Spontaneous release of GDP from G_i proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes

Abstract.

Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'-O-(2-thiodiphosphate; GDPβS). Here, we studied the mechanism underlying this unconventional activation.

Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg²⁺ concentration, decreased rapidly during incubation at 37°C. This decrease in enzyme activity was paralleled by a rapid release of GDP from G_i proteins, amounting to 75% release of total G_i-bound GDP within 10 min at 37°C. In contrast, no GDP release was observed at 4°C, and adenylyl cyclase activity remained stable for up to 20 min at 4°C. GDPβS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37°C or 4°C, but almost fully prevented the decrease in enzyme activity occurring at 37°C. Hence, after 10 min of incubation at 37°C, adenylyl cyclase activity in the presence of GDPβS was increased by 150%, while no difference in activity was observed at 4°C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory G_i proteins, i.e., at high concentrations of Mg²⁺ or Mn²⁺, adenylyl cyclase activity remained stable even at 37°C and GDPβS did not stimulate activity. In conclusion, G_i proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active G_i proteins.