Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

1 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. 2 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 ([Ca2+](i)) were monitored using FLIPR, before and after the addition of various agonists. 3 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). 4 Capsazepine, isovelleral and ruthenium red ail inhibited the capsaicin (100 nM)-induced Ca2+ 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. 5 The removal of extracellular Ca2+ abolished. whilst inhibition of protein kinase C with chelerythrine chloride (10 mu M) partially (similar to 20%) inhibited, the capsaicin (10 mu M)-induced Ca2+ response. However. tetrodotoxin (3 mu M), nimodipine (10 mu M), omega-GVIA conotoxin (100 mu M), thapsigargin (1 mu M), U73122 (3 mu M) or H-89 (3 mu M) had no effect on the capsaicin (100 nM)induced response. 6 In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca2+ channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.