The research examines the relationship between muscarinic cholinergic receptor subtypes as identified by binding criteria and second messenger systems in the rat brain. To accomplish this goal a limited number of muscarinic ligands, which have been selected on the basis of apparent selectivities for second messenger systems will be screened autoradiographically for the regional distribution of receptors having high affinities for the ligands. The most selective ligands--both agonists and antagonists--will be examined for their abilities to stimulate/antagonize muscarinic receptor-mediated stimulation of phosphatidylinositol turnover, inhibition of adenylate cyclase and stimulation of GTPase, comparing different brain areas. The hippocampus, brainstem and cerebellum have been selected as three prototypical brain areas that appear to possess relatively uniform but disparate populations of the receptor subtypes. Our preliminary data suggest that regional specificities of various agonists may not be related to their abilities to stimulate a particular second messenger system. Screening of ligands for regional selectivity by receptor autoradiography is performed indirectly by comparing the abilities of various ligands to compete for the universal antagonist (3H) quineclidinyl benzilate (3H)-QNB. The method has the advantage that a number of ligands can be compared directly in the same assay. The effects of ligands on second messenger systems are examined, comparing areas with different binding selectivities. Accumulation of inositol phosphates after prelabeling with (3H) inositol and accumulation of c-AMP have been selected because they represent the two major receptor-stimulated metabolic pathways leading to cellular responses. Receptor-stimulated GTP hydrolysis represents a step close to receptor occupancy involved in the muscarinic receptor-mediated inhibition of adenylate cyclase and most probably in the stimulation of phosphatidyl inositol (PI) turnover also. If the studies proceed as expected, we will be able to show clearly for the first time that regional selectivity based on binding is independent of the selectivity for the second messenger systems in the brain. This information should aid in rational drug design for cognitive and other deficits that result from the impairment of central cholinergic function.
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