High-affinity partial agonists of the vanilloid receptor

The vanilloid receptor VR1 is a polymodal nociceptor sensitive to capsaicin, protons, and heat. Because VR1 represents an attractive therapeutic target for conditions ranging from long-term pain to bladder hyperreflexia, we and other groups have sought to develop novel ligands with enhanced potencies and novel pharmacological properties. Here, we characterize two compounds, N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea (JYL827) and N-(4-tert-butylbenzyl)-N'-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea (JYL1511), that function as partial agonists for rat VR1 heterologously expressed in Chinese hamster ovary cells. Both compounds showed substantially enhanced potency, inhibiting [3H] resiniferatoxin binding with Ki values of 29.3 +/- 7.6 and 50.4 +/- 16.5 nM, respectively, compared with 1810 +/- 270 nM for capsaicin. The compounds showed different extents of partial agonism, 6.8 +/- 0.7% and 17.4 +/- 0.6%, respectively, and the expected corresponding degrees of partial antagonism (93.9 +/- 0.9 and 84.1 +/- 3.2%, respectively). Their IC50 values for antagonism of 45Ca2+ uptake in response to capsaicin were 67.3 +/- 24.9 nM and 3.4 +/- 0.5 nM, respectively. Protons, temperature, and protein kinase C all function as coactivators/modulators of rVR1. All enhanced the extent of partial agonism of JYL827 and JYL1511. Thus, at pH 5.5, for example, the extents of partial agonism increased to 54.9 +/- 2.5% and to 90.7 +/- 1.7%, respectively, relative to the response elicited by 300 nM capsaicin. The extents of partial antagonism decreased correspondingly. Compounds such as JYL827 and JYL1511 now permit exploration of the potential utility of partial agonists of rVR1 in animal models. Our results emphasize, moreover, the strong dependence of such partial agonists on other modulators of rVR1 and predict that their biological behavior will depend strongly on biological context.     

N-(3-acyloxy-2-benzylpropyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues: novel potent and high affinity antagonists and partial antagonists of the vanilloid receptor

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K(i) value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-2-benzylpropyl moieties in the C-region favored agonism.     

Pharmacological characterization of the TRPV1 receptor antagonist JYL1421 (SC0030) in vitro and in vivo in the rat

The TRPV1 capsaicin receptor is an integrator molecule on primary afferent neurones participating in inflammatory and nociceptive processes. The present paper characterizes the effects of JYL1421 (SC0030), a TRPV1 receptor antagonist, on capsaicin-evoked responses both in vitro and in vivo in the rat. JYL1421 concentration-dependently (0.1-2 microM) inhibited capsaicin-evoked substance P, calcitonin gene-related peptide and somatostatin release from isolated tracheae, while only 2 microM resulted in a significant inhibition of electrically induced neuropeptide release. Capsazepine (0.1-2 microM), as a reference compound, similarly diminished both capsaicin-evoked and electrically evoked peptide release. JYL1421 concentration-dependently decreased capsaicin-induced Ca(2+) accumulation in cultured trigeminal ganglion cells, while capsazepine was much less effective. In vivo 2 mg/kg i.p. JYL1421, but not capsazepine, inhibited capsaicin-induced hypothermia, eye wiping movements and reflex hypotension (a component of the pulmonary chemoreflex or Bezold-Jarisch reflex). Based on these data JYL1421 is a more selective and in most models also a more potent TRPV1 receptor antagonist than capsazepine, therefore it may promote the assessment of the (patho)physiological roles of the TRPV1 receptor.     

Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.     

Design of a high-affinity competitive antagonist of the vanilloid receptor selective for the calcium entry-linked receptor population

We describe the synthesis and characterization of N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (IBTU), a novel antagonist of the vanilloid receptor 1 (TRPV1 or VR1). IBTU competitively inhibited 45Ca2+ uptake into CHO cells heterologously expressing rat TRPV1, whether induced by capsaicin or resiniferatoxin (Ki = 99 +/- 23 and 93 +/- 34 nM, respectively). IBTU was thus somewhat more potent (5-fold) than capsazepine. In contrast to its antagonism of vanilloid-induced calcium uptake, IBTU (30 microM) inhibited [3H]resiniferatoxin binding to TRPV1 by less than 10%. We hypothesize that these dramatically distinct potencies reflect different fractions of TRPV1 in this system: namely, a minor plasma membrane fraction controlling 45Ca2+ uptake, and the predominant intracellular fraction that dominates the [3H]resiniferatoxin binding measurements. Intracellular Ca2+ imaging supports this explanation. IBTU antagonized the elevation in intracellular Ca2+ in response to 50 nM capsaicin with an IC50 of 106 +/- 35 nM. Likewise, 600 nM IBTU was able to antagonize the elevation in intracellular Ca2+ in response to 100 pM resiniferatoxin in the presence of normal (1.8 mM) extracellular Ca2+, where the increase in intracellular calcium reflects calcium influx. In contrast, in the absence of extracellular Ca2+, where in this system resiniferatoxin induces a modest increase in calcium from intracellular stores, IBTU was unable to block the response to resiniferatoxin, although the TRPV1 antagonist 5-iodoresiniferatoxin was able to do so. In summary, IBTU is a novel, potent TRPV1 antagonist with marked selectivity between subpopulations of TRPV1 and may permit the function of these distinct pools to be explored and potentially exploited.     

