title: Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology.
abstract: 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.
authors.affiliation: Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW.
authors.forename: J C
authors.initials: JC
authors.lastname: Jerman
authors.forename: S J
authors.initials: SJ
authors.lastname: Brough
authors.forename: R
authors.initials: R
authors.lastname: Prinjha
authors.forename: M H
authors.initials: MH
authors.lastname: Harries
authors.forename: J B
authors.initials: JB
authors.lastname: Davis
authors.forename: D
authors.initials: D
authors.lastname: Smart
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D004224:Diterpenes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010703:Phorbol Esters / Q000494:pharmacology; D000081223:Polycyclic Sesquiterpenes; D051381:Rats; D011955:Receptors, Drug / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics; D012430:Ruthenium Red / Q000494:pharmacology; D012717:Sesquiterpenes / Q000494:pharmacology; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D004224:Diterpenes; D008024:Ligands; D010703:Phorbol Esters; D000081223:Polycyclic Sesquiterpenes; D011955:Receptors, Drug; D012717:Sesquiterpenes; D012430:Ruthenium Red; C100823:phorbol 12-phenylacetate 13-acetate 20-homovanillate; C089286:isovelleral; C054256:olvanil; C024353:resiniferatoxin; C071423:capsazepine; D002211:Capsaicin; D002118:Calcium