The overall goal of this research project is to better understand the structure, function and regulation of muscarinic cholinergic receptors. It appears from our studies and studies from other laboratories, that at least in rat CNS in situ, two distinct binding sites can be distinguished that have the properties expected of a muscarinic receptor. One of these sites appears to mediate the breakdown of phosphoinositides while the other appears to mediate the inhibition of adenylate cyclase activity. The possibility that these binding sites and their associated responses can be regulated independently will be examined. Chronic alterations in the degree of receptor stimulation caused by the administration of receptor antagonists and agonists or by selective lesions will be made and their effects on the density and properties of each binding site and its associated response will be determined. Additionally, the ontogenetic profile for each binding site and its associated biochemical response will be determined to investigate the possibility that they do not develop concomitantly. Finally, quantitative autoradiographic techniques will be developed and used to examine the normal distribution of each site throughout the rat brain at 20 Mum resolution. The effects of lesions on the density of each site will be studied at high anatomical resolution with this technique. To determine if these binding sites are physically separate entities, a series of compounds will synthesized and utilized for affinity chromatographic purification of each site. Functional reconstitution of purified binding site preparations into clonal cells which have either a strong receptor-mediated inhibition of adenylate cyclase activity or stimulation of phosphoinositide breakdown but do not possess muscarinic receptors will be attempted. Additionally, functional reconstitution of purified binding sites with the purified guanine nucleotide-binding regulatory proteins that stimulate (Ns) and inhibit (Ni) adenylate cyclase will be performed in defined phospholipid vesicles.
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