Cloning and functional characterization of the guinea pig vanilloid receptor 1

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat.Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.     

The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons.

[(3)H]Resiniferatoxin (RTX) binding and calcium uptake by rat dorsal root ganglion (DRG) neurons show distinct structure-activity relations, suggestive of independent vanilloid receptor (VR) subtypes. We have now characterized ligand binding to rat VR1 expressed in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells and compared the structure-activity relations with those for calcium mobilization. Human embryonic kidney cells (HEK293/VR1 cells) and Chinese hamster ovary cells transfected with VR1 (CHO/VR1 cells) bound [(3)H]RTX with affinities of 84 and 103 pM, respectively, and positive cooperativity (Hill numbers were 2.1 and 1.8). These parameters are similar to those determined with rat DRG membranes expressing native VRs (a K(d) of 70 pM and a Hill number of 1.7). The typical vanilloid agonists olvanil and capsaicin inhibited [(3)H]RTX binding to HEK293/VR1 cells with K(i) values of 0.4 and 4.0 microM, respectively. The corresponding values in DRG membranes were 0.3 and 2.5 microM. HEK293/VR1 cells and DRG membranes also recognized the novel vanilloids isovelleral and scutigeral with similar K(i) values (18 and 20 microM in HEK293/VR1 cells; 24 and 21 microM in DRGs). The competitive vanilloid receptor antagonist capsazepine inhibited [(3)H]RTX binding to HEK293/VR1 cells with a K(i) value of 6.2 microM and binding to DRG membranes with a K(i) value of 8.6 microM. RTX and capsaicin induced calcium mobilization in HEK293/VR1 cells with EC(50) values of 4.1 and 82 nM, respectively. Thus, the relative potencies of RTX (more potent for binding) and capsaicin (more potent for calcium mobilization) are similar in DRG neurons and cells transfected with VR1. We conclude that VR1 can account for both the ligand binding and calcium uptake observed in rat DRG neurons.     

Selective antagonism of capsaicin by capsazepine: evidence for a spinal receptor site in capsaicin-induced antinociception.

1. Capsazepine has recently been described as a competitive capsaicin antagonist. We have used this compound to test the hypotheses that the in vitro and in vivo effects of capsaicin are due to interactions with a specific receptor. 2. In an in vitro preparation of the neonatal rat spinal cord with functionally connected tail, the activation of nociceptive afferent fibres by the application of capsaicin, bradykinin or noxious heat (48 degrees C) to the tail could be measured by recording a depolarizing response from a spinal ventral root. Application of capsaicin or substance P to the spinal cord also evoked a depolarizing response which was recorded in a ventral root. 3. When capsazepine (50 nM-20 microM) was administered to the tail or spinal cord it did not evoke any measurable response. However on the tail, capsazepine reversibly antagonized (IC50 = 254 +/- 28 nM) the responses to capsaicin but not to heat or bradykinin administered to the same site. Similarly capsazepine administration to the spinal cord antagonized the responses evoked by capsaicin (IC50 = 230 +/- 20 nM) applied to the cord but not responses evoked by substance P on the cord or by noxious heat and capsaicin on the tail. 4. In halothane anaesthetized rats, C-fibre responses evoked by transcutaneous electrical stimulation of the receptive field were recorded from single wide dynamic range neurones located in the spinal dorsal horn. C-fibre evoked discharges were consistently reduced by the systemic administration of capsaicin (20 mumol kg-1, s.c.) and this action of capsaicin was antagonized by capsazepine (100 mumol kg-1) administered by the same route.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cytotoxicity of capsaicin in monkey kidney cells: lack of antagonistic effects of capsazepine and Ruthenium red

Capsaicin is a natural product of Capsicum peppers, excitatory effects of which have been shown to be mediated by the recently cloned vanilloid receptor 1 (VR1). Since previous studies have shown that capsaicin inhibits protein synthesis, experiments were performed to investigate whether this effect is mediated by VR1 receptor on cultured monkey kidney cells (Vero cells). The capsaicin uptake was assessed in cellular homogenate and in medium by high-performance liquid chromatography (HPLC) separation and quantification on C18 reverse-phase column and fluorescence detection. Toxic effects were assessed by incorporation of [³H]L-leucine into cellular proteins in the presence of capsazepine, the VR1 vanilloid receptor antagonist and Ruthenium red or tyrosine or calcium. Capsazepine (1 to 256 μM) did not modify the uptake rate of capsaicin for incubation times up to 24 h and did not antagonize capsaicin-induced protein synthesis inhibition. It rather inhibited protein synthesis per se from 100 to 256 μM. Ruthenium red which blocks mitochondrial calcium uptake, inhibited protein synthesis and did not antagonise or increase synergistically the effects of capsaicin. Interestingly in a medium deprived of calcium and supplemented by calcium chloride (10–50 μM) the protein synthesis inhibition induced by capsaicin is antagonised somehow. There was no prevention of capsaicin diffusion into the cells. Tyrosine, which seems to be the best preventive agent of capsaicin inhibitory effects, prevents its metabolism but not its diffusion. Capsaicin might enter cells by diffusion and interfere with protein synthesis machinery by competition with tyrosine which in turn prevents the metabolism of capsaicin. The results of the present study suggest that cell responses to capsaicin may be transduced through at least two molecular pathways, one involving VR1, since the receptor antagonist capsazepine fails to prevent the inhibitory effect of capsaicin in Vero cells of renal origin. Received: 27 August 1999 / Accepted: 3 November 1999     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.     

Temperature-dependent activation of recombinant rat vanilloid VR1 receptors expressed in HEK293 cells by capsaicin and anandamide.

Capsaicin activates vanilloid (VR1) receptors found on sensory neurons. These ligand-gated ion channels are also sensitive to low pH, elevated temperature and the endocannabinoid, anandamide. In this study, we have measured capsaicin- and anandamide-induced elevations in intracellular calcium concentrations ([Ca(2+)](i)) in fura-2 loaded HEK293 cells stably expressing the rat VR1 receptor at 22, 37 and 50 degrees C. Both capsaicin and anandamide produced a concentration-dependent elevation in [Ca(2+)](i) at all temperatures. pEC(50) values were 7.74 and 5.69 at 22 degrees C and 6.90 and 5.15 at 37 degrees C for capsaicin and anandamide, respectively. At 50 degrees C, the pEC(50) value for capsaicin was 6.36 but the response to anandamide did not saturate. Responses to both agonists were sensitive to ruthenium red and capsazepine at all temperatures. This temperature-dependent reduction in potency may result from desensitization.     

Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection

Vanilloid receptors subtype 1 (VR1), a nonselective cation channel responsive to capsaicin, protons, and noxious heat, has been recently identified in not only neural but also non-neural cells. In the present study, we demonstrated the peripheral expression of VR1 in gastric mucosal epithelial cells and investigated the role of the receptor in cellular protection. The rat gastric mucosal epithelial cell line was used. The expression of VR1 was examined by Western blotting and RT-PCR. Cell damage was induced by immersion in 10% ethanol or acid (pH 4.0) for 30 min, and cell viability was determined by MTT assay. Capsaicin or resiniferatoxin was added 30 min before the challenge with ethanol or acid, while capsazepine or ruthenium red (a VR1 antagonist) was added simultaneously with capsaicin. The distinct expression of VR1 protein and mRNA was detected in rat gastric mucosal epithelial cell line as well as in the rat stomach and spinal cord by Western blotting and RT-PCR, respectively. The cDNA sequence of the PCR product was found to be almost identical to that of the authentic VR1 (99.8%) when the product was subcloned and sequenced. On the other hand, the cell damage induced by ethanol or acid was dose-dependently prevented by pretreatment with capsaicin. The protective effect of capsaicin was mimicked by resiniferatoxin and almost totally abolished by co-addition of capsazepine or ruthenium red. These findings suggest that VR1 is expressed peripherally in gastric mucosal epithelial cells and plays a cellular protective role.     

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.     

