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The transient receptor potential (TRP) ion channel family is composed of twenty-seven channel proteins that are ubiquitously expressed in the human body. The TRPV (vanilloid) subfamily has been a recent target of investigation within the cardiovascular field. TRPV1, which is sensitive to heat as well as vanilloids, is the best characterized TRPV channel and is the namesake for the subfamily that includes six members. Research into the function of TRPV2 has suggested that it plays an important role in cardiovascular function. Over the last twenty years a greater understanding of the differences among the TRPV channels has allowed for more precise experimentation and has opened various translational opportunities. TRPV2 has been found to be a both a mechanosensor and a mediator of calcium handling and has been found to play important roles in healthy and diseased cardiomyocytes. These roles have been translated into clinical studies in patients with muscular dystrophy (both agonism and antagonism) as well as in patients with cardiomyopathy and heart failure with reduced ejection fraction. Its role as a structural protein has also been elucidated, though the clinical significance of this finding has yet to be established. Despite the clinical progress that has been made there is still a need for large, prospective randomized studies with TRPV2 channel agonists and antagonists in order to bring these basic and translational science findings to the bedside.

Human monocytes and dendritic cells express transient receptor potential vanilloid 1 (TRPV1) which may play a role in mediating the inflammatory, immune and cancer surveillance responses of these cells. The aim of the present study was to investigate TRPV1 expression and function in THP-1 monocytic cells. RT-PCR and Western blot were used to detect TRPV1. The metabolic activity and viability of THP-1 cells following exposure to vanilloids was assessed using resorufin production from rezazurin. Cytokine release was measured using ELISA. TRPV1 was expressed in cultured THP-1 monocytic cells and naïve monocytes. Lower concentrations (250 μM) of capsaicin, but not other putative TRPV1 agonists, were shown to stimulate cell metabolic activity, whereas at concentrations250 μM, all agonists decreased metabolic activity. Capsaicin-stimulated THP-1 metabolic activity was blocked by the TRPV1 antagonist, 5-iodo-resiniferatoxin (5'-IRTX), whereas the decline in resorufin production by THP-1 cells at higher capsaicin concentrations (due to cell death), was not affected by 5'-IRTX. Finally, capsaicin (≤125 μM) significantly increased lipopolysaccharide-stimulated IL-6 and TNF-α release from THP-1 cells, whereas phytohaemagglutinin-stimulated IL-1β, TNF-α, MCP-1 and IL-6 release were concentration-dependently inhibited by capsaicin. Modulation of IL-1β release was not TRPV1 mediated. Overall, these results show that functional TRPV1 channels are present in naïve monocytes and THP-1 cells, and when activated, increase cell metabolic activity. In addition, capsaicin modifies cytokine release from THP-1 cells and induces cell death, most likely by a mechanism that is independent of TRPV1 activation.

Both a silent resident phosphatidylinositol lipid and a “hot” vanilloid agonist capsaicin or resiniferatoxin have been shown to share the same inter-subunit binding pocket between a voltage sensor like domain and a pore domain in TRPV1. However, how the vanilloid competes off the resident lipid for allosteric TRPV1 activation is unknown. Here, the in sillico research suggested that anchor-stereoselective sequential cooperativity between an initial recessive transient silent weak ligand binding site and a subsequent dominant steady-state strong ligand binding site in the vanilloid pocket may facilitate the lipid release for allosteric activation of TRPV1 by vanilloids or analogs upon non-covalent interactions. Thus, the resident lipid may play a critical role in allosteric activation of TRPV1 by vanilloid compounds and analogs., Highlights • Four active vanilloid binding pockets as revealed by the cryo-EM structure of TRPV1 have no cooperativity between subunits. • Allosteric activation of TRPV1 by vanilloid compounds and analogs is lipid-dependent and anchor-stereoselective. • The resident and occluded lipid must be competed off for allosteric activation of TRPV1 by vanilloid compounds and analogs. • The first ligand binding is needed to release the resident lipid for the second ligand binding in the vanilloid pocket. • A lipid-free anchor facilitates the vanilloid ligand binding to remove the resident lipid from the active site in TRPV1. • Site accessibility controls sequential cooperative interactions of TRPV1 with vanilloids or analogs to open the channel. • The anchor stereoselectivity depends on the formation of a vanilloid bridge between two separated residues at the active site. • Different anchor stereoselectivities produce diverse recessive transient reaction intermediates or steps for TRPV1 opening. • Membrane hyperpolarization and depolarization may stabilize and loosen the resident lipid, for TRPV1 gating, respectively. • Phospholipids at N- and C- terminal domains may affect the cooperativity or the potency of the vanilloid ligands.

Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium.

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na⁺ and Ca2 + influx via TRPV1 channels, which leads to mitochondrial Ca2 + accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist- induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid- sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau- type Ca2 + signals, mitochondrial Ca2 + accumulation and Na⁺- and Ca2 +-dependent membrane disorganization. Moreover, long- term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.

Cloning of the transient receptor potential vanilloid type 1 (TRPV1), the heat-gated cation channel/capsaicin receptor expressed by sensory neurons, has opened the door for development of new types of analgesics that selectively act on nociceptors. Here we summarize mutagenetic evidence for selective loss of responsiveness to vanilloids, protons, and heat stimuli to provide clues for avoiding on-target side effects of hyperthermia and burn risk. It is suggested that the complex chemoceptive thermosensor function of TRPV1 (which is modulated by depolarizing stimuli) can be attributed to multisteric gating functions. In this way, it forms the prototype of a new class of ion channels different from the canonical voltage-gated and ligand-gated ones. Several endogenous lipid ligands activate and inhibit TRPV1 and its gating initiates sensory transducer and mediator-releasing functions. Second generation TRPV1 antagonists that do not induce hyperthermia are under development, and a dermal capsaicin patch is already on the market for long-term treatment of neuropathic pain.

The structure–activity relationships of N-(3-acyloxy-2-benzylpropyl)-N′-4-[(methylsulfonylamino)benzyl] thioureas, which represent simplified RTX-based vanilloids, were investigated by varying the distances between the four principal pharmacophores and assessing binding and antagonistic activity on rTRPV1. The analysis indicated that a 3-pivaloyloxy-2-benzylpropyl C-region conferred the best potency in binding affinity and antagonism. The molecular modeling of this best template with the tetrameric homology model of rTRPV1 was performed to identify its binding interactions with the receptor.

Quinone reductase (QR) inducing activity, a biomarker for Phase II enzyme induction, was detected in a crude ginger rhizome ethanolic extract using a murine hepatoma cell (Hepa 1c1c7) bioassay. An ethyl acetate-partitioned fraction became enriched in QR inducing activity with a CD value (concentration required to double QR specific activity) of 7 μg/ml and was further separated by silica flash chromatography. The four major fractions recovered exhibited CD values ranging 10–20 μg/mL for QR inducing activity. 1 H NMR analysis revealed an abundance of gingerols in the first 3 fractions, and this led to a comparative assessment of QR inducing and anti-inflammatory activities among [6]-gingerol and structurally related vanilloids from ginger and other spices. [6]-Dehydroshogaol (DHSG), a minor component of fresh ginger, had QR inducing activity (CD = 8.4 μM) of comparable potency to the well known cancer preventive agent, curcumin (CD = 4.3 μM). In contrast, [6]-gingerol the major pungent component of ginger showed less potent QR inducing activity (CD = 55 μM) and capsaicin exhibited the weakest inducing activity (CD = 103 μM). [6]-Shogaol, [6]-DHSG and curcumin were also potent inhibitors of nitric oxide (NO) synthesis, inhibiting 50% NO evolution in activated macrophages at levels of 10–12 μM (IC 50 values). Comparatively, IC 50 values for [6]-gingerol and capsaicin were about an order of magnitude greater (less potent) at 90 μM and 169 μM, respectively. This appears to be the first report of biological activities of [6]-DHSG, and Phase II enzyme-inducing ability of shogoal and gingerol.

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.

Preclinical studies suggest that the vanilloid receptor (TRPV1) is an important component of several disease areas such as pain (inflammatory, visceral, cancer and neuropathic), airway disease (including chronic cough), inflammatory bowel disease (IBD), interstitial cystitis, urinary incontinence, pancreatitis and migraine. TRPV1 is a member of a distinct subgroup of the transient receptor potential (TRP) family of ion channels. The neuronally expressed TRPV1 is a non-selective, Ca(2+)-preferring, cation channel. In addition to capsaicin, this channel is activated by a number of different stimuli including heat, acid, certain arachidonic acid derivatives and direct phosphorylation via protein kinase C (PKC). Moreover, there is also evidence that various inflammatory mediators such as adenosine triphosphate (ATP), bradykinin, nerve growth factor (NGF) or prostaglandin E(2) (PGE(2)) may indirectly lead to activation of the TRPV1 channel via activation of their respective receptors. There is strong experimental evidence that the combination of direct and indirect mechanisms finely tune the TRPV1 activity. Each of the different known modes of direct TRPV1 activation (protons, heat and vanilloids) is capable of sensitising the channel to other agonists. Similarly, inflammatory mediators from the external milieu found in disease conditions can indirectly sensitise the receptor. It is this sensitisation of the TRPV1 receptor in inflammatory disease that could hold the key and contribute to the transduction of noxious signalling for normally innocuous stimuli, i.e. either hyperalgesia in the case of chronic pain or airway hyperresponsivness/hypertussive responses in patients with chronic cough. It seems reasonable to suggest that the various mechanisms for sensitisation provide a scenario for TRPV1 to be tonically active and this activity may contribute to the underlying pathology -- providing an important convergence point of multiple pain producing stimuli in the somatosensory system and multiple cough-evoking irritants in the airways. The complex mechanisms and pathways that contribute to the pathophysiology of chronic pain and chronic cough have made it difficult for clinicians to treat patients with current therapies. There is an increasing amount of evidence supporting the hypothesis that the expression, activation and modulation of TRPV1 in sensory neurones appears to be an integral component of pain and cough pathways, although the precise contribution of TRPV1 to human disease has yet to be determined. So the question remains open as to whether TRPV1 therapeutics will be efficacious and safe in man and represent a much needed novel pain and cough therapeutic.

Transient receptor potential V1 (TRPV1) is a nonselective cation channel expressed in nociceptors and activated by capsaicin. TRPV1 detects diverse stimuli, including acid, heat, and endogenous vanilloids, and functions as a molecular integrator of pain perception. Herein we demonstrate a novel regulatory role of extracellular Na+ ([Na+]o) on TRPV1 function. In human embryonic kidney 293 cells expressing porcine TRPV1, low [Na+]o evoked increases of [Ca2+]i that were suppressed by TRPV1 antagonists and facilitated responses to capsaicin, protons, heat, and an endovanilloid. [Na+]o removal simultaneously elicited a [Ca2+]i increase and outward-rectified current with a reversal potential similar to those of capsaicin. Neutralization of the two acidic residues which confer the proton sensitivity to TRPV1 resulted in a reduction of low [Na+]o-induced responses. In primary culture of porcine sensory neurons, the removal of [Na+]o produced a [Ca2+]i increase and current responses only in the cells responding to capsaicin. Low [Na+]o evoked a [Ca2+]i increase in sensory neurons of wild type mice, but not TRPV1-null mice, and in human embryonic kidney 293 cells expressing human TRPV1. The present results suggest that [Na+]o negatively regulates the gating and polymodal sensitization of the TRPV1 channel. [Na+]o surrounding several proton-sensitive sites on the extracellular side of the pore-forming loop of the TRPV1 channel may play an important role as a brake to suppress the excessive activity of this channel under physiological conditions.

The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S- and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoamino acids or vanilloids or may convert them to bioactive derivatives.

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.

Starting from ROPA (2), analogues of RTX (1a) modified on the acyl side chain were prepared and evaluated for vanilloid activity in HEK-293 cells over-expressing the human recombinant TRPV1. The ROPA motif provided an enhancement of potency sufficient to expand the range of vanillyl surrogates to structural elements (e.g., an unsubstituted phenyl ring) that afford inactive analogues in compounds from the capsaicin series.

The present review focuses on promising new opportunities for anti-inflammatory and analgesic therapy. The theoretical background is an original observation based on our own experimental results. These data demonstrate that somatostatin is released from capsaicin-sensitive, peptidergic sensory nerve endings in response to noxious heat and chemical stimuli such as vanilloids, protons or lipoxygenase products. It reaches distant parts of the body via the circulation and exerts systemic anti-inflammatory and analgesic effects. Somatostatin binds to G-protein-coupled membrane receptors (sst(1)-sst(5)) and diminishes neurogenic inflammation by prejunctional action on sensory-efferent nerve terminals, as well as by postjunctional mechanisms on target cells. It decreases the release of pro-inflammatory neuropeptides from sensory nerve endings and also acts on receptors of vascular endothelial, inflammatory and immune cells. Analgesic effect is mediated by an inhibitory action on peripheral terminals of nociceptive neurons, since circulating somatostatin cannot exert central action. Somatostatin itself is not suitable for drug development because of its broad spectrum and short elimination half life, stable, receptor-selective agonists have been synthesized and investigated. The present overview is aimed at summarizing the physiological importance of somatostatin and sst receptors, pharmacological significance of synthetic agonists and their potential in the development of novel anti-inflammatory and analgesic drugs. These compounds might provide novel perspectives in the pharmacotherapy of acute and chronic painful inflammatory diseases, as well as neuropathic conditions.

Recent findings show that the vanilloid receptor subtype 1 (TRPV1) is expressed by cochlear outer hair cells and spiral ganglion cells, and that its expression is up-regulated in ganglion cells after aminoglycoside treatment. This study tested the hypothesis that agents that act on TRPV1 receptors affect the spectrum of ensemble background activity (EBA). Consecutive intracochlear perfusions of the TRPV1 agonist, capsaicin (CAP 0.1, 1, and 10 parts per million), as well as its antagonist capsazepine (CZP), were used to test effects of TRPV1 activation on EBA recorded from the cochlear base. Perfusion with CAP alone produced a dose-dependent increase of the 900-Hz peak ratio (power normalized re the overall spectrum) of the EBA. The CAP effect was attenuated during concurrent perfusion with CZP. These findings are consistent with the hypothesis that TRPV1 activation increases background activity of spiral ganglion cells and support a role of TRPV1 in gating spontaneous and evoked auditory nerve excitability.

Vanilloid receptor subtype-1 (TRPV1), the founding member of the vanilloid receptor-like transient receptor potential channel family, is a non-selective cation channel that responds to noxious stimuli such as low pH, painful heat and irritants. In the present study, we show, as means of reverse transcriptase-polymerase chain reaction and Western blot analysis, that the vanilloid TRPV1 receptor is expressed in the prostate epithelial cell lines PC-3 and LNCaP as well as in human prostate tissue. The kinetic parameters inferred from [(125)I]-resiniferatoxin binding were in concordance with data of TRPV1 receptors expressed in other tissues. The contribution of the endogenously expressed TRPV1 channel to intracellular calcium concentration increase in the prostate cells was studied by measuring changes in Fura-2 fluorescence by fluorescence microscopy. Addition of capsaicin, (R)-methanandamide and resiniferatoxin to prostate cells induced a dose-dependent increase in the intracellular calcium concentration that was reversed by the vanilloid TRPV1 receptor antagonist capsazepine. These results indicate that the vanilloid TRPV1 receptor is expressed and functionally active in human prostate cells.

