ENDOGENOUS SEROTONIN-2A AND SEROTONIN-2C RECEPTORS IN BALB C-3T3 CELLS REVEALED IN SEROTONIN-FREE MEDIUM - DESENSITIZATION AND DOWN-REGULATION BY SEROTONIN/

We studied the endogenous expression of the serotonin-2A (5-hydroxytry ptamine(2A), 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C recept or in murine Balb/c-3T3 fibroblast cells that is revealed when these c ells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT2C receptor was exclusively at a single brain-specific site . Addition of 5-HT (EC50 = 23 +/- 29 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mo bilization displayed a 5-HT-(2)-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 +/- 2 fmol/mg protein) with a 5-HT2A-like pharmacology. Although the 5-HT2A receptor site was predominant, the smaller component of 5-HT2C recepto rs alone was sufficient to mediate a maximal calcium response. The 5-H T-induced increase in [Ca2+](i) was reversibly inhibited by >75% follo wing a 12-hr pretreatment (T-1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Ex tended treatment (24-96 hr) with 5-HT induced a complete functional de sensitization that was associated with a partial (60%) reduction in 5- HT2 receptor number, implicating both receptor down-regulation and pos t-receptor mechanisms in 5-HT-induced desensitization. Long-term (hour s to days) treatment with 5-HT did not modulate DNA synthesis, cell pr oliferation, or transformation in Balb/c-3T3 cells. These results demo nstrate that Balb/c-3T3 cells express endogenous 5-HT2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor res ponsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a ''safety valve'' to p revent abnormal cell growth during sustained 5-HT2 receptor activation .; BIOCHEM PHARMACOL 56;10:1347-1357, 1998. (C) 1998 Elsevier Science Inc.