Endogenous serotonin-2A and -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-hydroxytryptamine2A, 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells 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 +/- 2.9 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 mobilization 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 receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca2+]i was reversibly inhibited by >75% following a 12-hr pretreatment (T1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Extended treatment (24-96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate 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 responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a "safety valve" to prevent abnormal cell growth during sustained 5-HT2 receptor activation.
Saucier
C
C
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
Morris
S J
SJ
Albert
P R
PR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D015536:Down-Regulation; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D044402:Receptor, Serotonin, 5-HT2A; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012701:Serotonin / Q000378:metabolism*
D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D044402:Receptor, Serotonin, 5-HT2A; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin; D012701:Serotonin; D002118:Calcium