Capsaicin, the pungent ingredient of chilli peppers has become a "hot" topic in neuroscience with yearly publications over half thousand papers. It is outlined in this survey how this exciting Hungarian research field emerged from almost complete ignorance. From the initial observation of the phenomenon of "capsaicin desensitization", a long-lasting chemoanalgesia and impairment in thermoregulation against heat, the chain of new discoveries which led to the formulation of the existence of a "capsaicin receptor" on C-polymodal nociceptors is briefly summarized. Neurogenic inflammation is mediated by these C-afferents which are supplied by the putative capsaicin receptor and were termed as "capsaicin sensitive" chemoceptive afferents. They opened new avenues in local peptidergic regulation in peripheral tissues. It has been suggested that in contrast to the classical axon reflex theory, the capsaicin-sensitive sensory system subserves a "dual sensory-efferent" function whereby initiation of afferent signals and neuropeptide release are coupled at the same nerve endings. Furthermore, in the skin at threshold stimuli which do not evoke sensation elicit already maximum efferent response as enhanced microcirculation. In isolated organ preparations large scale of new type of peptidergic capsaicin-sensitive neurogenic smooth muscle responses were revealed after the first one was described by ourselves on the guinea-pig ileum in 1978. Recently the "capsaicin receptor" has been cloned and it is now named as the "transient receptor potential vanilloid 1" (TRPV1). Hence, capsaicin research led to the discovery of the first temperature-gated ion channel gated by noxious heat, protons, vanilloids and endogenous ligands as anandamide, N-oleoyldopamine and lipoxygenase products. Another recent achievement is the discovery of a novel "unorthodox" neurohumoral regulatory mechanism mediated by somatostatin. Somatostatin released from the TRPV1-expressing nerve endings reaches the circulation and elicits systemic antiinflammatory and analgesic "sensocrine" functions with counter-regulatory influence e.g. in Freund's adjuvant-induced chronic arthritis. Nociceptors supplied by TRPV1 and sst4 somatostatin receptors has become nowadays promising targets for drug development.

Capsaicin, the pungent ingredient in red pepper, has been used since ancient times as a spice, despite the burning sensation associated with its intake. More than 50 years ago, Nikolaus Jancso discovered that capsaicin can selectively stimulate nociceptive primary afferent neurons. The ensuing research established that the neuropharmacological properties of capsaicin are due to its activation of the transient receptor potential ion channel of the vanilloid type 1 (TRPV1). Expressed by primary afferent neurons innervating the gut and other organs, TRPV1 is gated not only by vanilloids such as capsaicin, but also by noxious heat, acidosis and intracellular lipid mediators such as anandamide and lipoxygenase products. Importantly, TRPV1 can be sensitized by acidosis and activation of various pro-algesic pathways. Upregulation of TRPV1 in inflammatory bowel disease and the beneficial effect of TRPV1 downregulation in functional dyspepsia and irritable bladder make this polymodal nociceptor an attractive target of novel therapies for chronic abdominal pain.

The cloning of the vanilloid receptor 1 opened a floodgate for discoveries regarding the function of this complex molecule. It has been found that, in addition to heat, protons and vanilloids, this receptor also responds to various endogenous ligands. Furthermore, it has been also emerged that, through associations with other molecules, the vanilloid receptor 1 plays an important role in the integration of various stimuli and modulation of cellular excitability. Although, originally, the vanilloid receptor 1 was associated with nociceptive primary afferent fibres, it has been gradually revealed that it is broadly expressed in the brain, epidermis and visceral cells. The expression pattern of the vanilloid receptor 1 indicates that it could be involved in various physiological functions and in the pathomechanisms of diverse diseases. Here, we summarise the molecular, pharmacological and physiological characteristics, and putative functions, of the vanilloid receptor 1, and discuss the therapeutic potential of this molecule.

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.

