Hilf_1989_Eur.J.Pharmacol_172_155

The mechanism of G-protein activation mediated by cardiac muscarinic acetylcholine receptors (mAcChR) was studied in porcine atrial membranes by measuring the agonist-stimulated hydrolysis of GTP and G-protein GTPase activity. The cholinergic agonist, carbachol, stimulated a high-affinity GTPase activity in mAcChR-enriched membranes by maximally about 70%. Carbachol increased the Vmax of GTP hydrolysis without affecting the Km for GTP (approximately 0.2 microM). The activity of low-affinity GTPase(s) was not altered by carbachol. Carbachol and the mAcChR agonist oxotremorine induced the same maximal response, with half-maximal activation occurring at about 5 and 0.5 microM, respectively. The stimulatory effect of carbachol was blocked by the mAcChR antagonist, atropine. The stimulation of GTP hydrolysis by cholinergic agonist, was strictly dependent on Mg2+, with half-maximal stimulation observed at 4-5 microM free Mg2+. The monovalent cations, in the potency order, Na+ greater than Li+ greater than K+, enhanced the GTP hydrolysis stimulated by carbachol at a maximally effective concentration. The increase in the maximal agonist response seen with NaCl was the result of NaCl decreasing the basal more than the carbachol-stimulated GTP hydrolysis. The data indicate that occupation of mAcChR in porcine atrial membranes by agonists leads to an increase in G-protein GTPase activity, apparently by increasing the amount of nucleotide-free G-protein to which GTP can bind without altering the apparent binding affinity for GTP. This receptor-induced G-protein activation is under the strict control of Mg2+ acting at low, microM concentrations, and is additionally modulated by monovalent cations.