Use of a fluorescent imaging plate reader-based calcium assay to assess pharmacological differences between the human and rat vanilloid receptor

Abstract

The cloned vanilloid receptor l (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 (rVRl) and a human orthologue (hVRl) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVRl or hVRl, exhibited dose-dependent increases in [Ca++]i when challenged with capsaicin (EC,50s = 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 rVRl potencies highly correlated to that of hVRl (R2 = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVRl compared to hVRl. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVRl, hVRl was more sensitive to blockade by both antagonists. For both rVRl and hVRl, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVRl but showed rapid responses with rVRl. Thus, FLIPR technology can be used to readily reveal differences between rVRl and hVRl pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands. © The Society for Biomolecular Screening.