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°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 n M )‐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 μ M ) partially (∼20%) inhibited, the capsaicin (10 μ M )‐induced Ca 2+ response. However, tetrodotoxin (3 μ M ), nimodipine (10 μ M ), ω‐GVIA conotoxin (1 μ M ), thapsigargin (1 μ M ), U73122 (3 μ M ) or H‐89 (3 μ M ) had no effect on the capsaicin (100 n M )‐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.British Journal of Pharmacology (2000) 130 , 916–922; doi: 10.1038/sj.bjp.0703390