1-2 weeks, gastric ulcers were induced by 45 s of luminal exposure of the stomach to 60% acetic acid injected into a clamped area of the distal stomach; control animals received saline. In whole-cell voltage-clamp recordings, all gastric DRG neurons and 55% of NG neurons exhibited transient, amiloride-sensitive, acid-sensing ion-channel (ASIC) currents. In the remaining 45% of NG neurons, protons activated a slow, sustained current that was attenuated by the transient receptor potential vanilloid subtype 1 antagonist, capsazepine. The kinetics and proton sensitivity of amiloride-sensitive ASIC currents differed between NG and DRG neurons. NG neurons had a lower proton sensitivity and faster kinetics, suggesting expression of specific subtypes of ASICs in the vagal and splanchnic innervation of the stomach. Effects of Zn2+ and N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine on acid-elicited currents suggest contributions of ASIC1a and ASIC2a subunits. Gastric ulcers altered the properties of acid-elicited currents by increasing pH sensitivity and current density and changing current kinetics in gastric DRG neurons. The distinct properties of NG and DRG neurons and their modulation after injury suggest differential contributions of vagal and spinal afferent neurons to chemosensation and chemonociception.

In situ hybridization evidence for the coexistence of ASIC and TRPV1 within rat single sensory neurons

The activation of nociceptors by protons plays a crucial role in the initiation and maintenance of acidosis-linked pain. Acid-sensing ion channel (ASIC) and transient receptor potential/vanilloid receptor subtype-1 (TRPV1) encode proton-activated cation channels expressed by nociceptors and the opening of these channels results in nociceptor excitation. Histological relations among ASIC clones and the colocalization of each ASIC subunit and TRPV1 within single sensory neurons were examined on serial sections of rat dorsal root ganglia (DRG) using in situ hybridization histochemistry. ASIC1a transcripts were expressed in 20–25% of the DRG neurons, and most of the neurons had small (<30 μm)-diameter cell bodies. ASIC1b transcripts and ASIC3 transcripts were expressed in ∼10% and 30–35% of the DRG neurons, respectively, and the greater part of each population was located in small-to-medium (30–50 μm)-diameter cells. The ASIC1a transcripts and ASIC1b transcripts were basically localized in the distinct populations of the DRG neurons, while ∼20% of the ASIC1a-positive neurons and ∼10% of the ASIC1b-positive neurons expressed ASIC3 transcripts. TRPV1 transcripts were expressed in 35–40% of the DRG neurons, and most of the TRPV1-positive neurons had small-diameter cell bodies. Intense expression signals for ASIC1a transcripts were detected in 40–45% of the TRPV1-positive neurons. Neurons expressing both ASIC1b and TRPV1 transcripts were barely detected in the DRG. Approximately 30% of the TRPV1-positive neurons expressed ASIC3 transcripts, and the double-labeled neurons were comprised of both small-diameter and medium-diameter cells. Approximately 13% of the TRPV1-positive neurons expressed both ASIC1a and ASIC3 transcripts.

Transient Receptor Potential Vanilloid Subtype 1 Mediates Cell Death of Mesencephalic Dopaminergic NeuronsIn VivoandIn Vitro

Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin, suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3 and that the caspase-3 inhibitor z-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.

Coexpression of Vanilloid Receptor Subtype-1 and Acid-sensing Ion Channel Genes in the Human Trigeminal Ganglion Neurons

Previous psychophysical experiments have shown that repeated applications of high concentrations of acids on one side of the dorsal surface of the human tongue evoke irritation or pain (Dessirier et al., 2000). Under acidification, protons dissociated from the acids probably activate excitatory cation channels expressed in local nociceptors that originate from trigeminal ganglia, leading to the generation of such sensations. Recent molecular investigations into sensory neurons have revealed that a transient receptor potential/vanilloid receptor subtype-1 (TRPV1) and an acid-sensing ion channel (ASIC) mediate the greater part of proton-induced irritation or nociception in mammals (Julius and Basbaum, 2001; Ugawa et al., 2003). Here we provide evidence for involvement of both channels in acid-evoked pain in humans and show their relative contributions to acid-evoked nociception. In our human pain model (approved by the Ethics Committee of Nagoya City University and conducted in accordance with the Declaration of Helsinki), direct infusion of acidic solutions (pH ≥ 6.0) into human skin caused localized pain, which was blocked by amiloride, an inhibitor of ASICs, but not by capsazepine, an inhibitor of TRPV1. Although the efficacy of amiloride was only partially attenuated under more severe acidification (pH 5.0), capsazepine produced some blocking effect on pH 5-evoked pain. Amiloride itself neither blocked capsaicin-evoked localized pain in human skin nor inhibited proton-induced currents in TRPV1expressing Xenopus oocytes (Ugawa et al., 2003). In situ hybridization histochemistry demonstrated that more than half of TRPV1-expressing dorsal root ganglion neurons were ASIC1a- or ASIC3-positive in the rat, and that approximately half of TRPV1expressing human trigeminal neurons were ASIC-positive (S. Ugawa, T. Ueda and S. Shimada, submitted for publication). These results suggest that ASICs are the leading acid sensors in human nociceptors and that both TRPV1 and ASIC channels are involved in acid-evoked oral irritation or pain.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.