Muscarinic cholinergic receptors (mAChRs) are present in high concentrations in the central nervous system and have been implicated in the etiology underlying a number of neuropsychiatric disorders. At least four mRNAs encoding mAChR subtypes are now known to exist and each gene product has its own distinct structural and pharmacological characteristics. While two of these subtypes (M1 and M2) have received considerable attention, relatively little is known of the properties of the other two and their mode of interaction with effector mechanisms. The human neuroblastoma cell line SK-N-SH expresses a large number of high MW mAChRs of the M3 subtype as defined pharmacologically. These receptors couple to enhanced phosphoinositide breakdown and Ca2+ signaling, but not to the inhibition of adenylate cyclase. The objectives of this proposal are three-fold. 1) To determine the contribution of glycosylation to the structural and functional characteristics of the receptor. Lectin affinity chromatography and endoglycosidase treatments will determine the extent of glycosylation of the receptor, while results obtained with cells grown in the presence of inhibitors of glycosylation will point to its functional significance. 2) To determine which guanine- nucleotide (G)-binding proteins can functionally interact with the M3 receptor and to probe the identity of the protein (Go), specifically linked to activation of phospholipase C. Using reconstitution assays, we will determine which G-proteins can restore high affinity agonist binding when added to membranes previously depleted of G-proteins. The identity of Gp will be assessed from the ability of muscarinic agonists to increase the labeling of un from guanine nucleotides. 3) To determine the relationship between mass of inositol trisphosphate (IP3) in cells and the IP3 requirements for occupancy of IP3 receptors and mobilization of Ca2+. A role for IP3 in mediation of Ca2+ influx will be assessed by measurement of IP3 binding sites in-plasma membranes and an analysis of their respective time-courses, agonist specificities and the effects of antagonists. The availability of a homogenous population of human neuronal cells that express an abundance of the M3 subtype provides a unique opportunity of studying this specific receptor subtype. It is likely that these studies will form the basis of an understanding of their physiological role and possible relevance to CNS disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS023831-08
Application #
2264951
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1986-09-01
Project End
1994-11-30
Budget Start
1993-09-01
Budget End
1994-11-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Psychiatry
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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