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.     

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.     

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.     

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.     

High affinity antagonists of the vanilloid receptor

The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tert-butylbenzyl)-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [(3)H]resiniferatoxin binding to rVR1 with an affinity of 53.5 +/- 6.5 nM and antagonized capsaicin-induced calcium uptake with an EC(50) of 9.2 +/- 1.6 nM, reflecting 25- and 60-fold greater potencies than capsazepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (<5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different activators could thus be differentially affected by different ligands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking response to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine.     

Capsaicin-like effects of N-arachidonoyl-dopamine in the isolated guinea pig bronchi and urinary bladder

A capsaicin-like endogenous ligand of vanilloid (VR1) receptors, N-arachidonoyl-dopamine, was recently identified in bovine and rat nervous tissue, and found to be almost as potent as capsaicin, and 5-10-fold more potent than anandamide, on these receptors, both in isolated cells and in vivo. Here we have investigated if N-arachidonoyl-dopamine also exerts other capsaicin-like effects at VR1 receptors in some isolated organ preparations. N-arachidonoyl-dopamine exerted a potent contractile response of guinea pig isolated bronchi (EC50=12.6 +/- 1.7 microM, Emax=69.2 +/- 2.4% of carbachol Emax), which was blocked by pre-treatment with capsaicin or with the VR1 antagonist capsazepine, as well as by a combination of tachykinin NK1 and NK2 receptor antagonists. In this assay, N-arachidonoyl-dopamine was less and more potent and/or efficacious than capsaicin (EC50=40.0 nM; Emax=93.5%) and anandamide (EC50=15.2 microM, Emax=38.0%), respectively. Unlike capsaicin and anandamide, forskolin or ethanol did not enhance N-arachidonoyl-dopamine effect in this preparation, whereas epithelial denudation resulted in a 2.5-fold increase in potency without affecting the efficacy. N-arachidonoyl-dopamine also contracted the isolated guinea pig urinary bladder, although in this preparation, as well as in the isolated rat urinary bladder, the potency (EC50=3.7 +/- 0.3 and 19.9 +/- 0.1 microM) and/or efficacy (Emax=12.0 +/- 0.1% and 20.7 +/- 0.7% of carbachol Emax) of the compound were significantly lower than those of both capsaicin and anandamide. These data suggest that the extent to which exogenous N-arachidonoyl-dopamine activates VR1 receptor in isolated organs is largely dependent on pharmacodynamics and bioavailability.     

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.     

TRPV1 activation results in disruption of the blood-brain barrier in the rat

We have examined the role of TRPV1 activation in disrupting the blood-brain barrier by measuring the permeability of single pial venular capillaries in anaesthetized rats. Capsaicin application to the brain surface resulted in increased permeability, maximal 2.1+/-0.12 x 10(-6) cm s(-1) (mean+/-s.e.m.) with log EC50 -4.5+/-0.10. Substance P methyl ester gave a similar response (maximal 2.0+/-0.07, n = 6, log EC50 -4.8+/-0.07), but the selective NK2 agonist, beta-Ala8-NKA(4-10) peptide, had no effect. Although CGRP decreased the permeability of venules (log EC50 10.3+/-0.11), its receptor antagonist CGRP(8-37) had no effect on the response to capsaicin. The TRPV1 antagonist capsazepine (1 mM) reduced the response to capsaicin (100 microM), from 1.78+/-0.15 to 0.63+/-0.10 (n = 4). The NK1 receptor antagonists GR205171 (100 microM) and SDZ NKT 376 (1 mM) also reduced the response to capsaicin (from 1.75+/-0.14 to 0.46+/-0.08; n = 6, and from 1.85+/-0.13 to 0.48+/-0.05; n = 5, respectively), indicating that capsaicin acts via TRPV1 in series with NK(1). Starch microspheres were used to produce transient focal ischaemia. Permeability was increased on reperfusion to a greater extent and more rapidly in vessels with diameter greater than 40 microm than those less than 15 microm. Capsazepine given intraperitoneally during ischaemia reduced the permeability increase in small venules from 5.9+/-0.3 to 2.4+/-0.1, and from 11.4+/-0.8 to 5.1+/-0.9 in large venules. In conclusion, the TRPV1 receptor is active in the brain microvasculature and has its permeability-increasing effect via substance P. It also plays a role in the immediate blood-brain barrier disruption following ischaemia-reperfusion.     

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)     

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.     

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.     

Vasorelaxant effects of oleamide in rat small mesenteric artery indicate action at a novel cannabinoid receptor

