Analysis of apparent noncompetitive responses to competitive H-1-histaminereceptor antagonists in fluorescent imaging plate reader-based calcium assays

We have examined the utility of high throughput fluorescent imaging plate r eader (FLIPR)-based calcium assays for pharmacological characterization of G-protein coupled receptors (GPCRs) using recombinant and native human H-1- histamine receptors (H-1-HR), expressed in HEK293 and HeLa S3 cells, respec tively, as model systems. For stably transfected HEK293 cell lines, the pot ency of histamine for elevating intracellular calcium increased (pD(2), 7.1 3 and 7.86) with increased H-1-HR density (about 0.8 and 14 pmol/mg protein , respectively), though histamine binding affinities were similar. The clas sic H-1-MR competitive antagonists diphenhydramine and chlorpheniramine app eared noncompetitive by causing depressions of the maximal histamine respon ses along with rightward shifts of histamine concentration-response curves, thus precluding Schild analysis. Applying the generalized Cheng-Prusoff eq uation to antagonist concentration-response curves for inhibition of fixed histamine concentrations yielded apparent pK(b) values that were consistent among recombinant and native receptors at different expression levels. The se pK(b) values for diphenhydramine and chlorpheniramine (e.g., 7.83 and 8. 77, respectively) were in good agreement with binding pK(i) values (e.g., 7 .98 and 8.52, respectively), Apparent antagonist affinities determined from FLIPR calcium and competition binding assays were also consistent for the competitive antagonists mepyramine, tripelennamine, and promethazine, In ph osphoinositide hydrolysis assays, chlorpheniramine exhibited insurmountable inhibition of histamine calcium responses, although to a lesser extent tha n that observed in calcium assays; pK(b) values were similar. These results demonstrate that competitive antagonist potencies can be attained from FLI PR-derived data by application of the generalized Cheng-Prusoff equation, d espite apparent noncompetitive antagonism under these assay conditions. App arent noncompetitive antagonist effects may in part be attributable to a la ck of equilibrium of histamine and antagonists with H-1-HR within the short duration of rapid transient effects of histamine on intracellular calcium.