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Title: 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 weremore » 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).« less
Authors:
Publication Date:
OSTI Identifier:
5722903
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Pharmacol. Exp. Ther.; (United States); Journal Volume: 249:3
Research Org:
Brigham and Women's Hospital, Boston, MA (USA)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENOSINE; RECEPTORS; CYCLASES; ENZYME ACTIVITY; MYOCARDIUM; CONTRACTION; CHEMICAL COMPOSITION; ANIMAL CELLS; BIOCHEMICAL REACTION KINETICS; CELL CULTURES; HEART; MATHEMATICAL MODELS; RADIOASSAY; TOXINS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; XANTHINES; ANTIGENS; AROMATICS; AZAARENES; BODY; CARDIOVASCULAR SYSTEM; ENZYMES; HETEROCYCLIC COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; LABELLED COMPOUNDS; LYASES; MATERIALS; MEMBRANE PROTEINS; MUSCLES; NUCLEOSIDES; NUCLEOTIDES; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANIC OXYGEN COMPOUNDS; ORGANS; PROTEINS; PURINES; REACTION KINETICS; RIBOSIDES; TOXIC MATERIALS 550201* -- Biochemistry-- Tracer Techniques