Vanilloids are pain evoking molecules that serve as ligands of the ‘heat and capsaicin receptor’ TRPV1. It is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat (>42°C) and various noxious chemicals. TRPV1 channel is formed by the symmetrical arrangement of four identical subunits around a central ion-conducting pore. Binding of vanilloids evokes channel opening and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation by vanilloids is not fully understood. Here, we combine the use of site-directed mutagenesis, concatemeric constructs harboring mutated binding sites with patch-clamp recordings and calcium imaging in order to determine the stoichiometry and activation mechanism of TRPV1 by the exo-vanilloid capsaicin, the ‘hot’ component of chili peppers. We show that a single capsaicin-bound subunit is sufficient to achieve a maximal open-channel lifetime, while the high sensitivity for this agonist requires all four subunits. Moreover, we defined the role of residues within TRPV1 vanilloid binding site in the channel activation mechanism, by acting to stabilize ligand-receptor interaction.

Temperature affects functions of all ion channels, but few of them can be gated directly. The vanilloid receptor VR1 provides one exception. As a pain receptor, it is activated by heat42 degrees C in addition to other noxious stimuli, e.g. acids and vanilloids. Although it is understood how ligand- and voltage-gated channels might detect their stimuli, little is known on how heat could be sensed and activate a channel. In this study, we characterized the heat-induced single-channel activity of VR1, in an attempt to localize the temperature-dependent components involved in the activation of the channel. At42 degrees C, openings were few and brief. Raising the ambient temperature rapidly increased the frequency of openings. Despite the large temperature coefficient of the apparent activity (Q(10) approximately 27), the unitary current, the open dwell-times, and the intraburst closures were all only weakly temperature dependent (Q(10)2). Instead, heat had a localized effect on the reduction of long closures between bursts (Q(10) approximately 7) and the elongation of burst durations (Q(10) approximately 32). Both membrane lipids and solution ionic strength affected the temperature threshold of the activation, but neither diminished the response. The thermodynamic basis of heat activation is discussed, to elucidate what makes a thermal-sensitive channel unique.

Capsaicin, the pungent ingredient of hot peppers, has long been used to identify nociceptors. Its molecular target, the vanilloid receptor VR1, was recently cloned and confirmed functionally as a polymodal detector of multiple pain stimuli: heat, acid, and vanilloids. Previous electrophysiology studies have focused on whole-cell characteristics of the receptor. Here, we provide the first in-depth single-channel kinetic study of VR1 to understand its activation mechanism. At low to medium concentrations, channel activity appeared as bursts. Not only did the durations of the interburst gaps vary with capsaicin, the bursts also appeared ligand-dependent, with high capsaicin prolonging bursts and stabilizing openings. Gating involved at least five closed and three open states, with strong correlations between short closures and long openings, and long closures and short openings. Increasing capsaicin reduced the long closures with little effect on short ones. The open time constants changed little with capsaicin concentration, though their relative proportions varied. These results suggest that 1), the channel contains multiple capsaicin binding sites; 2), both partial and full binding are capable of opening the channel; 3), when activated, multiple open states are accessible irrespective of the level of binding; and 4), capsaicin association occurs preferentially to the closed channel.

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca2+ uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKCα in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCβ, -δ, -e, and −ζ. Loss of PKCα correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKCα, but not PKCe. These studies suggest that PKCα in sensory neurons may elicit or enhance pain during inflammation or ischemia.

Vanilloid sensitivity is a functional signature of a subset of unmyelinated fibres innervating the urinary bladder. The role that these nerves have in the physiological control of storage and voiding is unclear. However, after the bladder has been disconnected by spinal injury from the pontine micturition centre, vanilloid-sensitive fibres assume a central role in the reflex emptying of the bladder that occurs at low volumes. Intravesical vanilloid (capsaicin or resiniferatoxin) administration is beneficial in this disorder by "desensitising" these nerves. Resiniferatoxin is superior to capsaicin in terms of its tolerability profile. Investigators are moving rapidly to identify the mechanisms by which desensitisation to vanilloids occurs. Vanilloids induce lasting, but fully reversible, changes in gene expression, including downregulation of the vanilloid receptor subtype 1. It is hoped that application of gene chip technologies will address the global profile of vanilloid-induced changes in gene expression and their relative contribution to desensitisation. Drugs that target signalling mechanisms that bring about these changes in gene expression have obvious therapeutic potential.