Oleamide (cis-9-octadecenoamide) exhibits some cannabimimetic responses despite its low affinities at the currently known cannabinoid receptors. Here we have investigated whether or not it is a vasorelaxant in rat small mesenteric arteries. Oleamide elicited vasorelaxation (EC50=1.2+/-0.2 microM, Rmax=99.1+/-3.9%, n=8) which was reduced by endothelial removal. Nitric oxide synthase inhibition reduced the response (EC50=5.3+/-1.6 microM, Rmax=59.2+/-7.7%, n=7; P<0.01) as did blockade of Ca2+-sensitive K+ channels (KCa) with apamin plus charybdotoxin (both 50 nM) (EC50=2.1+/-0.2 microM, Rmax=58.4+/-1.9%, n=5; P<0.05). Desensitisation of vanilloid receptors with capsaicin (10 microM for 30 min) shifted the oleamide concentration-response curve approximately 30-fold to the right (n=7; P<0.01). Pertussis toxin (400 ng ml-1 for 2 h) caused a two-fold shift in the response curve (EC50=2.2+/-0.4 microM, Rmax=66.8+/-4.5%, n=6; P<0.01). Rimonabant (CB1 cannabinoid receptor antagonist; SR141716A; 3 microM) significantly inhibited relaxation induced by oleamide (EC50=3.5+/-0.3 microM, Rmax=75.1+/-1.9%; n=8; P<0.05). In contrast, neither the more selective CB1 receptor antagonist, AM251 (1 microM), nor the CB2 antagonist, SR144528 (1 microM), had significant effects. O-1918 (10 microM), a putative antagonist at a novel endothelial cannabinoid receptor (abnormal-cannabidiol site), markedly reduced the relaxation to oleamide (n=7; P<0.01).It is concluded that oleamide responses in the rat isolated small mesenteric artery are partly dependent on the presence of the endothelium, activation of Ca2+-sensitive K+ channels (KC)) and involve capsaicin-sensitive sensory nerves. Oleamide may share a receptor (sensitive to rimonabant and O-1918, and coupled to KC) and Gi/o) with anandamide in this vessel. This might be distinct from both of the known cannabinoid receptors and the novel abnormal-cannabidiol site.     

Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

1. We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. 2. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. 3. The current-voltage relationship of piperine-activated currents showed pronounced outward rectification (25+/-4-fold between -70 and +70 mV) and a reversal potential of 0.0+/-0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. 4. Although piperine was a less potent agonist (EC50=37.9+/-1.9 microM) than capsaicin (EC50=0.29+/-0.05 microM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. 5. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230+/-20%, n=11) or capsaicin (284+/-32%, n=8) upon acidification to pH 6.5. 6. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t(1/2)=9.9+/-0.7 s) than capsaicin (t(1/2)>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. 7. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function.     

Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.     

Transient receptor potential vanilloid 1 (TRPV1) channels in cultured rat Sertoli cells regulate an acid sensing chloride channel

Sertoli cells provide a controlled microenvironment for regulation and maintenance of spermatogenesis for which an acidic milieu is crucial for male fertility. Sertoli cells also contribute to protection of spermatogenetic cells. Here, we showed that TRPV1 is expressed in rat Sertoli cells and regulates an acid sensing Cl(-) channel (ASCC). The expression of TRPV1 in rat Sertoli cells was demonstrated by RT-PCR, immunostaining and calcium measurement experiments. ASCC activity was inhibited by capsaicin (IC(50)=214.3+/-1.6 nM), olvanil (IC(50)=400+/-1.7 pM) and resiniferatoxin (IC(50)=9.3+/-1.5 nM) but potentiated by capsazepine (EC(50)=5.3+/-1.3 microM) and ruthenium red (EC(50)=2.3+/-1.5 microM). In the human airway epithelial cell line Calu-3 in which ASCC can be detected but not TRPV1, capsaicin and capsazepine were without any effect. Finally the application of the non-steroidal anti-inflammatory drug ibuprofen prevented the control of ASCC by TRPV1. Our study provides the first evidence for a regulation by TRPV1 of an acid sensing chloride channel in rat Sertoli cells. TRPV1 and ASCC may thus be considered as new potential physiological regulators of spermatogenesis and targets for pharmacological treatments of reproductive disorders as cryptorchidism, Sertoli cell tumors or torsion of the spermatic cord.     

Spiradoline, a kappa opioid receptor agonist, produces tonic- and use-dependent block of sodium channels expressed in Xenopus oocytes

Spiradoline, an arylacetamide kappa (kappa) opioid receptor agonist, produced a potent tonic block of rat neuronal (EC(50)= 34+/-5 microM) and heart (EC(50)= 183+/-13 microM) sodium channels and also blocked IFMQ3 mutant neuronal sodium channels (EC(50)= 130+/-34 microM) that lack fast inactivation when expressed in Xenopus oocytes. Spiradoline produced a hyperpolarizing shift in the voltage-dependence of sodium channel inactivation and exhibited a marked frequency-dependent component to blockade of sodium channels. The onset of open channel block of the IFMQ3 channel by spiradoline was best fit with a first-order blocking scheme, yielding an affinity constant of 116 +/- 33 microM. Thus, spiradoline blocks sodium channels by interacting with the major states of the channel which could result in local anesthetic action in nerves and antiarrhythmic action in the heart.     

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1. We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from rat isolated soleus muscle induced by capsaicin (1 microM), by superfusion with low pH medium (pH 5) or by KCl (80 mM). 2. Each one of the three stimuli tested produced a marked CGRP-LI release. Total evoked release (fmol g-1) was 482 +/- 69, 169 +/- 20 and 253 +/- 43 for capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 45 min preincubation) inhibited the evoked CGRP-LI release. Capsaicin-induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) capsazepine, respectively. 5. These findings demonstrate that capsazepine prevents low pH- and capsaicin-induced CGRP-LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP-LI release from peripheral endings of the capsaicin-sensitive primary afferent neurone.     

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.     

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)     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).