Rapid desensitization of serotonin 5-HT2C receptor-stimulated intracellular calcium mobilization in CHO cells transfected with cloned human 5-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 receptor protein as determined by mianserin-sensitive [3H]mesulergine binding (KD = 0.49 nM). The addition of 5-HT mobilized intracellular Ca2+ in a dose-dependent fashion, ranging from a basal level of 99 +/- 1.8 up to 379 +/- 18 nM, with an EC50 value for 5-HT of 0.029 microM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT2C 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 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 Ca2+ mobilization induced by 10 microM 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 Ca2+ 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Akiyoshi
J
J
Department of Neuropsychiatry, Oita Medical University, Japan.
Nishizono
A
A
Yamada
K
K
Nagayama
H
H
Mifune
K
K
Fujii
I
I
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology; D011985:Receptors, Serotonin / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection
D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013449:Sulfonamides; C051353:naphthalenesulfonamide; D011493:Protein Kinase C; D002118:Calcium