IDENTIFICATION OF AN ENDOGENOUS 5-HYDROXYTRYPTAMINE(2A) RECEPTOR IN NIH-3T3 CELLS - AGONIST-INDUCED DOWN-REGULATION INVOLVES DECREASES IN RECEPTOR RNA AND NUMBER

NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an incre ase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT 2A receptor that, when activated, mobilizes calcium from ionomycin-sen sitive intracellular stores via coupling to a pertussis toxin-insensit ive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5HT(2A) receptor RNA with a similar nucleot ide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2 A receptor in nontransfected cells was completely desensitized after c hronic treatment (half-time = 2 h) with 1 mu M 5-HT and resensitized o n removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2 A receptor cDNA under control of a viral promoter, the long-term agoni st-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA l evel. These results show that NIH-3T3 cells express an endogenous 5-HT 2A receptor that is desensitized by agonist via down-regulation of bot h receptor number and mRNA. The NIH-3T3 cells provide a novel system f or understanding 5-HT2A receptor regulation.