MUSCARINIC CHOLINERGIC RECEPTOR-BINDING SITES DIFFERENTIATED BY THEIR AFFINITY FOR PIRENZEPINE DO NOT INTERCONVERT

  • 1 May 1986
    • journal article
    • research article
    • Vol. 237  (2) , 577-582
Abstract
Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M2 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4.degree. C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner. Consistent with previously published studies, the Kd values of PZ for putative M1 and M2 binding sites were 12 nM and 168 nM, respectively, as determined by nonlinear least-squares regression analysis. When PZ was used to protect muscarinic receptor binding sites from irreversible blockade by PBCM, it was found to be more potent in protecting M1 sites than M2 sites. The potencies calculated for PZ protection of M1 and M2 sites from blockade by 20 mM PBCM were 3.7 nM and 205 nM, respectively. These data suggest that the M1 and M2 muscarinic receptor binding sites recognized by PZ in rat cerebral cortex are not interconvertible under the experimental conditions used.