The binding and regulation of selected muscarinic agonists to putative subtypes in rat cerebral cortex and heart was studied. Parallel inhibition studies of [3H]pirenzepine ([3H]PZ) and (-)-[3H]quinuclidinylbenzilate [(-)-[3H]QNB]-labeled membranes were done with and without 30 μM guanyl-5'-yl imidodiphosphate [Gpp(NH)p] at 25°C in 10 mM Na-K-phosphate buffer which enhances PZ binding affinity and in modified Krebs-phosphate buffer, which mimics physiological conditions. Classical agonists such as carbachol, oxotremorine and acetylcholine inhibited (-)-[3H]QNB binding to membranes with shallow Hill values (n(H) < 1), were better fit to a 2-state model, were Gpp(NH)p-regulated and showed lower affinity in modified Krebs-phosphate buffer than in 10 mM Na-K-phosphate buffer. Some agonists were not significantly better fit to a 2-state model in [3H]PZ-labeled cortical membranes, especially in 10 mM Na-K-phosphate buffer. Whereas putative M1 and M2 binding sites distinguished by PZ possessed multiple agonist affinity states, as judged by carbachol, and agonist binding to [3H]PZ-labeled sites were Gpp(NH)p modulated, the partial agonist pilocarpine and nonclassical agonist McN-A-343 [3-(m-chlorophenylcarbamoyloxy)-2-butynyl trimethylammonium chloride] showed little Gpp(NH)p-induced shift in [3H]PZ-labeled cortical membranes in physiological conditions. Agonist binding to (-)-[3H]QNB-labeled putative M2 cardiac sites was more sensitive to Gpp(NH)p than (-)-[3H]QNB-labeled cortical sites. Carbachol and acetylcholine showed significant selectivity for putative M2 sites. Whereas some selectivity remained with Gpp(NH)p present, M2 selectivity of these agonists was modified by guanine nucleotides, as Gpp(NH)p lowered agonist affinity in the heart to values comparable to the cortex. Pilocarpine and McN-A-343 showed significant selectivity for putative M1 sites in modified Krebs-phosphate buffer with Gpp(NH)p. Orders of potency and selectivity for agonists implied differences in muscarinic heterogeneity sensed by agonists vs. heterogeneity detected by selective antagonists like PZ, and suggested the existence of multiple agonist affinity states for high and low affinity PZ sites.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1986|
ASJC Scopus subject areas
- Molecular Medicine