The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca++]i), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca++]i when challenged with capsaicin (EC50s ≅ 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R2 = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.
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