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

Abstract

Abstract Vascular smooth muscle has been reported to contain the A 1 subtype of adenosine receptors, but the existence of such receptor(s) in coronary smooth muscle has not been established. In the present study, the 3 H-labeled A 1 -selective antagonist [ 3 H]8-cyclopentyl-1,3-dipropylxanthine ([ 3 H]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 - N 6 -(2-endo-norbornyl)adenosine (S-ENBA) 0.11= R (−)- N 6 -phenylisopropyladenosine 0.32>DPCPX 3.2=xanthine amine congener 2.4= N 6 -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 A 1 adenosine receptor. S-ENBA, a highly selective A 1 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 A 1 adenosine receptors linked to adenylyl cyclase in an inhibitory manner through pertussis toxin–sensitive G proteins in the coronary artery.