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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS023831-03
Application #
3407766
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1986-09-01
Project End
1989-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Fisher, Stephen K; Heacock, Anne M; Keep, Richard F et al. (2010) Receptor regulation of osmolyte homeostasis in neural cells. J Physiol 588:3355-64
Foster, Daniel J; Vitvitsky, Victor M; Banerjee, Ruma et al. (2009) Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells. J Neurochem 108:437-49
Fisher, Stephen K; Cheema, Tooba A; Foster, Daniel J et al. (2008) Volume-dependent osmolyte efflux from neural tissues: regulation by G-protein-coupled receptors. J Neurochem 106:1998-2014
Foster, Daniel J; Heacock, Anne M; Keep, Richard F et al. (2008) Activation of muscarinic cholinergic receptors on human SH-SY5Y neuroblastoma cells enhances both the influx and efflux of K+ under conditions of hypo-osmolarity. J Pharmacol Exp Ther 325:457-65
Cheema, Tooba A; Fisher, Stephen K (2008) Cholesterol regulates volume-sensitive osmolyte efflux from human SH-SY5Y neuroblastoma cells following receptor activation. J Pharmacol Exp Ther 324:648-57
Cheema, Tooba A; Pettigrew, Veryan A; Fisher, Stephen K (2007) Receptor regulation of the volume-sensitive efflux of taurine and iodide from human SH-SY5Y neuroblastoma cells: differential requirements for Ca(2+) and protein kinase C. J Pharmacol Exp Ther 320:1068-77
Heacock, Anne M; Foster, Daniel J; Fisher, Stephen K (2006) Prostanoid receptors regulate the volume-sensitive efflux of osmolytes from murine fibroblasts via a cyclic AMP-dependent mechanism. J Pharmacol Exp Ther 319:963-71
Heacock, Anne M; Dodd, Michael S; Fisher, Stephen K (2006) Regulation of volume-sensitive osmolyte efflux from human SH-SY5Y neuroblastoma cells following activation of lysophospholipid receptors. J Pharmacol Exp Ther 317:685-93
Cheema, Tooba A; Ward, Caroline E; Fisher, Stephen K (2005) Subnanomolar concentrations of thrombin enhance the volume-sensitive efflux of taurine from human 1321N1 astrocytoma cells. J Pharmacol Exp Ther 315:755-63
Heacock, Anne M; Kerley, Daniel; Gurda, Grzegorz T et al. (2004) Potentiation of the osmosensitive release of taurine and D-aspartate from SH-SY5Y neuroblastoma cells after activation of M3 muscarinic cholinergic receptors. J Pharmacol Exp Ther 311:1097-104

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