| Abstrakt: |
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 ([Ca2+]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 (pEC50values of 7.47±0.06, 7.16±0.06, 8.19±0.06 and 6.02±0.03 respectively at pH 7.4 vs7.71±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 Ca2+response in rVR1‐HEK293 cells, with pKBvalues 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 Ca2+abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 μM) partially (∼20%) inhibited, the capsaicin (10 μM)‐induced Ca2+response. However, tetrodotoxin (3 μM), nimodipine (10 μM), ω‐GVIA conotoxin (1 μM), thapsigargin (1 μM), U73122 (3 μM) or H‐89 (3 μM) 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 Ca2+channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor. |
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