SEROTONERGIC ANTAGONISTS DIFFERENTIALLY INHIBIT SPONTANEOUS ACTIVITY AND DECREASE LIGAND-BINDING CAPACITY OF THE RAT 5-HYDROXYTRYPTAMINE TYPE 2C RECEPTOR IN SF9 CELLS

The activities of serotonergic antagonists as inverse agonists at the rat 5-hydroxytryptamine (5-HT)(2C) serotonin receptor were compared wi th their potencies in promoting receptor ''down-regulation,'' after ex pression of the recombinant receptor in the baculovirus/Sf9 insect cel l system. Baculovirus expression yielded high levels of 5-HT2C recepto rs (up to 10(6) receptors/cell), which were functionally coupled to po lyphosphoinositide turnover in Sf9 cells through a pertussis toxin-ins ensitive pathway. The expressed receptor exhibited spontaneous activat ion of inositol phosphate production, which was inhibited in a dose-de pendent manner by serotonergic antagonists, consistent with inverse ag onist activity. The potencies of antagonists as inverse agonists corre lated with their respective binding affinities determined in competiti on binding studies with membrane preparations. The maximal inhibition of spontaneous activity ranged from 32% inhibition for mianserin to no effect for spiroxatrine, indicating that antagonists differ in their intrinsic inverse efficacies. Antagonist treatment of intact Sf9 cells or membranes containing the 5-HT2C receptor, followed by washout of r esidual drug, resulted in a decrease (up to 90%) in the number of bind ing sites for [H-3]mesulergine and [H-3]5-HT, with no change in the af finity for [H-3]mesulergine. The decrease in binding was irreversible, was not due to the presence of residual antagonist, and was not obser ved after treatment with agonists. This effect of antagonists in membr anes was dose dependent, but the rank order of potency was clearly dif ferent from that for inverse agonist activity, indicating that the two effects reflect distinct actions of antagonists at the 5-HT2C recepto r. The relative abilities of antagonists to produce loss of binding sh owed a good correlation with their reported abilities to down-regulate 5-HT2 receptors in vivo after chronic treatment, suggesting that thes e actions reflect the same underlying process.