Muscarinic receptors are known to couple to a number of effector systems in the CNS, one of which is the enhanced phosphodiesteratic breakdown of inositol phospholipids. The products of lipid hydrolysis, namely diacylglycerol and inositol trisphosphate may each act as intracellular second messengers. While the breakdown of inositol lipids following muscarinic receptor activation has been documented in a number of neural tissues, the series of events that intervene between receptor occupancy and activation of phospholipase C, and their regulation, remain largely unknown. Our objective is the elucidation of these events. 1) To determine the receptor occupancy requirements for the phosphoinositide response in discrete areas of the CNS and in chronically denervated tissue. Receptor alkylation studies will permit a direct comparison between receptor number and biochemical response. 2) To determine whether guanine nucleotide binding (G) proteins play a role in the transduction process. The effects of guanine nucleotides on agonist binding parameters in membranes and on the activation of phospholipase C in a permeabilized cell preparation will be assessed. The inhibitory effects of specific toxins or antibodies to G-proteins will be evaluated for their effects on inositol lipid hydrolysis. 3) To determine the temporal and stoichiometric relationship between inositol lipid hydrolysis and the generation of Ca2+ signals. Human neuroblastoma MC-NB-1 cells will be used as a model neuronal preparation. 4) To determine the relationship between an agonist's efficacy for phosphoinositide turnover, its ability to bind to multiple affinity forms of the receptor, and propensity to induce receptor desensitization by measurement of these parameters under the same conditions in intact dissociated cells from the CNS. Additional studies will focus on agonist-induced receptor loss from neural membranes. The measurement of inositol lipid hydrolysis is of sufficient magnitude to provide a convenient biochemical handle for studies on muscarinic receptor function and regulation in the CNS. A greater understanding of the molecular events underlying phosphoinositide hydrolysis may prove invaluable in unraveling the etiology of muscarinic receptor dysfunction known to occur in some neuropsychiatric disorders.
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