Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.     

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.     

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.     

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.     

Characterisation using microphysiometry of CRF receptor pharmacology.

We have assessed the utility of the Cytosensor microphysiometer for studying the pharmacology of recombinant CRF receptors. Chinese hamster ovary cells stably expressing the human CRF1 or CRF2 receptor were perfused in the Cytosensor with bicarbonate-free Hams F12 (pH 7.4) containing 0.2% bovine serum albumin. The rank order of potencies of agonist peptides were CRF = sauvagine = urocortin = urotensin at CRF1 (pEC50 values 11.16 +/- 0.17, 11.37 +/- 0.14, 11.43 +/- 0.09 and 11.46 +/- 0.13; n = 4), and urocortin = sauvagine > urotensin > CRF at CRF2 (pEC50 values 10.88 +/- 0.12, 10.44 +/- 0.05, 9.36 +/- 0.12 and 8.53 +/- 0.07; n = 7-9). alpha-Helical CRF (9-41) was a competitive antagonist at the CRF2 receptor (pK(B) = 6.99 +/- 0.08, n = 4), but was a partial agonist at the CRF1 receptor (pEC50 = 6.85 +/- 0.08, Emax = 33%, n = 3). CP 154,526 was a competitive antagonist at the CRF1 receptor (pK(B) = 8.17 +/- 0.05, n = 6), but was inactive at the CRF2 receptor. These data are consistent with established CRF receptor pharmacology and show that the Cytosensor is a viable method for assessing the functional activity of CRF-receptor agonists and antagonists.     

Physiology and pharmacology of the vanilloid receptor

The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia. Cumulative evidence is building a strong case for the involvement of this receptor in the etiology of both peripheral and visceral inflammatory pain, such as inflammatory bowel disease, bladder inflammation and cancer pain. The validation of TRPV1 receptor as a key therapeutic target for pain management has thrust intensive drug discovery programs aimed at developing orally active antagonists of the receptor protein. Nonetheless, the real challenge of these drug discovery platforms is to develop antagonists that preserve the physiological activity of TRPV1 receptors while correcting over-active channels. This is a condition to ensure normal pro-prioceptive and nociceptive responses that represent a safety mechanism to prevent tissue injury. Recent and exciting advances in the function, dysfunction and modulation of this receptor will be the focus of this review.     

Gingerols: a novel class of vanilloid receptor (VR1) agonists

1. Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin-activated VR1 (vanilloid) receptor. 2. [6]-Gingerol and [8]-gingerol evoked capsaicin-like intracellular Ca(2+) transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. 3. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. 4. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor.     

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.     

Role of vanilloid VR1 receptor in thermal allodynia and hyperalgesia in diabetic mice.

We examined the role of the vanilloid VR1 receptor in the thermal hyperalgesia and allodynia seen in diabetic mice. Tail-flick latencies at source voltages of 35 and 50 V for a 50-W projection bulb in diabetic mice were significantly shorter than those in non-diabetic mice. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with anti-vanilloid VR1 receptor serum. Intrathecal (i.t.) injection of anti-VR1 serum resulted in a significant increase in the tail-flick latency at 50 V in non-diabetic mice. However, i.t. pretreatment with anti-vanilloid VR1 receptor serum did not affect the tail-flick latency at a heat intensity of 35 V in non-diabetic mice. Thus, it seems likely that thermal allodynia and hyperalgesia in diabetic mice may be due to the sensitization of vanilloid VR1 receptors in primary sensory neurons in the spinal cord.     

Role of neuropeptide receptor systems in vanilloid VR1 receptor-mediated gastric acid secretion in rat brain

