RAPID DESENSITIZATION OF SEROTONIN 5-HT2C RECEPTOR-STIMULATED INTRACELLULAR CALCIUM MOBILIZATION IN CHO CELLS TRANSFECTED WITH CLONED HUMAN5-HT2C RECEPTORS

Serotonin 5-HT2C receptor-mediated intracellular Ca2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5- HT2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT2C receptor, expressed 287 fmol/mg of the rece ptor protein as determined by mianserin-sensitive [H-3] mesulergine bi nding (K-D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca 2+ in a dose-dependent fashion, ranging from a basal level of 99 +/- 1 .8 up to 379 +/- 18 nM, with an EC(50) value for 5-HT of 0.029 mu M. E xposure to 5-HT, 1-(3-chlorophenyl) piperazine dihydrochloride (a 5-HT 2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT 2C and 5-HT2A agonist) resulted in increased intracellular Ca2+ levels . Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT -mediated intracellular Ca2+ mobilization more effectively than spiper one. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myr istate 13-acetate, protein kinase C activators, weakly inhibited the i ntracellular Ca2+ mobilization induced by 10 mu M 5-HT. Furthermore, t he protein kinase C inhibitor H-7 partially prevented the protein kina se C activator-induced inhibition of the 5-HT-mediated increase in int racellular Ca2+ concentration, The desensitization induced by pretreat ment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinas es and weak analogue of W-7. Therefore, the 5-HT2C receptor may be con nected with protein kinase C and calcium/calmodulin turnover. These re sults suggest that 5-HT2C receptor activation mobilizes Ca2+ in CHO ce lls and that the acute desensitization of the receptor may be due to c almodulin kinase-mediated feedback.