Differences in agonist-independent and -dependent 5-hydroxytryptamine2C receptor-mediated cell division

Previous studies have shown that agonist activation of the 5-hydroxytryptamineC (5-HT2C) receptor expressed in NIH-3T3 fibroblasts results in development of a transformed phenotype. In light of recent evidence from our laboratory demonstrating constitutive 5-HT2C receptor activity, we examined the contribution of this agonist-independent activity to basal cell division. 5-HT2C receptor ligands modulated [3H]thymidine incorporation, DNA amounts, and cell number in serum-starved NIH-3T3 fibroblasts transfected with 5-HT2C receptor cDNA. Three classes of 5-HT2C receptor ligands were distinguished in transfected, but not nontransfected, fibroblasts. Basal [3H]thymidine incorporation was increased by agonists and decreased by inverse agonists, whereas neutral antagonists had little or no effect alone. Neutral antagonists did, however, block the effects of both agonists and inverse agonists. The rank order of potencies of inverse agonists to decrease basal [3H]thymidine incorporation was consistent with their rank order to decrease basal 5-HT2C receptor-mediated phosphoinositide hydrolysis. However, two antagonists previously classified as inverse agonists based on their ability to eliminate basal phosphoinositide hydrolysis did not elicit comparable reductions in basal [3H]thymidine incorporation. For example, mesulergine had no effect on basal cell division, even though it eliminates the phosphoinositide hydrolysis response. Pertussis toxin, which inactivates G proteins in the Gi and Go families, had no effect on basal [3H]thymidine incorporation or basal phosphoinositide hydrolysis but partially inhibited these responses when elicited by an agonist. Thus, agonist occupation of the 5-HT2C receptor apparently activates different or additional G proteins compared with constitutive 5-HT2C receptor activation. In conclusion, our findings suggest that constitutively active 5-HT2C receptors stimulate cell division in transfected fibroblasts in the absence of an agonist. In addition, the 5HT2C receptor may use multiple signaling pathways to mediate its effects.