Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.     

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.     

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.     

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.     

Respiratory actions of vanilloid receptor agonists in the nucleus of the solitary tract: comparison of resiniferatoxin with non-pungent agents and anandamide

1. Activation of vanilloid receptors on sensory nerve terminals in the commissural nucleus of the solitary tract (cNTS) of rats with capsaicin, produces respiratory slowing. In this study, we used microinjection techniques employing pungent and non-pungent vanilloids to further characterize vanilloid receptors in the cNTS. 2. Microinjection of the pungent vanilloid, resiniferatoxin (RTX), into the cNTS of urethane-anaesthetized rats, dose-dependently reduced respiratory rate without affecting tidal volume. RTX was 20 fold more potent at slowing respiration ( approximately ED(50), 100 pmol) than capsaicin ( approximately ED(50), 2 nmol). Doses of RTX greater than 100 pmol caused either irregular (dyspnoeic) breathing or terminal apnoea (>250 pmol). The respiratory slowing response to RTX (75 pmol), was dose-dependently attenuated by injecting RTX (but not vehicle) into the same site 60 min earlier. 3. The non-pungent phorbol derivative of RTX, phorbol 12-phenylacetete 13-acetate 20-homovanillate (PPAHV, 0.1-1 nmol), also slowed respiration (ED(50), approximately 1 nmol) and almost abolished response to RTX (75 pmol) injected into the same site 60 min later. 4. In contrast to RTX, PPAHV and capsaicin, the putative endogenous vanilloid receptor agonist, arachidonyl ethanolamide (AEA), and non-pungent capsaicin derivative, olvanil, had no direct effect on respiration. However, both AEA and olvanil dose-dependently reduced the respiratory response to injection of RTX (75 pmol) 60 min later into the same site (EC(50)s, for AEA and olvanil, approximately 2 and 0.2 nmol, respectively). 5. These studies suggest that both pungent and non-pungent vanilloids interact with vanilloid receptors in the cNTS. However, whereas RTX and PPAHV activate and subsequently desensitize vanilloid receptors on sensory nerve terminals in the cNTS, olvanil and AEA fail to activate despite readily desensitizing responses to RTX in this region.     

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.     

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.     

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.     

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.     

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.     

Effect of olvanil on the afferent and efferent function of capsaicin-sensitive C-fibers in guinea pig airways

The aim of the present study was to examine the ability of the nonpungent vanilloid VR1 receptor agonist, olvanil, to activate the afferent and efferent function of capsaicin-sensitive C-fibers in guinea pig airways. We found that while capsaicin (10 nM-10 microM) and resiniferatoxin (0.1 nM-1.0 microM) evoked a robust contraction of the guinea pig trachea in vitro, olvanil (10 nM-10 microM) was a weak spasmogen. In addition, pretreatment with olvanil caused only a minor reduction of subsequent responses to capsaicin or resiniferatoxin. Using single fiber recording from guinea pig airway C-fibers, we found that olvanil (10 microM) did not evoke action potential discharge although these fibers responded vigorously to capsaicin after prolonged treatment with olvanil (10 microM). These findings are indicative of significant differences in the relative sensitivity of vanilloid VR1 receptor-transfected cells and the peripheral terminals of airway C-fibers to pungent and nonpungent vanilloid VR1 receptor agonists.     

Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+]i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+]i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE > NADA), low pH and noxious heat (>42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.     

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.     

'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism

1. The abilities of a series of saturated N-acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) has been investigated. 2. The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+-influx, nor the influx in response to a submaximal concentration of AEA (1 microM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. 3. Lauroylethanolamide (C12:0) produced a leftward shift in the dose-response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1-HEK293 cells were 1.8, 1.5, 1.1 and 0.22 microM in the presence of 0, 1, 3 and 10 microM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose - response curves to capsaicin. 4. Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (K(i(slope)) 4.1 microM, K(i(intercept)) 66 microM) of [3H]-AEA metabolism by rat brain membranes. 5. The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 microM AEA. 6. There was no obvious relation between the abilities of the compounds to enhance the Ca2+ influx response to 1 microM AEA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. 7. It is concluded that although palmitoylethanolamide has entourage-like effects at VR1 receptors expressed on hVR1-HEK293 cells, other N-acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.     

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)     

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1)

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.     

Functional and desensitizing effects of the novel synthetic vanilloid-like agent 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) in the perfused rat hindlimb

1. In the present study, the effects of the novel vanilloid agonist, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), on oxygen consumption (VO(2)) and vascular resistance (perfusion pressure, PP) were investigated in the constant flow, perfused rat hindlimb. The acute desensitizing properties of this novel synthetic agent were also examined. 2. Maximum stimulation of VO(2) was produced by 0.2 microM PPAHV (delta VO(2), 0.83+/-0.06 micromol g(-1) h(-1)) and was accompanied by mild vasoconstriction (increase in PP; 8.0+/-1.1 mmHg). The highest concentration of PPAHV tested (2 microM) caused inhibition of VO(2) (delta VO(2), -2.73+/-0.51 micromol g(-1) h(-1)) and strong vasoconstriction (delta PP, 42.0+/-1.2 mmHg). 3. Capsazepine (10 microM) caused a parallel shift to the right of both VO(2) and PP concentration-response curves for PPAHV (pK(b)=5.00), indicative of competitive binding to vanilloid receptors. 4. The stimulation of VO(2) produced by 0.2 microM PPAHV decreased, but was not completely abolished, after repeated infusion of PPAHV (change in VO(2), first infusion, 0.66+/-0.18 micromol g(-1) h(-1); sixth infusion, 0.29+/-0. 08 micromol g(-1) h(-1), P<0.05), an acute tachyphylactic response not previously seen with the repeated infusion of other vanilloid analogues. Conversely, the PP response to repeated PPAHV infusion increased (delta PP, first infusion, 5.8+/-0.7 mmHg; sixth infusion, 9.0+/-0.6 mmHg, P<0.05). 5. In conclusion, PPAHV produces vasoconstriction and a biphasic effect on VO(2) in the perfused rat hindlimb very similar to that induced by naturally occurring vanilloids. Both effects are blocked by the competitive antagonist capsazepine. Since, the metabolic response to low concentrations of PPAHV (stimulation of VO(2)) undergoes tachyphylaxis, the present data suggest that PPAHV desensitizes putative vanilloid receptors in the hindlimb.     

Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6+/-7.5%, n=10 and 97.1+/-0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca(2+) channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro-l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca(2+)-free, EGTA solution. Phosphorylation of MLC(20) (a percentage of phosphorylated to total MLC(20)) was increased 1 min after application of 10 microM acetylcholine (7.8+/-2.0%, n=6 vs. 22.6+/-3.2%, n=6) and of 65.9 mM KCl (2.2+/-0.3%, n=8 vs. 10.7+/-1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC(20) phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC(20) phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC(20) phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca(2+) influx via non-L-type Ca(2+) channels, possibly via store-operated Ca(2+) channels and Ca(2+) release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC(20) phosphorylation.     

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.