Vanilloid Agonists Research Articles

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Structural snapshots of TRPV1 reveal mechanism of polymodal functionality

Many transient receptor potential (TRP) channels respond to diverse stimuli and conditionally conduct small and large cations. Such functional plasticity is presumably enabled by a uniquely dynamic ion selectivity filter that is regulated by physiological agents. What is currently missing is a "photo series" of intermediate structural states that directly address this hypothesis and reveal specific mechanisms behind such dynamic channel regulation. Here, we exploit cryoelectron microscopy (cryo-EM) to visualize conformational transitions of the capsaicin receptor, TRPV1, as a model to understand how dynamic transitions of the selectivity filter in response to algogenic agents, including protons, vanilloid agonists, and peptide toxins, permit permeation by small and large organic cations. These structures also reveal mechanisms governing ligand binding substates, as well as allosteric coupling between key sites that are proximal to the selectivity filter and cytoplasmic gate. These insights suggest a general framework for understanding how TRP channels function as polymodal signal integrators.

Limosilactobacillus reuteri DSM-17938 for preventing cough in adults with mild allergic asthma: A double-blind randomized placebo-controlled cross-over study.

Cough is a common troublesome symptom in asthma which is neuronally mediated. Limosilactobacillus reuteri DSM-17938 (L.reuteri DSM-17938) is a probiotic shown to be effective in pre-clinical models at suppressing neuronal responses to capsaicin, a transient receptor potential vanilloid agonist (TRPV1). Investigate the effects of DSM-17938 versus matched placebo on capsaicin-evoked coughs in mild allergic asthmatics. We performed a 4-visit, randomized, double-blind, placebo-controlled, two-way cross-over study comparing full dose cough responses with inhaled capsaicin in mild allergic asthmatics after 1month of treatment with DSM-17938 compared with matched placebo. Randomization and allocation to trial group were carried out by a central computer system. Histamine skin prick testing, airway hyper-responsiveness and inflammatory cells in induced sputum were measured at every visit. Blood was collected to extract PBMCs and stimulated with CD3/CD28 to ascertain the effects of DSM-17938 /placebo on T-cell cytokine responses. Seventeen subjects were recruited and 15 completed the study (8 females, mean age 27.3years). There was no difference in the change in maximum capsaicin-evoked coughs (Emax) after treatment with L.reuteri DSM-17938 compared with placebo [mean difference 2.07 coughs (95% CI -2.77 to 6.91, p=.38) or relative changes in geometric mean ratios for the dose evoking at least half the Emax (ED50) [1.05 (95% CI 0.31-3.58, p=.94)], concentration evoking 2 coughs (C2) [0.63 (0.26-1.53), p=.28] and 5 coughs (C5) [0.79 (0.25-2.50), p=.67]. There was no effect on histamine skin prick wheal size, intensity of itch sensation, methacholine PC20, airway inflammation or T-cell responses after stimulation with CD3/CD28. There were no serious adverse events. One subject developed a mild upper respiratory tract infection and another mild transient nausea whilst on DSM-17938. In this small study in adults with mild allergic asthma, we found no evidence that L.reuteri DSM-17938has any systemic effects on airway nerves, smooth muscle, sputum inflammatory cells, skin responses or T-cell responses after oral consumption. Clinicaltrials.gov Identifier: NCT03603522.

Investigating the Structure‐Function Relationship Between TRPV1 Channels and Pain

