PHARMACOLOGICAL CHARACTERIZATION OF REGULATION OF PHOSPHOINOSITIDE METABOLISM BY RECOMBINANT 5-HT2 RECEPTORS OF THE RAT

Transfection of 5-HT, receptor cDNA in 293 cells induced the expression of a protein binding domain exhibiting the classical 5-HT2 receptor transduction mechanism. Both [H-3]DOB and [H-3]spiperone high affinity binding sites were present in membranes of sense but not of antisense, 5-HT2 receptor cDNA transfected cells. Addition of 1-mu-M 5-HT induced a time-dependent increase of phosphoinositide (PI) metabolism in sense but not in antisense, S-HT2 receptor cDNA transfected cells. Graded concentrations of 5-HT and of different serotonergic agonists showed different potencies (DOI > 5-HT > quipazine > DOM > alpha-methyl-5-HT > 8-OH-DPAT > 2-methyl-5-HT > CGS-12066B) in stimulating turnover of PI in cells transfected with cDNA encoding for 5-HT2 receptors of the rat. The ability of different antagonists to inhibit S-HT-stimulated turnover of PI bore a direct relationship with their potency to inhibit 5-HT2 receptor binding in cells transfected with 5-HT2 receptor cDNA (spiperone > ketanserin > ritanserin > mianserin > haloperidol). Preincubation of transfected 293 cells with pertussis toxin failed to modify either 5-HT- or DOI-induced activation of metabolism of PI. Pretreatment of transfected 293 cells with DOI (100 nM) for 2 hr or more, significantly reduced activation of turnover of PI elicited by graded doses of 5-HT. When the transfected 293 cells were exposed to DOI (100 nM) for 12 hr and the challenge was performed after a 2-hr wash-out period, the desensitization of the response to 5-HT was virtually abolished. In conclusion, the receptors expressed in 293 cells, after transfection with 5-HT2 receptor cDNA, possessed biochemical and pharmacological properties, similar to those of the native receptors and they represent a useful model to investigate structural and functional characteristics of receptors and as a tool for discovery of drugs.