The goals of the current project are to study the mechanisms which regulate the activity and levels of guanine nucleotide regulatory proteins (G proteins) in embryonic chick heart and to determine the role of the regulation of G proteins in modulating the responsiveness of the heart to muscarinic stimulation. Specifically, we will test the hypothesis that treatment with phorbol esters, prolonged exposure to muscarinic agonist results in phosphorylation or other modification of G proteins which render them incapable of coupling the muscarinic receptor to a physiologic response. We have demonstrated that under conditions where levels of the 39 Kda(alpha 39) and 41 Kda(alpha) pertussis toxin substrate were limiting, in both atrial cells pretreated with pertusssis toxin and cultures of hears 31/2days in ovo. muscarinic stimulation resulted in a positive chronotropic response. Hence we will test the hypothesis that the levels of G proteins are important in controllin the extent of coupling of muscarinic receptors to muscarinic functions which are insensitive to pertussis toxin and which include the positive chronotropic response to muscarinic stimulation such as production of diacylglycerol, activation of protein kinase C, release of arachidonic acid, and activation of Na+/H antiporter. Furthermore, we will test the hypothesis that those stimuli which increse the responsiveness of the heart to muscarinic stimulaiton such as growth of heart cells in lipoproteindepleted serum, and coculture of embryonic chick hearts 31/2 days in ovo with ciliary ganglia, do so by stimulating the appearance of increased levels of low affinity muscarinic receptors followed by the synthesis of increased levels of G proteins and the subsequent interconversion of low affinity receptors to a haigh affinity form and that the time course of the increase in new receptors and G proteins correlates with the appearance of increasedmuscarinic inhibition of adenylate cyclase, increased stimulation of K efflux, and increased sensitivity of the inotropic and chronotropic response of the heart to muscarinic stimulation. We will further test the hypothersis that the appearance of the increased levels of G protein represents new protein synthesis as demonstrated by the time course of appearance of increased levels of messenger RNAs coding for alpha39 and/or alpha41. These studies will help us understand the mechanism of regulation of muscarinic responsiveness and the role of G proteins in setting the chromotropic and inotropic state of the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL036014-03
Application #
3350481
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1985-07-01
Project End
1992-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Gadbut, A P; Wu, L; Tang, D et al. (1997) Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for ras in regulating the expression of muscarinic receptors and G proteins. EMBO J 16:7250-60
Gadbut, A P; Riccardi, D; Wu, L et al. (1996) Specificity of coupling of muscarinic receptor isoforms to a novel chick inward-rectifying acetylcholine-sensitive K+ channel. J Biol Chem 271:6398-402
Loh, E; Barnett, J V; Feldman, A M et al. (1995) Decreased adenylate cyclase activity and expression of Gs alpha in human myocardium after orthotopic cardiac transplantation. Circ Res 76:852-60
Kilbourne, E J; Galper, J B (1994) Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus. Biochem J 297 ( Pt 2):303-8
Gadbut, A P; Toupin, D K; Kilbourne, E J et al. (1994) Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G alpha i2, and the acetylcholine-sensitive K+ chan J Biol Chem 269:30707-12
Kilbourne, E J; Galper, J B (1994) Cloning of cDNAs coding for the G alpha i1 and G alpha i2 G-proteins from chick brain. Gene 150:341-4
Gadbut, A P; Galper, J B (1994) A novel M3 muscarinic acetylcholine receptor is expressed in chick atrium and ventricle. J Biol Chem 269:25823-9
Barnett, J V; Taniuchi, M; Yang, M B et al. (1993) Co-culture of embryonic chick heart cells and ciliary ganglia induces parasympathetic responsiveness in embryonic chick heart cells. Biochem J 292 ( Pt 2):395-9
Tan, W; Barnett, J V; Pietrobon, D et al. (1993) Effect of low-density lipoproteins, mevinolin, and G proteins on Ca2+ response in cultured chick atrial cells. Am J Physiol 265:H191-7
Barnett, J V; Shamah, S M; Lassegue, B et al. (1990) Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling. Biochem J 271:443-8

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