Transient receptor potential, or TRP, ion channels are a superfamily of non-selective cation transmembrane proteins expressed in many tissues. These channels play a key role in the sensory signaling of taste, thermosensation, mechanosensation, and nociception. Transient receptor potential vanilloid 1, or TRPV1, a member of the TRP family of ion channels, specifically responds to both heat and spice. The activation of this channel occurs when a ligand, such as capsaicin, binds to the ligand-binding site, replacing phosphatidylinositol lipids. Capsaicin is a chemical compound commonly found in chili peppers that transmits sensation of taste and pain when it binds to TRPV1. Neuropathic pain, inflammation, and reduced ability to detect stimuli are all symptoms associated with dysfunctional TRPV ion channels. The Summit Country Day School MSOE Center for Biomolecular Modeling MAPS Team used 3D modeling and printing technology to examine the structure-function relationship of the interaction between the TRPV1 ion channel and capsaicin. TRPV ion channels are homotetramers, and individual polypeptide chains contain a transmembrane domain composed of the pore region and vanilloid binding pocket. Capsaicin molecules have been shown to bind to the vanilloid binding pocket located above the S4-S5 linker of one monomer of the TRPV1 tetramer. Two main restriction points in the ion permeation channel were identified. A vanilloid agonist displaces phosphatidylinositide, facilitating the formation of a salt bridge between Arg557 and Glu570, which pulls the S4-S5 linker away from the central axis, opening the lower gate of the pore. The binding of capsaicin to the pocket leaves the selective permeability filter closed but the lower ion gate partially open. This channel is considered as a possible target for the creation of therapeutic treatments for chronic pain. TRPV1 antagonists, such as capsaicin, may play a key role in blocking chronic pain. Capsaicin is already used as a component in topical pain patches and natural and synthetic molecules related to capsaicin have been tested for their ability to decrease response to pain. In the recent movement to decrease the prescription of brain-based pain-blocking medication in response to the opioid epidemic, TRPV1 channels may play a significant role in the development of new therapeutic options in the treatment of pain.

A single TRPV1 amino acid controls species sensitivity to capsaicin

Chili peppers produce capsaicin (a vanilloid) that activates the transient receptor potential cation channel subfamily V member 1 (TRPV1) on sensory neurons to alter their membrane potential and induce pain. To identify residues responsible for differential TRPV1 capsaicin sensitivity among species, we used intracellular Ca2+ imaging to characterize chimeras composed of capsaicin-sensitive rat TRPV1 (rTRPV1) and capsaicin-insensitive chicken TRPV1 (cTRPV1) exposed to a series of capsaicinoids. We found that chimeras containing rat E570-V686 swapped into chicken receptors displayed capsaicin sensitivity, and that simply changing the alanine at position 578 in the S4-S5 helix of the chicken receptor to a glutamic acid was sufficient to endow it with capsaicin sensitivity in the micromolar range. Moreover, introduction of lysine, glutamine or proline at residue A578 also elicited capsaicin sensitivity in cTRPV1. Similarly, replacing corresponding rTRPV1 residue E570 with lysine or glutamine retained capsaicin sensitivity. The hydrophilic capsaicin analog Cap-EA activated a cTRPV1-A578E mutant, suggesting that A578 may participate in vanilloid binding. The hydrophilic vanilloid agonist zingerone did not activate any A578 mutants with capsaicin sensitivity, suggesting that the vanilloid group alone is not sufficient for receptor activation. Our study demonstrates that a subtle modification of TRPV1 in different species globally alters capsaicin responses.

Substance P expression in the DRG of three murine models of arthritis pain: correlation with pain measures and effect of local neurotoxin analgesia

Purpose: Substance P (SP) release and binding to NK-1 produces pain transmission. Pain from arthritis is expressed as both tenderness and as loss of function. Different arthritis pain models express pain differently. This study evaluated the relationship of dorsal root ganglion (DRG) SP expression to pain using both measures of tenderness and function in 3 different models of arthritis pain. Neurotoxins (NT) that prevent release of SP, such as onabotulinum toxin (BoNT/A), and those that deplete SP, such as vanilloids (VAN), can produce analgesia in these models. We correlated effects on SP expression in the DRG with analgesic responses. Methods: C57/Bl6 male mice received intra-articular (IA) carrageenan, Complete Freund’s Adjuvant (CFA) or Collagenase (COL) to produce acute inflammatory, chronic inflammatory or chronic noninflammatory arthritis respectively. IA therapies were given at appropriate intervals before examination. Twelve-week-old mice were examined after arthritis induction. Evoked (tenderness) and spontaneous (functional) pain was quantitated. DRGs were harvested for immunohistochemistry (IHC) after pain assessment. SP expression was measured as % DRG neurons expressing SP. Results: Both Evoked and Spontaneous pain in arthritic and naïve mice correlated with SP expression (R2=0.799, β=1.242 and R2=0.751, β=0.649 respectively). IA vanilloid agonists and antagonist reduced SP expression in chronic inflammatory arthritis (CFA) but did not improve pain behavior. IA BoNT/A reduced SP expression in CFA arthritis but significantly increased SP expression in COL arthritis at 4 weeks after induction but not at 6 weeks. Vanilloid (VAN) treatment normalized evoked pain and weightbearing in mice with acute arthritis pain but did not change SP expression in the DRG. None of the neurotoxins altered pain expression or SP expression in non-arthritic mice. Conclusions: SP expression correlates with both evoked and spontaneous pain behaviors in murine models of arthritis pain. Depending on the NT mechanism of action, SP expression may either increase (BoNT/A) or decrease (VAN). The effect of NT on SP expression may also depend on pathophysiology of pain production and chronicity. SP expression does not necessarily correlate with NT-induced analgesia.

