Stable GDP analog-induced inactivation of Gi proteins promotes cardiac adenylyl cyclase inhibition by guanosine 5′-(βγ-imino) triphosphate and muscarinic acetylcholine receptor

AbstractLow concentrations of GDP and its stable analog guanosine 5′-O-(2-thio)diphosphate(GDPβS) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5′-(βγ-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 μmol/1 and 10 nmol/1, respectively. In the presence of GDPβS (5 μmol/1), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/1. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDPβS-stimulated adenylyl cyclase activity. The stimulatory effect of GDPβS and the inhibitory effect of Gpp(NH)p on GDPβS-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 μmol/1). These results suggest that the stimulatory effect of GDPβS on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of Gi proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors