guanosine-triphosphate and sulmazole

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.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Adipose Tissue; Animals; Cardiotonic Agents; Cholera Toxin; Colforsin; Cyclic AMP; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Imidazoles; In Vitro Techniques; Isoproterenol; Male; Pertussis Toxin; Rats; Rats, Inbred Strains; Virulence Factors, Bordetella

Although many of the new cardiotonic agents are known to increase cAMP and to inhibit with variable potency a low Km cAMP phosphodiesterase, there is still debate as to the mechanism(s) by which these agents act. In a rat adipocyte membrane model we demonstrate that only approximately 50% of the effect of the new cardiotonic agent sulmazole on cAMP accumulation can be attributed to phosphodiesterase inhibition and that the remaining production of cAMP involves stimulation of adenylate cyclase activity. Two distinct pathways for stimulation of adenylate cyclase are herein reported. Sulmazole, UD-CG 212 CL, enoximone, piroximone, amrinone, and milrinone are all shown to be competitive antagonists of inhibitory A1 adenosine receptors, with EC50 values of 11-909 microM. Elimination of the effects of endogenous adenosine with adenosine deaminase reveals a third distinct mechanism for activation of adenylate cyclase. This mechanism appears to involve Gi, the inhibitory guanine nucleotide-regulatory protein, in that sulmazole attenuates the capacity of GTP to inhibit adenylate cyclase activity, and covalent modification of Gi by pertussis toxin treatment abolishes the capacity of sulmazole to mediate stimulation. Thus, functional blockade of Gi activity is the likely mode of action. Restoration of sulmazole's stimulatory effect on adenylate cyclase activity in pertussis toxin-treated membranes can be accomplished by reconstituting purified preparations of either Gi or mixtures of Gi/Go into treated adipocyte membranes. Of note, this stimulatory effect is completely reversed by inhibitory receptor agonists. Thus, the new cardiotonic agent sulmazole mediates increases in cAMP accumulation by mechanisms other than phosphodiesterase inhibition, including A1 adenosine receptor antagonism and inhibition of Gi function.

Topics: Adenylate Cyclase Toxin; Adipose Tissue; Animals; Binding, Competitive; Cardiotonic Agents; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine Triphosphate; Imidazoles; Kinetics; Male; Pertussis Toxin; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Purinergic; Virulence Factors, Bordetella