Pirenzepine is a novel antagonist of muscarinic acetylcholine receptors, since it demonstrates considerable tissue selectivity. Although there is a substantial volume of literature related to the pharmacological and biochemical effects of pirenzepine, significantly less efforts have been devoted in order to characterize the nature of interaction of this drug with the muscarinic receptors and the relationship of its binding sites on the receptor to those of classical receptor antagonists. The present proposal is designed to provide better understanding of the features of binding of pirenzepine to brain muscarinic receptors. The relationship between pirenzepine binding sites in the brain and those of [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine will be investigated in detail. Several criteria will be used in order to define whether pirenzepine interacts with the binding sites of these ligands competitively or noncompetitively. These criteria will be assessed by studying: 1) the effect of pirenzepine on the saturation isotherms, in addition to the rates of association and dissociation of the ligands in rat brain, 2) displacement of increasing ligand concentrations by pirenzepine, 3) effects of preincubation with pirenzepine on subsequent ligand binding, and 4) the ability of pirenzepine to protect the binding sites of these ligands against irreversible alkylation. A second part of the project deals with investigating whether the hydrophilic nature of pirenzepine might explain its unique receptor binding features and pharmacological selectivity. To provide a satisfactory answer for this speculation, the binding profile of both pirenzepine and N-methylscopolamine, a quaternary muscarinic receptor ligand, will be compared. The proposed experiments include studying the regional distribution of the binding sites of both ligands and its relationship to [3H]quinuclidinyl benzilate binding sites. In addition, the functional selectively of N-methylscopolamine, if any, will be compared to that of pirenzepine in terms of their ability to block agonist-induced responses in mouse neuroblastoma cells. Taken together, the experiments suggested in the present proposal are expected to provide useful information concerning the mode of interaction of pirenzepine with brain muscarinic cholinergic receptors.
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