Biochemical, pharmacological and molecular biological approaches are integrated in an investigation of phosphoinositide (PPI)-mediated signal transduction in the CNS, with emphasis on PPI-linked muscarinic receptors (mAChR)s, toward the eventual goal of establishing the cellular basis of their regulation in normal and dysfunctional brain states. In vivo studies offer the advantage that physiological and anatomical relationships in the experimental preparation remain largely intact. They are complemented by studies in cultured cell lines of neural origin preparations that are vastly simpler and experimentally accessible. Together, the two approaches permit one to pose straightforward yet cogent experimental questions. Based on the known distribution of mRNAs for AChR isoforms in brain, we will explore their regulation using the combined approaches of receptor autoradiography, immunoprecipitation and mRNA hybridization methods. Diisopropylfluorophosphate- and trihexyphenidyl-treated rats will be used to study effects of chronic cholinergic stimulation. Cultured cell studies will explore distribution of mAChRs and the mechanism of homologous agonist-induced receptor sequestration, including the possible role for G-proteins in this process. A cell line expressing multiple PPI-linked receptors (in addition to adenyl cyclase-linked receptors) will be used to determine the extent of cross-talk among different receptors and intracellular signalling systems. We will explore to what degree IP3 receptor function may be regulated by cyclic AMP-dependent protein kinase. Effects of overexpression of key enzymes and receptors on signal transduction events will be investigated. Evidence in cultured cells will be brought to bear for or against the hypothesis that Li+ produces its therapeutic effect in manic depressive psychoses as a consequence of its inhibition of inositol monophosphate phosphatase (IP-Pase), which has been presumed to result in depletion of intracellular inositol available to phosphatidylinositol (PI) synthase. We will measure inositol and the inositol phosphates, the inositol lipids and their precursors: phosphatidate, DAG, and CDP-DAG, under conditions of rest and muscarinic stimulation in the presence and absence of Li+. Analytical and labeling studies on the PPI cycle lipids will take advantage of their known enrichment in the stearoyl arachidonoyl DAG species. We will also further explore in vivo and in vitro the regulation and properties of the key lipid enzyme, PI synthase. The gene for the latter will be cloned, antibodies will be prepared for immunohistochemistry studies, and the possibility of multiple genes encoding PI synthase will be explored. The enhancement or depletion of mRNA levels for this enzyme, as well as inositol 3P-synthase and IP-Pase, will be measured by northern blots, and where appropriate in sections of whole brain, by in situ hybridization. Together, the individual subprojects will contribute to the common goal of understanding regulatory mechanisms of signal transduction pathways in the CNS that underlie abnormal behavioral states.
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