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 and several selective antagonists are now available (including pirenzepine, AF-DX 116, and quaternary antagonists). Past characterizations of muscarinic responses may have been confounded by tissue-related differences, as the different responses were studied in different tissues or preparations. This proposal intends to correlated sophisticated assays of binding and response in a model in vitro system, so as to relate the responses to given subpopulations. We will evaluate multiple muscarinic responses and receptor binding in parallel, in identical preparations (CNS neurons in primary culture); thereby, the problems of between-tissue differences will be eliminated and the affinities of a given antagonists in the different response assays and binding assays can be compared directly. The ability to generate neuronal cultures from regions of the brain that possess very different distributions of subpopulations will test the uniformity of the relationship between response and receptor subtype. 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 blockage by alkylating antagonists. Autoradiographic studies will determine whether subpopulations of muscarinic receptors are localized to specific types of (immunocytochemically identified) neurons. Studies of phosphorylation of muscarinic receptors are aimed at determining whether different subpopulations are differentially phosphorylated, desensitized, and down-regulated. The interactions of different kinases in regulating the sensitivity of the receptor(s) will also be investigated. It is expected that these studies will lead to a better understanding of the functions and regulation of different subpopulations of muscarinic receptors.
Showing the most recent 10 out of 30 publications
