Identification of an endogenous 5-hydroxytryptamine2A 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 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 microM 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.
Saucier
C
C
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
Albert
P R
PR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015536:Down-Regulation; D007425:Intracellular Membranes / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D014158:Transcription, Genetic
D008024:Ligands; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; D012701:Serotonin; D002118:Calcium