The new positive inotrope sulmazole inhibits the function of guanine nucleotide regulatory proteins by affecting GTP turnover

Abstract

The effect of the new cardiotonic agent sulmazole on the guanine nucleotide regulatory protein-adenylate cyclase system was studied in rat adipocyte membranes. The inotrope enhanced basal adenylate cyclase activity by 46%. This stimulation occurred only at GTP concentrations (5 microM) sufficient to activate Gi. This stimulatory effect of sulmazole was abolished after functional inactivation of Gi, either by pertussis toxin or by using 10 nM GTP in the assay mixture, suggesting an important role of an active Gi in this process. Similarly, sulmazole enhanced isoproterenol-, forskolin-, and fluoride-stimulated adenylate cyclase activity by 33, 34, and 45%, respectively. However, when these latter experiments were performed after inactivation of Gi, sulmazole actually inhibited by approximately 25% adenylate cyclase activity stimulated by 1 and 10 microM isoproterenol. Under similar treatment conditions, enhancement of forskolin- and fluoride-stimulated activity by sulmazole was abolished. Sulmazole inhibited in a dose-dependent manner pertussis toxin- and cholera toxin-catalyzed labeling of Gi and Gs, respectively, with the respective inhibition observed at 100 microM of the inotrope being 29% and 56% of control. In addition, sulmazole inhibited PGE1 and isoproterenol-stimulated [3H]GDP release from Gi and Gs to 32% and 64% of control, respectively. Finally, the inotrope completely abolished PGE1-stimulated [3H]Gpp(NH)p binding with IC50 in the low micromolar range. These findings suggest that, whereas sulmazole inhibits the functioning of Gi and (to a lesser extent) Gs at low micromolar concentrations, expression of these effects on adenylate cyclase activity requires high micromolar to low millimolar concentrations of the drug. Thus, it appears sulmazole inhibits the function of Gi by decreasing its activation process, i.e., GTP-GDP exchange. Effects on Gs are manifested (at least in terms of adenylate cyclase activity) only after inactivation of Gi.