Conserved Allosteric Pathways for Activation of TRPV3 Revealed through Engineering Vanilloid-Sensitivity

The Transient Receptor Potential Vanilloid 1 (TRPV) channel is activated by an array of stimuli, including heat and vanilloid compounds. The TRPV1 homologues TRPV2 and TRPV3 are also activated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. It was recently discovered that four mutations in TRPV2 are sufficient to render the channel sensitive to the TRPV1-specific vanilloid agonist resiniferatoxin (RTx). Here, we show that mutation of six residues in TRPV3 corresponding to the vanilloid site in TRPV1 is sufficient to engineer RTx binding. However, robust activation of TRPV3 by RTx requires facilitation of channel opening by introducing mutations in the pore, temperatures > 30°C, or sensitization with another agonist. Our results demonstrate that the energetics of channel activation can determine the apparent sensitivity to a stimulus and suggest that allosteric pathways for activation are conserved in the TRPV family.

Conserved allosteric pathways for activation of TRPV3 revealed through engineering vanilloid-sensitivity.

The Transient Receptor Potential Vanilloid 1 (TRPV) channel is activated by an array of stimuli, including heat and vanilloid compounds. The TRPV1 homologues TRPV2 and TRPV3 are also activated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. It was recently discovered that four mutations in TRPV2 are sufficient to render the channel sensitive to the TRPV1-specific vanilloid agonist resiniferatoxin (RTx). Here, we show that mutation of six residues in TRPV3 corresponding to the vanilloid site in TRPV1 is sufficient to engineer RTx binding. However, robust activation of TRPV3 by RTx requires facilitation of channel opening by introducing mutations in the pore, temperatures > 30°C, or sensitization with another agonist. Our results demonstrate that the energetics of channel activation can determine the apparent sensitivity to a stimulus and suggest that allosteric pathways for activation are conserved in the TRPV family.

Expression of functional TRPV1 receptor in primary culture of canine keratinocytes.

The interest for the endovanilloid system and for transient receptor potential vanilloid 1 (TRPV1) is continuously increasing, due to their involvement in inflammation, nociception and pruritus. Even if TRPV1 enrolment was highlighted in both physiological and pathological conditions, some aspects remain unclear, mostly in veterinary medicine. This study aimed to verify the expression and functionality of TRPV1 in canine keratinocytes to investigate in vitro the role of TRPV1 in these cells that are involved in different cutaneous pathologies. Keratinocytes primary cultures were isolated from bioptical samples and cultivated. Binding assay (using 3 [H]-resiniferatoxin), displacement assay (in the presence of 1.2nM 3 [H]-resiniferatoxin) and functional assays (in the presence of 1μCi/45 Ca2+ ) with vanilloid agonists and antagonists, specifically addressed to TRPV1 receptor, were performed. Binding assay demonstrated the presence of measurable concentrations of TRPV1 (Bmax =1,240±120fmol/mg protein; Kd =0.01±0.004nM). Displacement assay highlighted the highest affinity for resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX), among agonists and antagonists, respectively. The same compounds results as the most potent in the functional assays. This study demonstrated the identification and the characterization of TRPV1 receptor in primary canine keratinocytes cultures. The results are promising for a clinical use, but further in vivo investigations are required.

