Rapid desensitization of serotonin 5-HT2C receptor-stimulated intracellular calcium mobilization in CHO cells transfected with cloned human 5-HT2C receptors

Serotonin 5-HT 2C receptor-mediated intracellular Ca 2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT 2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT 2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT 2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [ 3 H] mesulergine binding (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 μM. Exposure to 6-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-HT 2A agonist) resulted in increased intracellular Ca 2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca 2+ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca 2+ mobilization induced by 10 μM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca 2+ concentration. The desensitization induced by pretreatment 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 kinases and weak analogue of W-7. Therefore, the 5-HT 2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT 2C receptor activation mobilizes Ca 2+ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback