Regulation of GDP and GTP binding in cardiac sarcolemma by muscarinic receptor agonists.

Regulation of GTP and GDP binding and GTPase activity of cardiac sarcolemmal guanine nucleotide-binding proteins was investigated. In purified sarcolemmal membranes, carbachol and a variety of other muscarinic receptor (MR) agonists induced increases in [3H]GTP, [gamma-32P]GTP, and [3H]GDP binding to relatively high affinity sites. Carbachol-dependent GTP and GDP binding changes were maximal within 5 sec at 30 degrees and thereafter remained at steady state. Carbachol increased GTP binding to two sites with apparent Kapp values of 50 nM and 250 nM and GDP binding to a single site with a Kapp of 100 nM. N-Ethylmaleimide attenuated carbachol-dependent GDP and GTP binding, tentatively identifying the binding sites as Gi and/or Go. Further studies showed that [3H]GDP and [3H]GTP bound to Gi/Go in the presence of carbachol rapidly exchanged with GTP and GDP in the medium. In membranes preincubated with carbachol and [gamma-32P]GTP or carbachol and [3H]GDP, postaddition of atropine resulted in complete hydrolysis of [gamma-32P]GTP bound to Gi/Go, to unlabeled GDP and 32Pi, by GTPase, within 10 sec, whereas [3H]GDP remained bound. This study also showed that bound [3H]GDP did not exchange with GDP or GTP in the absence of an MR agonist. Under identical conditions, atropine reversed adenylate cyclase (AC) inhibition by carbachol and GTP or GDP in 5-10 sec. MR agonists appear to increase the rate of dissociation of GDP from Gi/Go, which results in rapid GTP turnover on these sites by a combination of GTPase and GDP/GTP exchange reactions. Furthermore, MR-Gi/Go may be tightly coupled during AC inhibition, so that GTP hydrolysis as well as MR-Gi/Go uncoupling may be required to reverse AC inhibition.