Analysis of Apparent Noncompetitive Responses to Competitive H(1)-Histamine Receptor Antagonists in Fluorescent...

We have examined the utility of high throughput fluorescent imaging plate reader (FLIPR)-based calcium assays for pharmacological characterization of G-protein coupled receptors (GPCRs) using recombinant and native human H1-histamine receptors (H(1)-HR), expressed in HEK293 and HeLa S3 cells, respectively, as model systems. For stably transfected HEK293 cell lines, the potency of histamine for elevating intracellular calcium increased (pD(2), 7.13 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 classic H(1)-HR competitive antagonists diphenhydramine and chlorpheniramine appeared noncompetitive by causing depressions of the maximal histamine responses along with rightward shifts of histamine concentration-response curves, thus precluding Schild analysis. Applying the generalized Cheng-Prusoff equation 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. These 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 phosphoinositide hydrolysis assays, chlorpheniramine exhibited insurmountable inhibition of histamine calcium responses, although to a lesser extent than that observed in calcium assays; pK(b) values were similar. These results demonstrate that competitive antagonist potencies can be attained from FLIPR-derived data by application of the generalized Cheng-Prusoff equation, despite apparent noncompetitive antagonism under these assay conditions. Apparent noncompetitive antagonist effects may in part be attributable to a lack of equilibrium of histamine and antagonists with H(1)-HR within the short duration of rapid transient effects of histamine on intracellular calcium.