Regulation of guanine nucleotide turnover on G(i)/G(o) by agonist-stimulated and spontaneously active muscarinic receptors in cardiac membranes

Muscarinic receptor regulation of guanine nucleotide turnover on G(i)/G(o) proteins in ventricular sarcolemma was investigated. In the absence of a mu scarinic receptor (MR) agonist, GTP bound to background sites with a K-app value of 60 nM and a B-max of 50 pmol/mg. The addition of the MR agonist, c arbachol, further increased GTP binding by 50 pmol/mg to sites with the sam e K-app value of 60 nM. Pertussis toxin treatment reduced GTP binding to ca rbachol-regulated and background binding sites, thus identifying both sites as G(i)/G(o). The identity of the carbachol-regulated GTP binding sites wa s further confirmed by demonstrating that carbachol stimulated GTP binding and inhibited adenylyl cyclase with an EC50 value of 200 nM. Background and carbachol-regulated guanine nucleotide binding sites bound GDP with a K-ap p value of 150 nM. However, maximal background GDP binding was 50 pmol/mg, whereas maximal carbachol-regulated GDP binding was only 12-15 pmol/mg. In sarcolemma preloaded with [H-3]GDP, carbachol-regulated [H-3]GDP release wa s strictly dependent on the presence of guanine nucleotides. The K-app valu es for GTP and GDP to support carbachol-regulated [H-3]GDP release were 60 nM and 150 nM, respectively. Guanosine 5'-O-(3-thiotriphosphate) (GDP beta S) facilitated carbachol-regulated [H-3]GDP release with a K-app value of 2 mu M. However, GTP was two times more efficacious than GDP or GDP beta S i n facilitating carbachol-regulated [H-3]GDP release. Mn2+ also stimulated [ H-3]GDP release from carbachol-regulated sites by a mechanism not requiring guanine nucleotides. These studies indicate that two pools of muscarinic r eceptors, carbachol regulated and spontaneously active, regulate guanine nu cleotide turnover on pertussis toxin sensitive G(i)/G(o). These studies fur ther suggest that guanine nucleotide binding provides the signal to stimula te GDP release from receptor activated G(i)/G(o) proteins. A quaternary mec hanism involving G-protein interactions may be necessary to promote guanine nucleotide exchange on G(i)/G(o). (C) 1999 Academic Press.