Abstract
Both muscarinic m2 receptors that inhibit adenylyl cyclase and m3 receptors that stimulate the hydrolysis of inositol phospholipids are expressed in cerebellar granule cells. In order to determine whether a reserve population of either of these receptors is present in this cell culture, the irreversible muscarinic receptor antagonist, propylbenzilylcholine mustard (PBCM), was used at different concentrations to bind various proportions of available muscarinic receptors. After pretreating the cell cultures with low concentrations of PBCM (< 1 nM), the muscarinic m2 receptor-mediated response decreased. Higher concentrations of PBCM (1-3 nM) were needed to reduce the muscarinic m3 receptor-mediated response. These results suggested that either a reserve population of muscarinic m3 receptors is present or that PBCM shows greater specificity for muscarinic m2 receptors. Because the muscarinic m2 receptor is a 66 kDa protein, whereas the muscarinic m3 receptor is a 92 kDa protein, these receptors can be separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after being labeled with [3H]PBCM. PBCM appears to specifically bind the 66 kDa m2 receptor at concentrations lower than those required to bind to the 92 kDa m3 receptor. A linear correlation was found between the increased binding of [3H]PBCM to each receptor and the proportional loss of that receptor-mediated response. Thus, a reserve population of either muscarinic m2 or m3 receptors does not appear to exist in cerebellar granule cells. These studies also show that PBCM has greater affinity for the muscarinic m2 receptor than the muscarinic m3 receptor.
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