Previously, we reported that the injection of capsaicin into the lateral cerebroventricle (i.c.v.) stimulated gastric acid secretion via vanilloid VR1 receptors and the vagal cholinergic pathways in anesthetized rats. In the present study, we investigated the involvement of receptor systems for neurokinin A, calcitonin gene-related peptide (CGRP) and glutamate in the vanilloid VR1 receptor-mediated response. The i.c.v. injection of neurokinin A (30 nmol) stimulated gastric acid secretion in the presence of cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1-azabicyclo[2.2.2]octan-3-amine oxalate (L-703606, a tachykinin NK1 receptor antagonist, 30 nmol) and the effect was inhibited by cyclo[Gln-Trp-Phe-Gly-Leu-Met] (L-659877, a tachykinin NK2 receptor antagonist, 30 nmol); the values were 145.9 +/- 32.3 and 21.1 +/- 16.6 microEq HCl per 120 min, respectively. The value in the control group was 14.3 +/- 3.8 microEq HCl. The tachykinin NK2 receptor-mediated secretion was inhibited by i.c.v. injections of antagonists of the CGRP1 receptor (human CGRP fragment 8-37, 15 nmol) and non-N-methyl-D-aspartate (non-NMDA)-type glutamate receptor (6-cyano-7-nitroquinoxaline-2,3-dione, 10.9 nmol); the values were 30.8+/-29.8 and 5.7+/-16.9 microEq HCl, respectively. Gastric acid secretion induced by the i.c.v. injection of 30 nmol capsaicin (178.4 +/- 34.0 microEq HCl) was inhibited by antagonists of tachykinin NK2 (23.7 +/- 6.2) and CGRP1 (21.2 +/- 8.5), but not tachykinin NK1 (181.4 +/- 37.0), receptors. The gastric acid secretion induced by capsaicin was decreased by the i.c.v. pre-injection of low doses of neurokinin A or CGRP, which alone had no effect on the secretion. These findings suggest the involvement of tachykinin NK2, CGRP and non-NMDA receptor systems in the vanilloid VR1 receptor-mediated regulation of gastric acid secretion in the rat brain regions close to the lateral cerebroventricle.     

Hot channels in airways: pharmacology of the vanilloid receptor

Airway hyperresponsiveness of the tracheobronchial path is recognized as the critical feature of bronchial asthma. Sensory nerves in the airway are implicated strongly in this hyperresponsiveness. The vanilloid VR1 receptor, a cloned capsaicin receptor and a nociceptor-specific cation channel, is known to detect and transduce various harmful stimuli to electrical signals. Recent findings suggest that bradykinin can activate VR1 through generation of lipoxygenase products and that protein kinase C and phospholipase C mediate the sensitization of VR1 by many key inflammatory mediators. Such findings will lead to a better understanding of the enigmatic etiology of asthma.     

辣椒素受体研究进展

辣椒素受体（vanilloid receptor subtypel，VR1）是机体的一个重要的伤害性感受分子，除了参与中枢和外周水平疼痛的形成外，还参与了多种生理活动和病理过程。激活神经纤维末端的辣椒素受体，可调节外周血管的舒缩，影响血压；对缺血心肌起到保护作用；辣椒素受体还参与哮喘的发生，调节胃酸的分泌，促进胃肠蠕动。另外，激活辣椒素受体，还可抑制毛干的增殖，并介导肿瘤细胞的凋亡。本文主要就辣椒素受体近几年的研究情况作一简要综述。     

Cloned human EP1 prostanoid receptor pharmacology characterized using radioligand binding techniques

Prostaglandins such as prostaglandin E2 (PGE2) interact with EP-class prostanoid receptors including EP1, EP2, EP3 and EP4 subtypes. We have conducted a detailed pharmacological characterization of the binding of [3H]-PGE2 to recombinant human EP1 prostanoid receptors expressed in human embryonic kidney (HEK-293) cells using a broad panel of natural and synthetic prostanoids. The receptor displayed high affinity (Kd = 16.0 +/- 0.69 nM; n = 3) for [3H]-PGE2, and was expressed at high levels (Bmax =3.69 +/- 0.30 pmol (mg protein)(-1)) in cell membranes of HEK-293 cells. Specific binding constituted 97.5 +/- 1.4% (n = 12) of the total binding. In competition assays, the rank order of affinities of natural prostanoids for the receptor was PGE2 > PGE1 > PGF2 > PGI2 > PGD2. PGE2 was more effective than PGE1 at displacing bound [3H]-PGE2 (Ki for PGE2 = 14.9 +/- 2.2 nM; Ki for PGE1 = 165 +/- 29 nM). The affinities of enprostil (Ki = 14.5 +/- 3.1 nM) and 17-phenyl-omega-trinor-PGE2 (Ki = 7.3 +/- 2.7 nM) for the receptor were quite similar to that of PGE2, while that of sulprostone (Ki = 137 + 13 nM) more closely resembled PGE1. Some compounds historically classified as specific for DP prostanoid receptors bound with relatively high affinity to the recombinant human EP1 receptor (e.g. ZK118182 (K = 73.4 +/- 8.6 nM) and ZK110841 (K = 166 +/- 20 nM)). All FP (e.g. travoprost acid, fluprostenol), IP (iloprost) and TP (SQ29548) receptor-specific ligands exhibited low affinity (Ki > or = 1 microM).     

