EFFECTS OF CLOZAPINE ON RAT STRIATAL MUSCARINIC RECEPTORS COUPLED TO INHIBITION OF ADENYLYL-CYCLASE ACTIVITY AND ON THE HUMAN CLONED M4 RECEPTOR

1. Clozapine has recently been claimed to behave as a selective and fu ll agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have in vestigated whether clozapine could activate the rat striatal muscarini c receptors coupled to the inhibition of adenylyl cyclase activity, co nsidered as pharmacologically equivalent to the m4 gene product. In ad dition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2. In rat striatum, clozapine (1 nM-10 mu M) caused a s light inhibition of forskolin-stimulated adenylyl cyclase activity, wh ich was not counteracted by 10 mu M atropine. On the other hand, cloza pine. antagonized the inhibitory effect of acetylcholine with a pA(2) value of 7.51. Moreover, clozapine (1 mu M) failed to inhibit dopamine DI receptor stimulation of adenylyl cyclase activity, but counteracte d the inhibitory effect of carbachol (CCh). Clozapine displaced [H-3]- N-methylscopolamine ([H-3]-NMS) bound to striatal M-4 receptors with a monophasic inhibitory curve and a pK(i) value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5'-O-(thio)tr iphosphate (GTP gamma S). 3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC50 of 31 nM. Th is effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 mu M (EC50= 50 nM) and stimulating at higher concentrations (EC50=7 mu M). Clozapi ne (0.3-5 mu M) antagonized the CCh stimulation of cyclic AMP with a p K(i) value of 7.47. Similar results were obtained when the adenylyl cy clase activity was assayed in CHO cell membranes. 4. In CHO cells pret reated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (10 mu M), the maximal inhibitory effect of clozapi ne on cyclic AMP formation was markedly reduced, whereas the CCh inhib itory curve was shifted to the right with no change in the maximum. 5. As in rat striatum, in CHO cell membranes the displacement of [H-3]-N MS binding by clozapine yielded a monophasic curve which was not affec ted by GTP gamma S. 6. Clozapine (10 nM-10 mu M) had a small stimulant effect (similar to 20%) on the binding of [S-35]-GTP gamma S to CHO c ell membranes, whereas CCh caused a 250% increase of radioligand bindi ng. Moreover, clozapine (50 nM-5 mu M) antagonized the CCh-stimulated [S-35]-GTP gamma S binding with a pA(2) value of 7.48. 7. These result s show that at the striatal M-4 receptors clozapine is a potent and co mpetitive antagonist, whereas at the cloned m4 receptor it elicits bot h agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, w ith intrinsic activity changing as a function of the coupling efficien cy of the receptor to effector molecules.