This proposal concerns muscarinic receptor mechanisms in the brain and the development of new therapies for brain diseases in which there is a significant change in cholinergic functions. In Alzheimer's disease (AD) the predominant ml, m3 and m4 receptors of the cortex and hippocampus are extensively denervated and the development of new selective agonists for cholinergic replacement therapy appears warranted. In Parkinson's disease (PD) the predominant ml and m4 receptors in the striatum are believed to be overactivated, and new selective antagonists could be significantly better than existing, non selective drugs. Therapy for PD might also be directed at m5 receptors which are unusually prevalent in the substantia nigra and globus pallidus and which may help control the activity of dopaminergic nerves. This laboratory has shown that ml receptors (binding sites for 1 nM 3H-pirenzepine) remain nearly normal in numbers, affinity for agonists, coupling to endogenous G protein, and ability to promote phosphoinositide hydrolysis, in AD and/or after chronic experimental cholinergic denervation. Hence selective agonists should work in AD, provided that they can be developed, and provided that the cellular location of receptors is not too abnormal. The design and testing of new agonists and antagonists is greatly complicated by evidence for at least 3 ligand binding sites on various receptor subtypes. This laboratory has found equal numbers of high (H) and low (L) affinity sites for agonists on ml and m2 receptors, suggesting bivalent or dimeric receptors, plus at least one more site for allosteric antagonists, Some antagonists also appear to bind to equal numbers of high and low affinity sites. To address the physical meaning of two sites, attempts will be made to show two binding sites for 3H-QNB on bivalent receptors in membranes and in solution. If receptors appear dimeric, further studies will involve crosslinking of the monomers and G protein, and measurements of the sizes of the two monomers. To address the functional meaning of two sites, either H or L sites on ml-m4 receptors will be blocked in membranes and in cultured cells, with antagonists, and the other site will be characterized for its binding of agonists and antagonists, functional coupling, its pK for blockade by protonation, and for the contribution of the site to allosteric phenomena. The probable identity of H and L sites, sites showing high and low affinity for certain antagonists, and sites showing two different pK values will then be examined, using ml and m2 receptors with 4 point-mutated aspartate residues. When it is clear how each site contributes to the binding and function of agonists and antagonists, new screening procedures for ml-m5 agonists and antagonists will be developed, based on these methods. To address the cellular location of receptors in normal, AD and PD brains, autoradiography will be carried out using a new toxin which shows marked ml + m3 > > M2 + m4 selectivity. Novel morphological data should be obtained for AD and PD, and these data should provide a good basis for physiological work with the toxin, and for understanding which cells are most likely to be controllable with muscarinic ligands.
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