Reduced cholinergic function in the central nervous system contributes to memory disturbances, including those associated with senile dementia of the Alzheimer type. Previous studies by ourselves and others have demonstrated conclusively that distinct subtypes of muscarinic receptors are present in varying proportions in different brain regions. Classical receptor theory dictates that the most useful receptor classification schemes be based on the affinities of antagonists. The only antagonist that displays heterogeneous binding profiles and has been extensively characterized in response assays is pirenzipine; therefore, there is some reason for concern that the results obtained may reflect anomalous properties of this single compound, rather than an underlying pharmacology. Further, the characterizations may have been confounded by tissue-related differences, as the different responses were studied in different tissues. This proposal intends to correlate sophisticated assays of binding and response in model in vitro systems so as to relate the responses to given subpopulations. One line of investigation will evaluate multiple muscarinic responses and receptor binding in parallel, in identical preparations (neurally derived cell lines); thereby, the problems of between-tissue differences will be eliminated and the affinities of a given antagonist in the different response assays and binding assays can be compared directly. A second approach will examine a single response in regions of the brain that possess very different distributions of subpopulations, as a test of the uniformity of the relationship between response and receptor subtype. In both of these approaches, receptor subtypes will be defined according to the affinities of pirenzipine and other antagonists (e.g. gallamine, clozapine), characterized in our ongoing evaluation of potentially selective muscarinic ligands. Furthermore, the results of classical pharmacological methods, such as Schild analysis of competitive antagonists, will be supplemented by an independent procedure: selective protection of given subpopulation(s) of receptors from irreversible blockade by alkylating antagonists. Selective protection is a powerful technique that can also be used to determine the relationships between the subpopulations defined by different selective ligands and to evaluate the response characteristics of agonist-defined subpopulations. It is expected that these studies will lead to the development of agents with greater selectivity toward muscarinic subpopulations and that this knowledge may foster the development of newer and safer approaches to the enhancement of cognitive performance.
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