Evaluation of the antinociceptive and anti-inflammatory activities of piperic acid: Involvement of the cholinergic and vanilloid systems

Piperin is the active compound of black pepper (Piper nigrum). From the piperine was obtained the molecule of the piperic acid (PAC). The objective of this study was to evaluate the antinociceptive and anti-inflammatory of the compound. The antinociceptive effects of PAC were evaluated by abdominal writhing, formalin, capsaicin and tail-flick tests; while the anti-inflammatory effects were evaluated by paw oedema and air pouch tests, and in vitro COX inhibition assay. The possible action mechanism of PAC was evaluated using naloxone, L-NAME, glibenclamide and atropine in tail flick test and by Cholinesterase activity assay and production of TNF-α and IL-1β. PAC significantly reduced the nociceptive effects induced by acetic acid or formalin in mice. PAC also demonstrated an antinociceptive effect in the tail-flick model. The muscarinic receptor antagonist, atropine reduced the antinociceptive effect of PAC in the tail-flick model. PAC was able to inhibit capsaicin-induced nociception, showing involvement of TRPV1. The compound did not alter the motor capacity of the animals, not interfering in the nociceptive response. PAC also showed anti- inflammatory activity by inhibiting the formation of carrageenan-induced paw oedema, leukocyte migration, and cytokine production / release. Atropine reduced the activity of PAC on leukocyte migration, and cytokine production. The compound showed to be able to reduce the cytokine production stimulated by capsaicin. PAC inhibited the COX activity. The results presented suggest that the possible cholinomimetic action and vanilloid agonist of the piperic acid may be responsible by antinociceptive and anti- inflammatory effects; these effects are devoid of toxicity.

The effect of intra-articular vanilloid receptor agonists on pain behavior measures in a murine model of acute monoarthritis.

Arthritis is the most common cause of disability in the US, and the primary manifestation of arthritis is joint pain that leads to progressive physical limitation, disability, morbidity, and increased health care utilization. Capsaicin (CAP) is a vanilloid agonist that causes substance P depletion by interacting with vanilloid receptor transient receptor potential V1 on small unmyelinated C fibers. It has been used topically for analgesia in osteoarthritis with variable success. Resiniferatoxin (RTX) is an ultra potent CAP analog. The aim of this study was to measure the analgesic effects of intra-articular (IA) administration of CAP and RTX in experimental acute inflammatory arthritis in mice. Evoked pain score (EPS) and a dynamic weight bearing (DWB) device were used to measure nociceptive behaviors in a murine model of acute inflammatory monoarthritis. A total of 56 C57B16 male mice underwent EPS and DWB testing – 24 nonarthritic controls and 32 mice with carrageenan-induced arthritis. The effects of pretreatment with 0.1% CAP, 0.0003% RTX, or 0.001% RTX were measured. Nociception was reproducibly demonstrated by increased EPS and reduced DWB measures in the affected limb of arthritic mice. Pretreatment with 0.001% RTX resulted in statistically significant improvement in EPS and DWB measures when compared with those observed in carrageenan-induced arthritis animals. Pretreatment with IA 0.0003% RTX and IA 0.01% CAP resulted in improvement in some but not all of these measures. The remaining 24 mice underwent evaluation following treatment with 0.1% CAP, 0.0003% RTX, or 0.001% RTX, and the results obtained were similar to that of naïve, nonarthritic mice.

Interaction between paired-pulse facilitation and long-term potentiation during the stimulation of the cannabinoid and vanilloid systems in the dentate gyrus

Synaptic plasticity includes short-term and long-term changes in synaptic strength. Short-term plasticity can be used to assess the site mediating the long-lasting forms of synaptic plasticity such as long-term potentiation (LTP). The endogenous endocannabinoid systems can modulate LTP, and similarly, the activation of the vanilloid system has been shown to mediate synaptic plasticity in the hippocampus. In this study, we examined the interaction between short-term and long-term plasticity during the stimulation of the cannabinoid and vanilloid systems in the hippocampus of rats in vivo. Forty male Wistar rats, divided into four groups, were treated with the following compounds: control (saline+dimethyl sulfoxide), WIN55,212-2, capsaicin, and WIN55,212-2+capsaicin. The animals were anesthetized with urethane and then recording and stimulating electrodes were positioned at the dentate gyrus(DG) and perforant pathway(PP), respectively. Population spike (PS) amplitudes were measured before and after the induction of LTP, which was induced with high-frequency stimulation (HFS). The paired-pulse ratio (PPR) was measured before and after the induction of LTP in all groups.We showed that WIN55,212-2 reduced the PS amplitude after HFS, whereas the vanilloid agonist increased the induction of LTP compared with the control treatment. In the present study, we found that in the presence of WIN55,212-2 and capsaicin, the induction of LTP changed the PPR. Additionally, we showed that the co-administration of cannabinoid and vanilloid agonists modulate the PPR. These findings suggest the presynaptic expression of this LTP form, and therefore, this form of LTP is caused by the increase of neurotransmitter release.

