The goal is to elucidate at the molecular level the factors involved in the development, function, and regulation of muscarinic acetylcholine receptors (mAChR) in the heart. We will study chick cardiac mAChR during embryonic development in vivo and in cardiac muscle cell culture. We will determine the molecular basis of the diminished physiological responsiveness of newly synthesized mAChR to confirm our hypothesis that the mAChR is synthesized in an immature form and is subsequently converted to a more physiologically active form. We have also obtained preliminary evidence by quantitative immunoblotting that at least 2 of the subunits of the GTP regulatory proteins (N Alpha and Beta) are increased greatly in amount in embryonic atria but not ventricles between days 10 and 15. It has been reported that an atrial-specific increase in mAChR number which is dependent on innervation also occurs at this time. This proposal will test the hypothesis that the mAChR and its GTP-binding effector proteins are coordinately regulated during embryonic development by innervation. We have obtained preliminary evidence that treatment of cultured cardiac cells with phorbol esters (which activate protein kinase C) prevents the mAChR-mediated increase in K+ permeability. We will test the hypothesis that short-term desensitization of the mAChR-induced negative chronotropic response is mediated by protein kinase C. We will use two different methods to isolate antibodies specific for the mAChR: by immunizations with purified mAChR preparations, and by immunization with several monoclonal antibodies specific for mAChR ligands to isolate anti-idiotype antibodies which recognize the ligand binding site on the mAChR. These antibodies will be used in our subsequent studies on mAChR regulation. This research should identify the factors involved in cardiac mAChR regulation, development, and function, and may aid in the understanding of a number of cardiac abnormalities.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL030639-08
Application #
3341652
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1983-08-01
Project End
1992-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Nathanson, Neil M (2008) Synthesis, trafficking, and localization of muscarinic acetylcholine receptors. Pharmacol Ther 119:33-43
Goin, Juan C; Nathanson, Neil M (2002) Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells. J Neurochem 83:964-72
Creason, S; Tietje, K M; Nathanson, N M (2000) Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene. J Neurochem 74:882-5
Belmonte, K E; McKinnon, L A; Nathanson, N M (2000) Developmental expression of muscarinic acetylcholine receptors in chick retina: selective induction of M2 muscarinic receptor expression in ovo by a factor secreted by muller glial cells. J Neurosci 20:8417-25
Rosoff, M L; Nathanson, N M (1998) GATA factor-dependent regulation of cardiac m2 muscarinic acetylcholine gene transcription. J Biol Chem 273:9124-9
McKinnon, L A; Gunther, E C; Nathanson, N M (1998) Developmental regulation of the cm2 muscarinic acetylcholine receptor gene: selective induction by a secreted factor produced by embryonic chick retinal cells. J Neurosci 18:59-69
Fischer, A J; McKinnon, L A; Nathanson, N M et al. (1998) Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick. J Comp Neurol 392:273-84
McKinnon, L A; Rosoff, M; Hamilton, S E et al. (1997) Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice. Life Sci 60:1101-4
Jackson, D A; Nathanson, N M (1997) Regulation of expression and function of m2 and m4 muscarinic receptors in cultured embryonic chick heart cells by transforming growth factor-beta 1. Biochem Pharmacol 54:525-7
Hamilton, S E; McKinnon, L A; Jackson, D A et al. (1995) Molecular analysis of the regulation of muscarinic receptor expression and function. Life Sci 56:939-43

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