PIRENZEPINE DISTINGUISHES BETWEEN MUSCARINIC RECEPTOR-MEDIATED PHOSPHOINOSITIDE BREAKDOWN AND INHIBITION OF ADENYLATE-CYCLASE

Abstract

Subtypes of muscarinic cholinergic receptors were proposed to exist, but the biochemical responses mediated by the putative subtypes are unknown. Muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase activity were characterized in rat brain as well as rat parotid and heart. These responses were studied to determine if they are mediated by separate subtypes of muscarinic receptors, the potencies of agonists and antagonists were determined in both assays. Antagonist potencies were calculated by Schild analysis. In the brain, the putatively selective muscarinic receptor antagonist, pirenzepine, exhibited Ki values of 21 nM in the assay of phosphoinositide breakdown and 310 nM in the assay of adenylate cyclase activity. Using radioligand binding techniques, it distinguished 2 binding sites with Kd values of 12 and 168 nM. The antagonist, atropine, on the other hand, was equipotent in the 2 biochemical assays and the radioligand binding assay with Ki values of .apprx. 1-2 nM. In peripheral tissues with robust muscarinic receptor-mediated phosphoinositide (parotid) and adenylate cyclase (heart) responses, pirenzepine exhibited a similar selectivity (19-fold) for the phosphoinositide assay that was seen in the brain, but it was 6- to 7-fold less potent in both peripheral tissues than in the CNS. The potencies of pirenzepine in binding and functional studies in each tissue were not as well correlated as in the brain. Atropine and other antagonists were 4- to 9-fold selective for inhibiting oxotremorine-stimulated phosphoinositide breakdown in the peripheral tissues. McN A343 [4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride], considered to be a selective ganglionic muscarinic receptor agonist, was an antagonist or partial agonist in these studies. It was only 4-fold selective for the phosphoinositide assay in the periphery and pirenzepine exhibited a similar potency at antagonizing either McN A343 for oxotremorine-mediated phosphoinositide breakdown. All the agonists, except pilocarpine, which had low efficacy for both responses, were not selective for either assay. The site in the CNS that has high affinity (10-20 nM) for pirenzepine in radioligand binding studies mediates phosphoinositide breakdown and the site that has low affinity (200-300 nM) mediates inhibition of adenylate cyclase. The observed properties of muscarinic receptors are not identical in the CNS and peripheral tissues. It remains to be seen whether the putative muscarinic receptor subtypes are different proteins or if the observed differences are induced by the biochemical effector system to which the receptor is coupled.