Receptor-Independent Activation of Cardiac Adenylyl Cyclase by GDP and Membrane-Associated Nucleoside Diphosphate Kinase. A New Cardiotonic Mechanism?

Regulation of adenylyl cyclase activity by guanine nucleoside tri- and diphosphates as well as by stimulatory and inhibitory receptors was studied in canine cardiac sarcolemmal membranes. Guanosine triphosphate (GTP) increased adenylyl cyclase activity by a maximum of 80%, with an EC50value of 0.7 μmol/l. The addition of theβ-adrenoceptor agonist, isoprenaline (100 μmol/l), caused a further, about 100%, increase in GTP-stimulated activity. The nucleoside diphosphate (GDP) also activated cardiac adenylyl cyclase, but in a biphasic manner. At low concentrations (EC500.12 μmol/l), GDP increased enzyme activity by about 80%, followed by a plateau at 0.5–2 μmol/l and a second increase to a maximum of 60% with an EC50value of 14 μmol/l. The stable GDP analog, guanosine 5′-O-(2-thio)diphosphate (GDPβS), also increased cardiac adenylyl cyclase activity, but in a monophasic manner, by a maximum of 150%, with an EC50of 0.4 μmol/l. Addition of uracil diphosphate (UDP) (3 mmol/l), which completely inhibited transphosphorylation of GDP to GTP, did not reduce adenylyl cyclase stimulation by low concentrations of GDP, whereas enzyme stimulation by high GDP concentrations was almost completely attenuated. Furthermore, pretreatment of the membranes with cholera toxin led to an increased stimulation of adenylyl cyclase activity by high concentrations of GDP. These findings suggest that the second phase of adenylyl cyclase stimulation by GDP is due to transphosphorylation of GDP to GTP, associated with activation of Gsproteins, and that stimulation by GDP itself (first phase) and endogenously formed GTP (second phase) is additive. However, in contrast to exogenously added GTP, β-adrenoceptor activation did not enhance GDP-stimulated adenylyl cyclase activity. Furthermore, in the presence of 1 μmol/l GDP, the addition of GTP did not cause any further increase in enzyme activity. On the other hand, the muscarinic acetylcholine receptor agonist carbachol inhibited both GTP- and GDP-activated adenylyl cyclase. The inhibition of GDP-stimulated activity was lost when formation of GTP from GDP was blocked. The contrasting effects of endogenously formed GTP and exogenous GTP suggest that the formation of GTP from GDP is closely linked to the activation site of adenylyl cyclase, i.e. the stimulatory Gsprotein. This receptor-independent activation can apparently bypassβ-adrenoceptor-dependent activation of cardiac adenylyl cyclase.