| Subtypes of muscarinic cholinergic receptors, termed M(,1) and M(,2), that have high affinity (10-20 nM) and low affinity (200-400 nM), respectively, for the antagonist, pirenzepine, have been postulated, but the biochemical responses mediated by the putative subtypes are unknown. In several receptor systems (eg. (alpha)-adrenergic), separate receptor subtypes modulate phosphoinositide metabolism and adenylate cyclase activity. Muscarinic cholinergic receptors also regulate these biochemical responses and analogy with other receptors suggests that muscarinic receptors may exist as two subtypes, each modulating a distinct biochemical response. The potencies of muscarinic receptor drugs, therefore, were determined in assays of phosphoinositide metabolism and adenylate cyclase inhibition. Antagonist potencies were emphasized and were calculated by Schild analysis.;In the rat forebrain, pirenzepine exhibited K(,i) values of 21 nM in the assay of phosphoinositide breakdown and 310 nM in the assay of adenylate cyclase inhibition. Similarly, using radioligand binding techniques, it distinguished two binding sites with K(,d) values of 12 and 168 nM. The antagonist, atropine, on the other hand, was equipotent in the two biochemical assays and the radioligand binding assay with K(,i) values of approximately 1 to 2 nM. In peripheral tissues with robust muscarinic receptor-mediated phosphoinositide (parotid gland) and adenylate cyclase (heart) responses, pirenzepine exhibited a similar selectivity (19-fold) for the phosphoinositide assay that was seen in the forebrain, but it was 6- to-7-fold less potent in both peripheral tissues than in the forebrain.;To test the hypothesis that the binding sites for pirenzepine are interconvertible conformational states of the same protein, pirenzepine was used to protect muscarinic receptors from irreversible blockade by the antagonist, propylbenzilylcholine mustard. The potencies calculated for pirenzepine protection of M(,1) and M(,2) binding sites were 3.7 nM and 205 nM, respectively. These values were similar to potencies found in functional and radioligand binding studies. Thus, in the rat forebrain, the M(,1) site appears to mediate phosphoinositide breakdown and the M(,2) site appears to mediate inhibition of adenylate cyclase. These sites do not appear to interconvert, but their properties are not entirely conserved in other tissues. |
