High Concentrations Of Capsaicin Research Articles

Experimental evidence for alleviating nociceptive hypersensitivity by single application of capsaicin.

The single application of high-concentration of capsaicin has been used as an analgesic therapy of persistent pain. However, its effectiveness and underlying mechanisms remain to be further evaluated with experimental approaches. The present study provided evidence showing that the single application of capsaicin dose-dependently alleviated nociceptive hypersensitivity, and reduced the action potential firing in small-diameter neurons of the dorsal root ganglia (DRG) in rats and mice. Pre-treatment with capsaicin reduced formalin-induced acute nocifensive behavior after a brief hyperalgesia in rats and mice. The inhibitory effects of capsaicin were calcium-dependent, and mediated by the capsaicin receptor (transient receptor potential vanilloid type-1). We further found that capsaicin exerted inhibitory effects on the persistent nociceptive hypersensitivity induced by peripheral inflammation and nerve injury. Thus, these results support the long-lasting and inhibitory effects of topical capsaicin on persistent pain, and the clinic use of capsaicin as a pain therapy.

Capsicum annuum extracts/β-cyclodextrin complexes

In this study, thermal analyses and Karl Fischer titration correlations on the new varieties of Capsicum annuum L. extracts/β-cyclodextrin complexes were performed. Two of seven studied Capsicum varieties containing the highest concentration of capsaicin (0.7 and 1.1 mg g−1 of dry sample) were used for obtaining Capsicum extracts/β-cyclodextrin complexes by co-crystallization method. Thermogravimetric analysis (TG) indicates mass losses with 5–5.7 % lower for the complexes in comparison with β-cyclodextrin, while differential scanning calorimetry analysis (DSC) shows a temperature peak shift to lower values, from 378.2 K (105 °C) for β-cyclodextrin to 355.2–361.2 K (82–88 °C) for complexes. On the other hand, Karl Fischer water titration (KFT) indicates a water concentration of 10.9–11.8 %, with 3.1–4.1 % lower than for β-cyclodextrin (14.9 %). The antioxidant activity of Capsicum extracts/β-cyclodextrin complexes has values comparable with standard antioxidant compound solutions, being significant longer time than the starting extracts. Statistically significant correlations between TG, DSC, and KFT main parameters [TG mass loss up to 423.2 K (150 °C), DSC peak temperature corresponding to water dissociation and KFT water content, and water reaction rate for “strongly-retained” water molecules partially replaced from the cyclodextrin cavity in the complexation process] were obtained, proving the formation of guest/host (Capsicum bioactive compounds/β-cyclodextrin) supramolecular systems.

TRPs in the kidney - location, location, location

TRPs in the kidney - location, location, location

THU0347 Clinical Effectiveness of 8 % Capsaicin Dermal Patch in the Treatment of Carpal Tunnel Syndrome

BackgroundThe name of carpal tunnel syndrome (CTS) refers to a complex of symptoms resulting from the pressure exerted on the median nerve in the carpal tunnel area. CTS is the...

Supercritical carbon dioxide extraction of Capsicum peppers: Global yield and capsaicinoid content

The objective of this study was to select a variety of pepper with high concentration of capsaicin and subject it to supercritical fluid extraction (SFE), in order to determine the best conditions of temperature (40, 50 and 60°C) and pressure (15, 25 and 35MPa) in terms of global yield (X0) and capsaicinoids content of the extracts. The influence of drying process (freeze and oven drying) on X0, capsaicin (C) and dihydrocapsaicin (DHC) contents and total phenolics was also analyzed. Capsicum frutescens showed the highest levels of capsaicinoids (1516μg/g fresh fruit). For the responses C and DHC, the extraction conditions of 15MPa and 40°C provided the highest concentrations (C ⿿ 42mg/g extract and DHC ⿿ 18.5mg/g extract). The freeze drying process resulted in extracts with the highest concentration of capsaicinoids (61mg/g extract), but in contrast, the phenolics were less susceptible to different drying processes, with a mean concentration of 35mg GAE/g extract. The kinetics experiments indicated that the extraction rate of oleoresin was slightly slower than that of capsaicinoids at the operation conditions (40°C and 15MPa).

INTRODUCTION - Targeting TRP Channels for Pain Relief: TRPV1 and Beyond

Management of chronic and pathological pain without incurring systemic side effects is a major medical challenge.Currently available drugs, such as non-steroidal anti-inflammatory drugs or opioid agonists, are efficacious through peripheral and central mechanisms. However, various complications and development of tolerance are serious problems. Other classes of drugs, such as anti-depressants and anti-convulsants, are often used for multiple pain syndromes. However, the efficacy of these drugs is commonly unsatisfactory, and their mechanism of action is not clear. For establishing novel, selective anti-hyperalgesic therapeutic approaches, targeted inhibition of pain-specific pathways or molecules would be ideal, and these approaches suggest straightforward strategies. A new era of exploring such “straightforward” approaches was opened with regard to peripheral nociceptors by the identification of the vanilloid receptor-1 (VR-1), which was designated transient receptor potential channel vanilloid subtype 1 (TRPV1). TRPV1 is a receptor for capsaicin, proton, and noxious heat. Capsaicin has long been known to be a natural compound capable of evoking an intense burning sensation and pain in human and experimental animals. It has been hypothesized that specific manipulation of TRPV1 may selectively relieve pain under injury or inflammatory conditions. Interfering with TRPV1 has been a central focus of these efforts during the 15 years following the cloning of TRPV1. Numerous pharmacological compounds have been developed targeting TRPV1. The characteristics and roles of TRPV1 have been rigorously studied using multiple approaches ranging from biophysical characterization to clinical trials in human subjects. Meanwhile, other members of the TRP channel family in addition to TRPV1 have been suggested to be also involved in nociception under pathophysiological conditions. These studies have identified targets in addition to TRPV1 as potential candidates for selective anti-hyperalgesic treatment free from complications. In this special issue of The Open Pain Journal, current knowledge regarding the roles of various TRP channels in pain is reviewed. Multiple scientists in academia and the pharmaceutical industry took part in this exciting project, and have shared their opinions regarding the prospects for relieving pain through targeting TRP channels. In the opening chapter, “Changes in TRP channels expression in painful conditions,” Bishnoi and Premkumar summarize the involvement of various members of the TRP channel family in nociception. The five following chapters are devoted to a discussion of the role of various TRP channels in specific pathological conditions. Blackshaw and colleagues review the evidence for the roles of “TRP channels in visceral pain.” They discuss the contribution of TRP channels, especially TRPV1, TRPV4, TRPA1, and TRPM8, to pain signaling in visceral systems in various visceral pathological models. The roles of “TRP channels in dental pain” are reviewed by Chung and Oh. The expression of various TRP channels in pulpal afferents and odontoblasts is summarized, and their potential contribution to dental pain by thermal and mechanical stimuli is discussed. In “The role of TRP channels in migraine,” Oxford and Hurley review the potential roles of TRP channels in a trigeminovascular system, their involvement in migraine attack, and recent clinical trials. Fernandes et al. discuss “TRP receptors in arthritis, gaining knowledge for translation from experimental models”. They review current knowledge pertaining to the therapeutic potential of TRP channels, primarily TRPV1 and TRPA1, for treating pain in osteoarthritis and other arthritic conditions. The roles of “Transient Receptor Potential channels in chemotherapy-induced neuropathy” are discussed by Nassini et al. In this chapter, the current understanding of the involvement of TRPV1, TRPV4, TRPM8, and TRPA1 in chemotherapy-induced neuropathy is highlighted. The next three chapters analyze the status of therapeutic approaches targeting TRPV1 using specific agonists and antagonists. In effects of topical capsaicin on cutaneous innervation: Implications for pain management Bley reviews the potential mechanisms of topical application of high concentrations of capsaicin to skin, and suggests pharmacokinetic and pharmacodynamic considerations of this therapy. Iadarola and colleagues discuss how site-specific administration of resiniferatoxin, an ultrapotent capsaicin analog, can overcome the limitations of topical capsaicin therapy, and they give an insight into the clinical trials using resiniferatoxin for permanent pain relief in cancer patients with intractable pain. Trevisani and Gatti discuss “TRPV1 antagonists as analgesic agents.” The authors review preclinical and clinical studies addressing the application of small molecule TRPV1 antagonists as anti-hyperalgesic thera-pies, and update current status of clinical trials

Capsaicin-induced activation of ERK1/2 and its involvement in GAP-43 expression and CGRP depletion in organotypically cultured DRG neurons.

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.

