FIG. 4. Serotonin antagonists have negative intrinsic activity in cells transfected with the 5-HT2C receptor. NIH-3T3 fibroblasts were transfected with 5-HT1C receptor cDNA (obtained from Dr. David Julius, University of California, San Francisco), and a clonal line expressing a receptor density of about 600 fmol/mg protein was isolated. Cells were labeled overnight with [3H]inositol in medium without serum. Phosphoinositide hydrolysis was evaluated by quantitating the formation of [3H]inositol monophosphate (3H-IP) in the presence of lithium as described previously (15). The values plotted are means ± SEM of triplicate determinations. Basal refers to 3H-IP formation in the absence of agonist—that is, constitutive activity (basal values were about 1000 cpm in a typical experiment). The putative antagonists mianserin and cyproheptadine produced a dose-dependent reduction in basal activity. In other experiments, a maximum concentration of 5-HT produced a four- to fivefold increase in 3H-IP formation. In nontransfected fibroblasts, basal activity was much lower than that found in transfected cells and was resistant to the negative effects of mianserin and cyproheptadine. These studies were done in collaboration with a graduate student, Eric Barker (9a).