Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding.
Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-diproylxanthine ([3H]CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that [3H] CPX is an antagonist radioligand. Competition curves for [3H] CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific [3H]CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid). These data indicate that the adenosine receptor coupled to inhibition of adenylate cyclase activity and to the negative inotropic effect is the A1 subtype. Pertussis treatment uncoupled the adenosine receptor from both inhibition of adenylate cyclase activity and negative inotropic effect. Taken together, the present study indicates that adenosine receptors of the A1 subtype are present on the spontaneously contracting atrial myocytes and are negatively coupled to adenylate cyclase and to the contractile state. The cultured embryonic chick atrial myocyte preparation represents a useful model system for characterizing the cardiac A1 adenosine receptor.
Liang
B T
BT
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D002642:Chick Embryo; D006325:Heart Atria / Q000187:drug effects / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D037342:Pertussis Toxin; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014970:Xanthines
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D011983:Receptors, Purinergic; D010566:Virulence Factors, Bordetella; D014970:Xanthines; D014316:Tritium; C051360:1,3-dipropyl-8-cyclopentylxanthine; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases; D007545:Isoproterenol