Resiniferatoxin-type phorboid vanilloids display capsaicin-like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1.

1 Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities. 2 Phorbol 12,13-didecanoate 20-homovanillate (PDDHV) evoked 45Ca(2+)-uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]-resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin-type pharmacology. 3 45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors. 4 PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]-RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non-transfected CHO cells, confirming its action through VR1. 5 We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin-type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure-activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin-type vanilloid pharmacology.     

TRPV1b, a functional human vanilloid receptor splice variant

Transient receptor potential (TRP) genes encode a family of related ion-channel subunits. This family consists of cation-selective, calcium-permeable channels that include a group of vanilloid receptor channels (TRPV) implicated in pain and inflammation. These channels are activated by diverse stimuli, including capsaicin, lipids, membrane deformation, heat, and protons. Six members of the TRPV family have been identified that differ predominantly in their activation properties. However, in neurons, TRPV channels do not account for the observed diversity of responses to activators. By probing human and rat brain cDNA libraries to identify TRPV subunits, we identified a novel human TRPV1 RNA splice variant, TRPV1b, which forms functional ion channels that are activated by temperature (threshold, approximately 47 degrees C), but not by capsaicin or protons. Channels with similar activation properties were found in trigeminal ganglion neurons, suggesting that TRPV1b receptors are expressed in these cells and contribute to thermal nociception.     

Discovering orphan receptor function using human in vitro pharmacology

mRNA encoding several recently discovered orphan receptors and their putative ligands has been identified in human tissues, suggesting the existence of new transmitter systems that may be important in the regulation of human cardiac and vascular function. Data obtained from in vitro pharmacological studies using healthy and pathological surgical samples of human arteries, veins and heart muscle are proving advantageous in the identification and characterisation of orphan receptor systems, such as urotensin-II, ghrelin, apelin and relaxin, which are most likely to provide novel targets for drugs to treat human cardiovascular disease.     

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.     

Agonist-dependent potentiation of vanilloid receptor transient receptor potential vanilloid type 1 function by stilbene derivatives

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.     

香草酸受体激动剂的研究进展

疼痛是很多疾病的常见症状，香草酸受体激动剂为治疗疼痛提供了一个新方向，部分药物已经进入临床研究阶段。现简单介绍了香草酸受体亚型1的结构及组织分布，并总结了近年来部分香草酸受体激动剂的研究状况。因为香草酸受体激动剂激动受体会产生一定的不良反应，因此目前用香草酸受体阻滞剂来治疗疼痛也开始受到关注。     

Functional characterisation of the S512Y mutant vanilloid human TRPV1 receptor

1 Mammalian transient receptor potential (TRP) channels include the nonselective cation channel TRPV1, which is activated by a range of stimuli including low pH, vanilloids and heat. Previously, selective mutagenesis experiments identified an intracellular residue (S512Y) critical to discriminating between pH and vanilloid (capsaicin) gating of the rat TRPV1 receptor. 2 In this study, switching the equivalent residue in the human TRPV1 (which has some significant differences with the rat TRPV1) also rendered this channel relatively insensitive to activation by capsaicin and proved critical in determining the receptor's sensitivity to the putative endovanilloid N-arachidonoyl-dopamine (NADA), suggesting a similar mode of activation for these two agonists. 3 Potency of pH gating was reduced; however, voltage-dependent outward rectification properties of the pH-dependent current and gating by heat and pH sensitisation of the S512Y heat response remained unaffected. 4 Surprisingly, residual capsaicin gating was detected and could be sensitised by pH even in the presence of a competitive antagonist. Taken together, these findings indicate that effective functional interaction of capsaicin with the S512Y channel still occurred, although the vanilloid-dependent gating per se was severely compromised. 5 This observation provides additional evidence for capsaicin interacting at multiple sites, distinct from the S512 residue located close to the intracellular face of the pore.