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

The potential of resiniferatoxin and capsaicin to modulate emesis and genital grooming was investigated in Suncus murinus. Resinifertoxin (3-30 nmol, i.c.v.), E-capsaicin (10-100 nmol, i.c.v.) and Z-capsaicin (100 nmol, i.c.v.) induced emesis (P0.05) and subsequently antagonised the emetic response induced by intragastric copper sulphate (480.6 micromol/kg; P0.05). However, resiniferatoxin failed to affect nicotine-induced (30.7 mol/kg, s.c.) emesis (P0.05). Only resiniferatoxin induced genital grooming that was antagonised (P0.05) by capsazepine (300-600 nmol, i.c.v.) and ruthenium red (3 nmol, i.c.v.). E-capsaicin-induced emesis was antagonised by capsazepine (300-600 nmol, i.c.v.; P0.05) and ruthenium red (3 nmol, i.c.v.; P0.05) but resiniferatoxin-induced emesis was resistant to capsazepine (30-600 nmol, i.c.v.; P0.05). The emetic action of resiniferatoxin but not E-capsaicin was subject to tachyphylaxis. In cross-tachyphylaxis experiments, E-capsaicin reduced the genital grooming induced by resiniferatoxin (P0.05). The data are discussed in relation to the classification of vanilloid receptors and mechanisms involved in emesis and genital grooming.

The real time dynamics of vanilloid-induced cytotoxicity and the specific deletion of nociceptive neurons expressing the wild-type vanilloid receptor (VR1) were investigated. VR1 was C-terminally tagged with either the 27-kDa enhanced green fluorescent protein (eGFP) or a 12-amino acid epsilon-epitope. Upon exposure to resiniferatoxin, VR1eGFP- or VR1epsilon-expressing cells exhibited pharmacological responses similar to those of cells expressing the untagged VR1. Within seconds of vanilloid exposure, the intracellular free calcium ([Ca(2+)](i)) was elevated in cells expressing VR1. A functional pool of VR1 also was localized to the endoplasmic reticulum that, in the absence of extracellular calcium, also was capable of releasing calcium upon agonist treatment. Confocal imaging disclosed that resiniferatoxin treatment induced vesiculation of the mitochondria and the endoplasmic reticulum ( approximately 1 min), nuclear membrane disruption (5-10 min), and cell lysis (1-2 h). Nociceptive primary sensory neurons endogenously express VR1, and resiniferatoxin treatment induced a sudden increase in [Ca(2+)](i) and mitochondrial disruption which was cell-selective, as glia and non-VR1-expressing neurons were unaffected. Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory ganglia, may be employed to rapidly and selectively delete nociceptive neurons.

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. Here, we describe specific vanilloid responses in rat C6 glioma cells. Capsaicin and RTX stimulated 45 Ca uptake in a similar fashion to that found for cultured rat dorsal root ganglion neurons (DRGs); this response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of C6 cells with capsaicin or RTX produced desensitization to subsequent stimulation of 45 Ca uptake. The potency for desensitization by RTX in the C6 cells corresponded to that for 45 Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high affinity [ 3 H]RTX binding site. Consistent with this difference, in C6 cells we were unable to detect [ 3 H]RTX binding. These characteristics suggest the presence of C-type but not R-type vanilloid receptors on C6 cells. After 2 day treatment, capsaicin but not RTX inhibited the proliferation and altered the differentiation of the cells and produced apoptosis. In the long term experiments, capsazepine, instead of antagonizing the effect of capsaicin, acted as an agonist. Moreover, capsazepine displayed these effects with higher potency than that of capsaicin. The different potencies and structure activity relations suggest a distinct mechanism for these long-term vanilloid effects. Our finding that C6 cells can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of response to vanilloids, and highlights the importance of the neuron–glial network.