Albaconol from the mushroom Albatrellus confluens induces contraction and desensitization in guinea pig trachea

The contraction and desensitization induced by albaconol and the influence of capsazepine, capsaicin and extracellular Ca2+ were investigated to see whether the actions were mediated via a specific VR receptor in guinea pig trachea spiral strips in vitro. Both albaconol and capsaicin were contractors of tracheal smooth muscle, but albaconol was not so potent as capsaicin, with -log (M) EC50 values of 4.23 +/- 0.18 (n = 10) and 7.33 +/- 0.21 (n = 10) respectively. 2.5 microM capsazepine competitively antagonized the contractile response to albaconol and capsaicin. Albaconol increased the contraction induced by a low dose of capsaicin (10(-10) to 10(-9) M), but non-competitively antagonized the contraction induced by a high dose of capsaicin (10(-8) to 10(-3) M). Either albaconol (1 or 100 mM) or capsaicin (3 or 10 microM) was able to desensitize the isolated guinea pig bronchi to subsequent addition of albaconol. Capsazepine (5.0 microM) significantly prevented the desensitization induced by either albaconol (1 or 100mM) or capsaicin (3 or 10 microM). Extracellular Ca2+ was essential for albaconol to induce excitation, but it did not affect albaconol- or capsaicin-induced desensitization. In summary, the results from the present study suggest that albaconol induces contraction and desensitization of guinea pig trachea in vitro as a partial agonist for VR.     

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.     

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1. Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2. Capsazepine (10 microM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 microM gamma-aminobutyric acid (GABA) and 5 microM adenosine 5'-triphosphate (ATP) were unaffected. 3. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 +/- 46 nM (mean +/- s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 microM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 +/- 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4. Capsazepine also inhibited the capsaicin- and RTX-evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of endothelial cell-derived CGRP in heat stress-induced protection of endothelial function

Previous studies have shown that heat stress possesses cardioprotection, which is related to the synthesis and release of calcitonin gene-related peptide (CGRP) via activation of capsaicin receptor (vanilloid receptor subtype 1, VR1) on the capsaicin-sensitive sensory neurons. The VR1 exists in human umbilical vein endothelial cells (HUVECs). Endothelial cells can synthesize CGRP and CGRP could protect against endothelial dysfunction induced by lysophosphatidylcholine (LPC) or oxidized low-density lipoprotein. In the present study, we explored whether the endothelial cell-derived CGRP is involved in the effect of heat stress on endothelial function in vivo and in vitro. Our results indicated that heat stress significantly increased the plasma concentration of CGRP, which was abolished by pretreatment with capsazepine, a VR1 antagonist. Immunohistochemistry and in situ hybridization showed that the endothelium of mesenteric artery and aorta expressed CGRP. And heat stress increased the expression of CGRP, which was also abolished by capsazepine. LPC attenuated the endothelium-dependent relaxation responses of aorta rings, which were improved by pretreatment with heat stress. In cultured HUVECs, the CGRP secretion was increased after heat stress. LPC increased the lactate dehydrogenase (LDH) activity in the cultured medium and decreased the cell viability, suggesting that LPC injured the HUVECs. However, pretreatment with heat stress attenuated the injurious effects of LPC on HUVECs. And this beneficial effect of heat stress on HUVECs was inhibited by capsazepine or CGRP(8-37), the CGRP receptor antagonist. The present results suggest that the endothelial cell-derived CGRP contributes to the protective effects of heat stress on endothelial function. Our study provides a potential mechanism to explain the protective effect of heat stress on cardiovascular system.     

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.     

Vanilloid (subtype 1) receptor-modulatory drugs inhibit [3H]batrachotoxinin-A 20-alpha-benzoate binding to Na+ channels

This investigation was conducted to provide further insight into the effects of vanilloid (subtype 1) receptor (VR1) drugs at voltage-gated sodium channels and examine the potential of this interaction to influence release of neurotransmitters from synaptosomes prepared from mammalian brain. The VR1 modulatory drugs capsaicin, olvanil and capsazepine inhibited the binding of batrachotoxinin-A 20-alpha-benzoate ([(3)H]BTX-B) to receptor site 2 of voltage-gated sodium channels. All drugs reduced the affinity of radioligand for sodium channels, and capsazepine also decreased the number of [(3)H]BTX-B binding sites. In kinetic experiments, no reduction in radioligand association rate was found, but capsaicin, olvanil and capsazepine all enhanced the dissociation rate of [(3)H]BTX-B. All drugs inhibited veratridine-evoked release of L-glutamic acid, gamma-amino butyric acid and L-aspartic acid from synaptosomes; however, their inhibitory effects on transmitter release were much weaker when 35 mM potassium chloride was used to depolarize synaptosomes. The study compounds, in common with other central nervous system depressants, interact with a region on the voltage-gated sodium channel that permits negative allosteric coupling with receptor site 2 and this mechanism likely accounts for blockade of sodium channel-activated transmitter release.