Premium
Rapid Desensitization of Serotonin 5‐HT 2C Receptor‐Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5‐HT 2C Receptors
Author(s) -
Akiyoshi Jotaro,
Nishizono Akira,
Yamada Kenji,
Nagayama Haruo,
Mifune Kumato,
Fujii Isao
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64062473.x
Subject(s) - receptor , protein kinase c , ritanserin , biology , agonist , intracellular , chinese hamster ovary cell , microbiology and biotechnology , 5 ht receptor , serotonin , endocrinology , chemistry , medicine , signal transduction , biochemistry
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 n M ). 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 n M , with an EC 50 value for 5‐HT of 0.029 µ M . Exposure 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‐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 µ M 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.