Inhalation cough challenge is a useful tool in the physiological and pharmacological investigation of the cough reflex. There are three types of challenge used, capsaicin and the vanilloids, organic acids, and distilled water challenge. In addition, challenge may be administered in a single dose or cumulative dose fashion. The correct combination of inhalation technology and tussive stimulus produces a sensitive and specific method for estimating the cough reflex.

The TRPV1 channel in sensory neurons integrates pain-producing stimuli, including noxious temperature, acidosis, pungent vanilloid compounds and pro-inflammatory lipids. Measurements of TRPV1 channel activity at extreme positive and negative voltages in the presence of other stimuli suggest that the channel has a unique opening transition that is allosterically modulated by independent stimulus-sensing domains. On the contrary, recently obtained near-atomic resolution structures of TRPV1 in different states suggest that different agonists promote distinct open states, by differentially modulating the inner pore gate or the selectivity filter. Finally, it has been proposed that the opening of the pore may confer the channel with high temperature-sensitivity. Here we show that extracellular sodium ions tune both temperature-dependent activation and inactivation in TRPV1 channels to maintain sensitivity to noxious stimuli at physiological temperatures. Exploration of temperature-dependent activation over a wide range of temperatures in the presence of distinct modulators demonstrates that the gating mechanism involves temperature-sensitive and -insensitive transitions that are allosterically coupled and differentially modified by sodium, pH, vanilloids and tarantula toxins. We observe that the channels exhibit non-maximal open probability and temperature-independent gating in the absence of external sodium or in the presence of double knot tarantula toxin DkTx, indicating that the opening transition is temperature-independent and supporting the modular allosteric view of this channel protein. This unique gating mechanism provides a framework for understanding how the temperature sensitivity of TRPV1 is tuned by a wide array of regulators.

Specific [3H]resiniferatoxin binding is thought to represent the postulated vanilloid (capsaicin) receptor. We have previously characterized [3H]resiniferatoxin binding to membranes from rat and pig dorsal root ganglia, which contain the cell bodies of capsaicin-sensitive primary afferent neurons. We now demonstrate specific binding of [3H]resiniferatoxin to particulate preparations from pig dorsal horn, which contains the central nerve endings of the capsaicin-sensitive primary afferent neurons. The Kd was 0.27 ± 0.03 nM; the Bmax was 370 ± 40fmol/mg protein. Vanilloids of the capsaicin class (capsaicin, piperine, zingerone) and resiniferatoxin class (tinyatoxin, 12-deoxyphorbol 13-phenylacetate 20-homovanillate) inhibited binding with affinities consistent with their relative in vivo potencies. Given the interest in vanilloids as potential non-narcotic analgesic agents, this binding assay affords an attractive approach for characterization of structure-activity relations at spinal vanilloid receptors.

Capsaicin, the pungent constituent of chili peppers, represents the paradigm for the capsaicinoids or vanilloids, a family of compounds shown to stimulate and then desensitize specific subpopulations of sensory receptors, including C-polymodal nociceptors, A-delta mechanoheat nociceptors and warm receptors of the skin, as well as enteroceptors of thin afferent fibers. An exciting recent advance in the field has been the finding that resiniferatoxin (RTX), a naturally occurring diterpene containing a homovanillic acid ester, a key structural motif of capsaicin, functions as an ultrapotent capsaicin analog. For most of the responses characteristic of capsaicin, RTX is 100-10,000 fold more potent. Structure/activity analysis indicates, however, that RTX and related homovanillyl-diterpene esters display distinct spectra of activity. Specific [3H]RTX binding provides the first direct proof for the existence of vanilloid receptors. We expect that the RTX class of vanilloids will promote rapid progress in understanding of vanilloid structure/activity requirements and mechanism.