Low-concentration capsaicin promotes colorectal cancer metastasis by triggering ROS production and modulating Akt/mTOR and STAT-3 pathways

Colorectal cancer (CRC), one of the most common human malignancies, is a major public health problem in the developed world. Capsaicin, widely used as a food additive and as an analgesic agent, is a major pungent ingredient of red pepper. Though capsaicin-induced apoptosis was previously reported in cancer cells, relatively little is known about the impact of capsaicin on other aspect of cancer cell behavior. In this study, we demonstrated that treatment with high-concentration of capsaicin (≥ 200 µM for SW480 and CT-26 cell lines; ≥ 25 µM for HCT116 cell line) inhibited CRC cell proliferation in a dose-dependent manner. In spite of no anti-proliferative effect, notably, low-concentration of capsaicin (100 µM for SW480 and CT-26 cell lines; 12.5 µM for HCT116 cell line) enhanced both migratory and invasive capability of these cells, which was validated by both in vitro and in vivo model. Further, we showed that 100 µM capsaicin induced epithelial-to-mesenchymal (EMT), up-regulated expression of MMP-2 and MMP-9, and activated Akt/mTOR and STAT-3 pathways in SW480 cells. Finally, we showed that capsaicin-induced metastasis of CRC cells was mediated by modulating reactive oxygen species (ROS) production. Our findings are considered a significant step toward a better understanding of capsaicin-associated regulatory network on CRC cells.

Facial skin blood flow responses to irritant stimuli in the oral cavity

To investigate whether capsaicin and menthol stimuli elicit characteristic responses in facial skin blood flow (SkBF), we observed the facial SkBF response to low and high concentrations of capsaicin and menthol stimuli of 1-ml solution applied to the oral cavity for 20s in 17 healthy subjects. High concentration of capsaicin significantly increased the SkBF in all of the facial areas monitored. High concentration of menthol stimulus significantly decreased SkBF in the nose and increased that in the eyelid, and upper and lower lips. These results demonstrated that capsaicin and menthol stimuli in the oral cavity elicit characteristic responses in facial SkBF.

Urothelial TRPV1: TRPV1-Reporter Mice, a Way to Clarify the Debate?

Urothelial TRPV1: TRPV1-Reporter Mice, a Way to Clarify the Debate?

Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.

Nanomechanical analysis of insulinoma cells after glucose and capsaicin stimulation using atomic force microscopy

Glucose stimulates insulin secretion from pancreatic islet β cells by altering ion channel activity and membrane potential in the β cells. TRPV1 channel is expressed in the β cells and capsaicin induces insulin secretion similarly to glucose. This study aims to investigate the biophysical properties of the β cells upon stimulation of membrane channels using an atomic force microscopic (AFM) nanoindentation system. ATCC insulinoma cell line was used. Cell stiffness, a marker of reorganization of cell membrane and cytoskeleton due to ion channel activation, was measured in real time using an integrated AFM nanoindentation system. Cell height that represented structural changes was simultaneously recorded along with cell stiffness. After administration of glucose (16, 20 and 40 mmol/L), the cell stiffness was markedly increased in a dose-dependent manner, whereas cell height was changed in an opposite way. Lower concentrations of capsaicin (1.67 × 10(-9) and 1.67 × 10(-8) mol/L) increased the cell stiffness without altering cell height. In contrast, higher concentrations of capsaicin (1.67 × 10(-6) and 1.67 × 10(-7) mol/L) had no effect on the cell physical properties. A unique bio-nanomechanical signature was identified for characterizing biophysical properties of insulinoma cells upon general or specific activation of membrane channels. This study may deepen our understanding of stimulus-secretion coupling of pancreatic islet cells that leads to insulin secretion.

Dissociation of the neural correlates coding stimulus intensity and perception of capsaicin urge‐to‐cough in humans

Inhalations of capsaicin are associated with increasing ratings of urge-to-cough. We hypothesised that brain regions involved in urge-to-cough intensity discrimination would show increased activation at higher compared to lower capsaicin concentrations. Functional brain images were acquired with a Siemens 3T scanner from healthy subjects (n=16) during repeated 18s blocks of inhalation via nebuliser of saline, a low concentration or a high concentration of capsaicin. General linear modelling was used to identify variance in signals associated with inhalation events, and contrasts were generated to identify capsaicin intensity-dependent activation. Capsaicin challenges produced activations in a widely distributed brain network which included sensory, motor, midbrain, limbic and cerebellar regions. Regional brain activations were classified as (a) stimulus intensity dependent (anterior insula, anterior midcingulate and primary motor cortices), (b) all or nothing (prefrontal, posterior parietal cortices), or (c) dependent upon the perceived level of urge-to-cough experienced (primary sensory cortex). These data suggest that the actual intensity and the subjective experience are differentially represented in the brain urge-to-cough network. Furthermore, posterior parietal and prefrontal regions likely play a role in spatial discrimination rather than intensity detection.

Capsaicin Dermal Patch

Capsaicin dermal patch is an adhesive patch containing a high concentration (8% w/w) of synthetic capsaicin. It is indicated in the EU for the treatment of peripheral neuropathic pain in non-diabetic adults using a single 30- or 60-minute application repeated every 90 days, as required, and in the US for the treatment of neuropathic pain associated with postherpetic neuralgia (PHN). In pivotal, randomized, double-blind, multicentre trials in adults with PHN, a single 60-minute application of capsaicin dermal patch reduced the mean Numeric Pain Rating Scale (NPRS) scores from baseline to a significantly greater extent than a low-concentration (0.04% w/w capsaicin) control patch during weeks 2-8. In randomized, double-blind, multicentre trials in patients with HIV-associated neuropathy, capsaicin dermal patch reduced the mean NPRS scores from baseline significantly more than control in one study for the 30- and 90-minute, but not the 60-minute, application during weeks 2-12. In another study, the differences between capsaicin (30- and 60-minute applications) and control did not reach statistical significance. An integrated analysis of both studies showed that the 30-minute application of capsaicin dermal patch was significantly better than control for the reduction from baseline in mean NPRS scores during weeks 2-12. The efficacy of capsaicin dermal patch was maintained for up to 1 year in extension studies in which patients could receive up to three or four repeat treatments. Capsaicin dermal patch was generally well tolerated in clinical trials. The most common adverse events were transient, mostly mild to moderate, application-site reactions.

Variability in hot pepper for phytochemicals offers promising tools in plant-breeding programmes

Screening of natural biodiversity for the variation in quality traits is of prime importance for quality-breeding programmes. The objective of this investigation was to select candidate accession of hot pepper having high concentrations of ascorbic acid, capsaicin, β-carotene, and total phenols for use as parents in breeding for these compounds. Forty-two accessions of pepper (Capsicum annuum) were field grown and their mature fruits were analysed for their functional and nutritional composition. Wide variations were observed in most of the measurements, e.g. ascorbic acid (25–217mg per 100g), total phenolics (38.4–188.1mg per 100g catechol eq.), and capsaicin (0.08–0.67%), suggesting that there are considerable levels of genetic diversity. Across all accessions the concentration of ascorbic acid was negatively correlated with that of β-carotene (r=−0.33, p <0.05). Concentrations of L-ascorbic acid were significantly greater in VLC 22-I-2-1, VLC-29-II-1-1, VLC-30-I-1, and Janjeera Mirch compared with other accessions analysed. Total capsaicin concentrations were greatest (0.67%) in VLC-30-II-1 and lowest (0.08%) in VLC-32-3. Four factors were computed by principal-component analysis to explain 67% of the variation in the traits. The great variability for these phytochemicals suggests that these selected accessions may be useful as parents in breeding programmes to produce fruits with value-added traits.

TRPV1 Modulates Acetylcholine Release From Motor Nerve Terminals

Transient receptor potential (TRP) proteins are expressed ubiquitously throughout the body. Previous studies demonstrated expression of TRPV1, the capsaicin receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve endings (MNEs; J. Pharmacol. Exp. Ther. Aug 04. 2009) where we observed that capsaicin protected MNEs from botulinum neurotoxin A (BoNT/A). We hypothesized that capsaicin reduced clathrin coated pit (CCP) dependent endocytosis of BoNT/A and demonstrate the regulatory influence of TRPV1 in exo-endocytic processes of MNEs. Phrenic nerve diaphragm muscle preparations isolated from isoflurane anesthetized adult mice were analyzed for the nerve-evoked twitch and transmitter release (TR). Capsaicin produced a concentration-dependent decline of twitch tension (TT), an effect attributed to suppression of stimulus-evoked acetylcholine release (SEAR) since these capsaisin concentrations reduced the amplitude of endplate currents. These effects of capsaicin were antagonized by capsazepine, the TRPV1 antagonist. To understand the mechanism whereby capsaicin reduced TR, we studied cholinergic Neuro 2a cells. Acute exposure to capsaicin altered the subcellular distribution of clathrin heavy chain (CHc) and AP2, two proteins essential to CCP formation. Wortmannin, (non selective PI3K/PI5K inhibitor), inhibited the TT and SEAR of the isolated nerve-muscle preparations and delocalized CHc and AP2 in Neuro 2a cells. Chlorpromazine, an inhibitor of CCP dependent endocytic pathway [Cell Mol. Biol. Lett. 2004; 9 (3): 475-81], mimicked the effects of capsaicin on AP2 delocalization. These data suggest that endogenous TRPV1 proteins are coupled to the exo-endocytic mechanisms that regulate neuromuscular transmission and that activation of TRPV1 with high capsaicin concentrations reduces exocytosis of acetylcholine by down regulating the compensatory CCP dependent endocytic pathways.

