Changes in expression of a functional Gi protein in cultured rat heart cells.
The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (Gi) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol (0.1 mM). However, in cells cultured for 11 days, carbachol (0.1 mM) inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for Gi in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although Gi is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional Gi is dependent on culture conditions. Furthermore, the ANG II receptor can couple to Gi in heart.
Allen
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Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
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Rogers
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D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000096:biosynthesis*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism
D011976:Receptors, Muscarinic; D000804:Angiotensin II; D006165:Guanylyl Imidodiphosphate; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D007545:Isoproterenol