Functional activation of G-proteins coupled with muscarinic acetylcholine receptors in rat brain membranes.
The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) 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 [(35)S]GTPγS 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 (αR)-α-cyclopentyl-α-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 [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 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 Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.
Department of Psychiatry, Faculty of Medicine, Saitama Medical University, Japan.
Yuji
Y
Odagaki
Masakazu
M
Kinoshita
Ryoichi
R
Toyoshima
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D003342:Corpus Striatum / Q000166:cytology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D013268:Stimulation, Chemical
D011976:Receptors, Muscarinic; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D019204:GTP-Binding Proteins; D000109:Acetylcholine