Functional Activation of G-Proteins Coupled With Muscarinic Acetylcholine Receptors in Rat Brain Membranes

The functional activation of G(i/o) proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [S-35]GTP gamma S binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (alpha R)-alpha-cyclopentyl-alpha-hydroxy-N[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [S-35]GTP gamma S binding. The highly selective allosteric potentiator at the M-4 mAChR subtype, 3-amino-N-[(4-chlorophenyl) methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of G(i/o) proteins. Collectively, all of the data suggest that functional activation of G(i/o) proteins coupled to mAChRs, especially the M-4 subtype, is detectable by means of CCh-stimulated [S-35]GTP gamma S binding assay in rat discrete brain regions.