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(Circulation Research. 1995;77:194-198.)
© 1995 American Heart Association, Inc.

Articles

Binding of A₁ Adenosine Receptor Ligand [³H]8-Cyclopentyl-1,3-Dipropylxanthine in Coronary Smooth Muscle

Tahir Hussain, S. Jamal Mustafa

From the Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC.

Abstract Vascular smooth muscle has been reported to contain the A₁ subtype of adenosine receptors, but the existence of such receptor(s) in coronary smooth muscle has not been established. In the present study, the ³H-labeled A₁-selective antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine ([³H]DPCPX) was used to demonstrate the specific binding in porcine coronary artery smooth muscle membranes. The binding was saturable with a B_max of 6.43±1.02 fmol/mg protein. Scatchard analysis of the binding data provided a single binding site with a K_d of 0.21±0.025 nmol/L. In the competition experiments, adenosine receptor agonists and antagonists showed the following order of potency (nmol/L): S-N⁶-(2-endo-norbornyl)adenosine (S-ENBA) 0.11=R(-)-N⁶-phenylisopropyladenosine 0.32>DPCPX 3.2=xanthine amine congener 2.4=N⁶-cyclopentyladenosine 2.67>5'-(N-ethylcarboxamido)-adenosine 7.35>>2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-(N-ethylcarboxamido)-adenosine 1000>theophylline 83 000. This order of potency fits the criteria for the A₁ adenosine receptor. S-ENBA, a highly selective A₁ receptor agonist, was used to investigate the effect on isoproterenol-mediated vasorelaxation and cAMP accumulation. S-ENBA (0.1 to 10 nmol/L) dose-dependently shifted the isoproterenol-mediated (10^-8 to 10^-5 mol/L) vasorelaxation to the right in vascular rings. S-ENBA (10 nmol/L) inhibited the basal cAMP levels by 36% and attenuated the isoproterenol (10^-5 mol/L)–stimulated cAMP by 25% in the coronary rings. These inhibitory effects of S-ENBA on isoproterenol-mediated cAMP-accumulation and vasorelaxation were abolished by pertussis toxin (100 ng/mL, overnight) treatment of the arteries. Similarly, the effects of S-ENBA on isoproterenol-mediated responses were antagonized by DPCPX. This study provides the evidence suggesting the existence of A₁ adenosine receptors linked to adenylyl cyclase in an inhibitory manner through pertussis toxin–sensitive G proteins in the coronary artery.

Key Words: A₁ adenosine receptor • coronary artery • adenosine receptor antagonist • cAMP • G proteins

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