Sperm Release From the Oviductal Epithelium Depends on Ca(2+) Influx Upon Activation of CB1 and TRPV1 by Anandamide.

The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway.

The interactive role of cannabinoid and vanilloid systems in hippocampal synaptic plasticity in rats

Long-term potentiation (LTP) has been most thoroughly studied in the hippocampus, which has a key role in learning and memory. Endocannabinoids are one of the endogenous systems that modulate this kind of synaptic plasticity. The activation of the vanillioid system has also been shown to mediate synaptic plasticity in the hippocampus. In addition, immunohistochemical studies have shown that cannabinoid receptor type 1 (CB1) and vanilloid receptor 1 (TRPV1) are closely located in the hippocampus. In this study, we examined the hippocampal effects of co-administrating WIN55-212-2 and capsaicin, which are CB1 and TRPV1 agonists, respectively, on the induction of LTP in the dentate gyrus (DG) of rats. LTP in the hippocampal area was induced by high-frequency stimulation (HFS). Our results indicated that the cannabinoid agonist reduced both field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude after HFS with respect to the control group, whereas the vanilloid agonist increased these parameters along with the increased induction of LTP as compared to the control group. We also showed that the co-administration of cannabinoid and vanilloid agonists had different effects on fEPSP slope and PS amplitude. It seems that agonists of the vanilloid system modulate cannabinoid outputs that cause an increase in synaptic plastisity, while in contemporary consumption of two agonist, TRPV1 agonist can change production of endocannabinoid, which in turn result to enhancement of LTP induction. These findings suggest that the two systems may interact or share certain common signaling pathways in the hippocampus.

The Role of the Capsaicin 8 % Patch (Qutenza®) in the Treatment of Post-herpetic Neuralgia

Post-herpetic neuralgia (PHN) occurs as a complication of acute herpes zoster. The capsaicin 8 % (w/w) dermal patch (Qutenza®) is a transient receptor potential vanilloid agonist that increases intracellular calcium concentration, triggering calcium-dependent protease enzymes to cause cytoskeletal breakdown. This action is thought to lead to loss of cellular integrity and defunctionalization of nociceptor epidermal nerve fibers. The capsaicin 8 % patch is indicated in the US for the treatment of neuropathic pain associated with PHN. In pivotal, randomized, doubleblind, multicenter trials in patients with PHN, a single 60-minute application of the capsaicin 8 % dermal patch reduced mean numeric pain rating scale scores between baseline and weeks 2–8 to a significantly greater extent than the low-dose comparator patch (capsaicin 0.04 % w/w). The capsaicin 8 % (w/w) dermal patch was found to be safe and generally well-tolerated. Most commonly reported side effects were local, comprising dermal irritation, erythema, and pain at the site of application. These effects were transient and mild to moderate in severity. Transient patch application-related pain was not a barrier to use and was managed with local cooling or oral analgesics in nearly all cases. Thus, the capsaicin 8 % dermal patch appears to have a good safety profile, is well tolerated, and offers effective topical therapy in some patients with PHN. This review aims to characterize the use of the capsaicin 8 % dermal patch in the treatment of PHN.

Characterization of synapses in the rat subnucleus centralis of the nucleus tractus solitarius.

