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The novel benzopyranopyrrolidine and potential antipsychotic, S16924 ((+)-2-{1-[2-(2,3-dihydro-benzo[1,4] dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl}-1-(4-fluoro-phenyl)-ethanone), displays marked affinity for serotonin (5-HT)1A, 5-HT2A and dopamine D2 receptors. Herein, we show that it also possesses high affinity for the cloned, INI isoform of h5-HT2C receptors (pKi=8.28) stably expressed in CHO cells. Similarly, clozapine (8.04) was a potent ligand, whereas haloperidol (<6.0) showed low affinity. As demonstrated by fura2-detection, S16924 concentration-dependently abolished (pKb=7.93) the 5-HT-induced elevation in intracellular levels of Ca2+ ([Ca2+]i) in a CHO cell line stably expressing the INI isoform of 5-HT2C receptors. Further, as determined by depletion of membrane-bound levels of pre-labelled [3H]phosphatidylinositols ([3H]PI), S16924 concentration-dependently, surmountably and competitively blocked the activation of phospholipase C by 5-HT. This action was expressed with a pA2 of 7.89 according to Schild analysis. Clozapine likewise inhibited 5-HT-induced alterations in [Ca2+]i and [3H]PI levels with pKbs of 7.43 and 7.84, respectively, whereas haloperidol was inactive (<5.0 in each case). Applied alone, S16924, clozapine and haloperidol modified levels of neither [Ca2+]i nor [3H]PI. In conclusion, in analogy to clozapine, and in contrast to haloperidol, S16924 behaves as a potent and competitive antagonist at h5-HT2C receptors, the blockade of which may contribute to its distinctive functional profile of activity. [ABSTRACT FROM AUTHOR]

6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-ylcarbamoyl] indoline (SB242,084) is a novel, selective 5-HT2C receptor antagonist, but its actions at these sites have been little characterised at the cellular level. We employed a rapid and innovative approach to investigate its functional activity at phospholipase C (PLC)-coupled human 5-HT2C receptors expressed in CHO cells. PLC activity was determined as a decrease in the [3H]phosphatidylinositol ([3H]PI) content of cell membranes. Serotonin (5-HT) stimulated [3H]PI depletion (pEC50=8.74), and SB242,084, like mesulergine, completely reversed this action of 5-HT (pKB=9.25 and 9.01, respectively). Further, in Schild analysis, SB242,084 behaved as a high affinity competitive antagonist, inducing a parallel, rightward displacement of the 5-HT stimulation isotherm without loss of maximum efficacy. The pA2 of 9.50 was similar to its binding affinity (pKi=9.38). SB242,084 also displayed antagonist properties when PLC activity was examined by conventional determination of [3H]inositol phosphate generation. Employing this parameter, the potency of SB242,084 (pKB=9.21) and that of mesulergine (pKB=9.06) closely resembled those determined by [3H]PI depletion. In conclusion, determination of [3H]PI depletion constitutes a useful and novel technique to characterise agonist and antagonist properties of ligands at PLC-coupled receptors. [ABSTRACT FROM AUTHOR]

Abstract: Cloned, human dopamine D1 receptors recruit multiple effectors but the G-protein subtype(s) activated by cerebral populations remain poorly defined, a question addressed using a rapid immunocapture technique. In rat striatum, dopamine (DA) and four selective, benzazepine agonists at D1 receptors concentration-dependently enhanced [35S]GTPγS binding to Gαs/olf. For all drugs, Gαq was also recruited with similar potencies and efficacies. Comparable observations were made in the cortex wherein profiles of Gαs/olf vs Gαq activation were also highly correlated. In contrast to Gαs/olf and Gαq, Gαo and Gαi were activated neither in the striatum nor in the cortex, except for SKF82958. As compared to DA, both SKF81297 and SKF82958 were full agonists at Gs/olf and Gq in cortex and striatum, whereas SKF38393 behaved as a partial agonist. Likewise, the “atypical” agonist, SKF83959 only partially activated Gαq and also Gs/olf in these two regions. In both striatum and cortex, the selective D1 receptor antagonist, SCH23390, abolished the recruitment of Gαq and Gαs by DA, and the action of DA was partially attenuated by SKF83959. These findings demonstrate that, in native CNS tissue, DA and other D1 receptor agonists activate Gαs and Gαq with similar potencies and efficacies, suggesting their recruitment via pharmacologically-indistinguishable populations of D1 receptors, and show that SPA technology is well-adapted to study the coupling of native DA receptors. [Copyright &y& Elsevier]