Identification of an Endogenous 5‐Hydroxytryptamine2A Receptor in NIH‐3T3 Cells: Agonist‐Induced Down‐Regulation Involves Decreases in Receptor RNA and Number

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Abstract
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 increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH‐3T3 cells endogenously express a 5‐HT2A receptor that, when activated, mobilizes calcium from ionomycin‐sensitive intracellular stores via coupling to a pertussis toxin‐insensitive pathway. Using reverse transcriptase‐PCR cloning and northern blot analysis, the presence of 5‐HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH‐3T3 cells. Responsiveness of the endogenous 5‐HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half‐time = 2 h) with 1 µM 5‐HT and resensitized on removal of 5‐HT. In contrast to NIH‐3T3 cells transfected with 5‐HT2A receptor cDNA under control of a viral promoter, the long‐term agonist‐induced functional desensitization in nontransfected NIH‐3T3 cells was paralleled by a decrease in both 5‐HT2A receptor density and RNA level. These results show that NIH‐3T3 cells express an endogenous 5‐HT2A receptor that is desensitized by agonist via down‐regulation of both receptor number and mRNA. The NIH‐3T3 cells provide a novel system for understanding 5‐HT2A receptor regulation.

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