MUSCARINIC RECEPTORS & SELECTIVE PSYCOTROPIC DRUGS

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Pirenzepine (PZ) is a novel muscarinic acetylcholine receptor (mAChR) antagonist. It is selective for some central nervous system (CNS) mAChR's and is used clinically. Studies of (3H) pirenzepine ((3H)PZ) reveal differences in CNS and cardiac mAChR's. Another new antagonist, AF-DX 116, has the inverse selectivity of PZ, showing high affinity for a subset of mAChR's that bind PZ with low affinity. (3H)PZ and (3H)AF-DX 116 can be used to study putative mAChR subtypes in the CNS and heart directly. Using rapid filtration assays and nonlinear least squares regression analyses, the binding and regulation of M1 (high affinity (3H)PZ and M2 (high affinity (3H)AF-DX 116) mAChR's in these tissues and in cultured neuronal and cardiac cells will be studied. Quantitative autoradiography (qARG) allows sublocalization of mAChR subtypes in tissue slices of CNS and heart. The hypothesis that M1 mAChR's are functionally coupled to phosphatidylinositol (PI) turnover will be evaluated by comparing potencies of mAChR agonists and antagonists in stimulating or inhibiting PI turnover with potencies in binding assays in parallel conditions. Other studies will try to correlate cAMP generation selectively to M2 mAChR's. Differences in ontogeny of CNS and heart M1 and M2 mAChR's in fetal through adult rats will be characterized homogenates and visualized by qARG. Primary recognition sites for (3H)PZ (M1) and (3H)AF-DX 116 (M2) in solubilized mAChRs will be studied in adult and neonatal tissues, as will the time of onset and nature of coupling to PI turnover and cAMP during postnatal ontogeny. Differences in binding and coupling characteristics after chronic drug administration will also be studied. Mechanistic aspects of PI turnover and cAMP generation, and their relationship to mAChR binding and possible associations with different guanine nucleotide binding proteins will be studied in cultured intact human neuroblastoma (SH-SY 5Y) cells in dissociated heart cells (cardiomyocytes) to exploit these more homogeneous systems. The selective involvement of M1 or M2 mAChRs in aging and in Senile Dementia of the Alzheimer's Type (SDAT) will be determined in binding, functional and behavioral studies of aged rats, and in an ibotenic acid induced nucleus basalis of Meynert (magnocellularis) lesioned rat model of SDAT and also in age- matched post-mortem human brain tissue. These studies will enable us to precisely define the molecular basis of M1/M2 mAChR subtypes and provide useful data for the rational use of current mAChR drugs including minimizing side effects, as well as aid in the future development of efficacious and more highly M1 and M2 selective drugs.