The Endoplasmic Reticulum of Dorsal Root Ganglion Neurons Contains Functional TRPV1 Channels

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.

Voltage is a partial activator of rat thermosensitive TRP channels

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20-25 degrees C) membrane depolarization evokes responses that saturate at approximately 50-60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 microm), resiniferatoxin (5 microm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.

The activation of transient receptor potential vanilloid receptor subtype 1 by capsaicin without extracellular Ca2+ is involved in the mechanism of distinct substance P release in cultured rat dorsal root ganglion neurons

Using a highly sensitive substance P (SP) radioimmunoassay, we attempted to elucidate the molecular mechanism of SP release from cultured adult rat dorsal root ganglion (DRG) neurons triggered by capsaicin. As a result, we found that capsaicin can induce SP release in the absence of extracellular Ca2+ by activating transient receptor potential vanilloid receptor subtype 1 (TRPV1). Therefore, we compared the pharmacological profile of SP release involved in several intracellular effectors (phosphoinositide 3-kinase (PI3K), Ca2+ release from intracellular stores, and mitogen-activated protein (MAP) kinases) in the presence/absence of extracellular Ca2+ by stimulating DRG neurons with various concentrations (10 to 1,000 nM) of capsaicin. In the presence of extracellular Ca2+, a capsaicin-induced maximal release of SP obtained at 100 nM capsaicin was attenuated by either the phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY294002), the inositol 1,4,5-triphosphate (IP3)-induced Ca2+ release blocker (2-aminoethyl diphenylborinate, 2-APB) or a specific antagonist of TRPV1 (capsazepine). In contrast, in the absence of extracellular Ca2+, only a high concentration (1 microM) of capsaicin induced a significant increase in the SP release, which was then completely abolished by either a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or capsazepine, and significantly inhibited by either thapsigargin (a Ca2+-ATPase inhibitor) or BAPTA-AM (a rapid Ca2+ chelator). In summary, the activation of TRPV1 by capsaicin modulates the SP release from DRG neurons via two different mechanisms, one requiring extracellular Ca2+, the activation of PI3K and the IP3-dependent intracellular Ca2+ release, and the other which is independent of extracellular Ca2+ but involves the activation of MEK. These data suggest that a distinct SP release mechanism exists on DRG through the activation of TRPV1 without extracellular Ca2+ by capsaicin to facilitate neuropeptide release.

Dual Regulation of TRPV1 by Phosphoinositides

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PIP2] regulates many ion channels. There are conflicting reports on the effect of PtdIns(4,5)P2 on transient receptor potential vanilloid 1 (TRPV1) channels. We show that in excised patches PtdIns(4,5)P2 and other phosphoinositides activate and the PIP2 scavenger poly-Lys inhibits TRPV1. TRPV1 currents undergo desensitization on exposure to high concentrations of capsaicin in the presence of extracellular Ca2+. We show that in the presence of extracellular Ca2+, capsaicin activates phospholipase C (PLC) in TRPV1-expressing cells, inducing depletion of both PtdIns(4,5)P2 and its precursor PtdIns(4)P (PIP). The PLC inhibitor U73122 and dialysis of PtdIns(4,5)P2 or PtdIns(4)P through the patch pipette inhibited desensitization of TRPV1, indicating that Ca2+-induced activation of PLC contributes to desensitization of TRPV1 by depletion of PtdIns(4,5)P2 and PtdIns(4)P. Selective conversion of PtdIns(4,5)P2 to PtdIns(4)P by a rapamycin-inducible PIP2 5-phosphatase did not inhibit TRPV1 at high capsaicin concentrations, suggesting a significant role for PtdIns(4)P in maintaining channel activity. Currents induced by low concentrations of capsaicin and moderate heat, however, were potentiated by conversion of PtdIns(4,5)P2 to PtdIns(4)P. Increasing PtdIns(4,5)P2 levels by coexpressing phosphatidylinositol-4-phosphate 5-kinase inhibited TRPV1 at low but not at saturating capsaicin concentrations. These data show that at low capsaicin concentrations and other moderate stimuli, PtdIns(4,5)P2 partially inhibits TRPV1 in a cellular context, but this effect is likely to be indirect, because it is not detectable in excised patches. We conclude that phosphoinositides have both inhibitory and activating effects on TRPV1, resulting in complex and distinct regulation at various stimulation levels.