The nucleus tractus solitarius (NTS) receives subdiaphragmatic visceral sensory information via vagal A- or C-fibers. We have recently shown that, in contrast to cardiovascular NTS medialis neurons, which respond to either purinergic or vanilloid agonists, the majority of esophageal NTS centralis (cNTS) neurons respond to vanilloid agonists, whereas a smaller subset responds to both vanilloid and purinerigic agonists. The present study aimed to further investigate the neurochemical and synaptic characteristics of cNTS neurons using whole cell patch-clamp, single cell RT-PCR and immunohistochemistry. Excitatory postsynaptic currents (EPSCs) were evoked in cNTS by tractus solitarius stimulation, and in 19 of 64 neurons perfusion with the purinergic agonist αβ-methylene ATP (αβMeATP) increased the evoked EPSC amplitude significantly. Furthermore, neurons with αβMeATP-responsive synaptic inputs had different probabilities of release compared with nonresponsive neurons. Single cell RT-PCR revealed that 8 of 13 αβMeATP-responsive neurons expressed metabotropic glutamate receptor 8 (mGluR8) mRNA, which our previous studies have suggested is a marker of glutamatergic neurons, whereas only 3 of 13 expressed glutamic acid dehydroxylase, a marker of GABAergic neurons. A significantly lower proportion of αβMeATP-nonresponsive neurons expressed mGluR8 (2 of 30 neurons), whereas a greater proportion expressed glutamic acid dehydroxylase (12 of 30 neurons). Esophageal distension significantly increased the number of colocalized mGluR8- and c-Fos-immunoreactive neurons in the cNTS from 8.0 ± 4% to 20 ± 2.5%. These data indicate that cNTS comprises distinct neuronal subpopulations that can be distinguished based on their responses to purinergic agonists and that these subpopulations have distinct neurochemical and synaptic characteristics, suggesting that integration of sensory inputs from the esophagus relies on a discrete organization of synapses between vagal afferent fibers and cNTS neurons.

Vanilloids induce oral cancer apoptosis independent of TRPV1

To investigate the mechanisms of vanilloid cytotoxicity and anti-tumor effects in oral squamous cell carcinoma (OSCC). Immunohistochemistry and qPCR analyses demonstrated expression of the TRP vanilloid type 1 (TRPV1) receptor in OSCC. Using cell proliferation assays, calcium imaging, and three mouse xenograft models, prototypical vanilloid agonist (capsaicin) and antagonist (capsazepine) were evaluated for cytotoxic and anti-tumor effects in OSCC. OSCC cell lines treated with capsaicin displayed significantly reduced cell viability. Pre-treatment with capsazepine failed to reverse these effects. Moreover, capsazepine alone was significantly cytotoxic to tumor cells, suggesting the mechanism-of-action is independent of TRPV1 activation. This was further confirmed by calcium imaging indicating that TRPV1 channels are not functional in the cell lines tested. We then examined whether the observed vanilloid cytotoxicity was due to the generation of reactive oxygen species (ROS) and subsequent apoptosis. Induction of ROS was confirmed by flow cytometry and reversed by co-treatment with the antioxidant N-acetyl-cysteine (NAC). NAC also significantly reversed vanilloid cytotoxicity in cell proliferation assays. Dose-dependent induction of apoptosis with capsazepine treatment was demonstrated by FACS analyses and c-PARP expression in treated cells. Our in vivo xenograft studies showed that intra-tumoral injections of capsazepine exhibited high effectiveness in suppressing tumor growth with no identifiable toxicities. These findings confirm TRPV1 channel expression in OSCC. However anti-tumor effects of vanilloids are independent of TRPV1 activation and are most likely due to ROS induction and subsequent apoptosis. Importantly, these studies demonstrate capsazepine is a potential therapeutic candidate for OSCC.

High-resolution views of TRPV1 and their implications for the TRP channel superfamily.

The first high-resolution structures of a near-full-length TRP channel were recently described, structures of the noxious heat receptor TRPV1 in the absence or presence of vanilloid agonists and a spider toxin. Here we briefly review the salient features, including the overall architecture, agonist binding sites, and conformational changes related to channel pore gating. We also discuss some of the structures' implications for the TRP channel family and a few of the many questions still left unanswered.

TRPV1 structures in distinct conformations reveal activation mechanisms

TRP channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (S1-S4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiologic modulation exhibited by TRPV1 and other TRP channels.

Divalent Heavy Metal Cations Block the TRPV1 Ca2+ Channel

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co2+ [half-maximal inhibitory concentration (IC50) = 13 μM] > Cd2+ (IC50 = 38 μM) > Ni2+ (IC50 = 62 μM) > Cu2+ (IC50 = 200 μM). Zn2+ proved to be a weak (IC50 = 27 μM) and only partial inhibitor of the channel function, whereas Mg2+, Mn2+ and La3+ did not exhibit any substantial effect. Co2+, the most potent channel blocker, was able not only to compete with Ca2+ but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co2+ accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co2+ applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked 45Ca2+ influx assays in cells expressing TRPV1. The IC50 of Co2+ on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca2+.