Abstract

Coronary arteries are known to contain adenosine receptors that elicit vasodilation. Past attempts to characterize these receptors by radioligand binding have been unsuccessful. In the present study, a newly synthesized iodinated adenosine analogue, [125I]2-[2-(4-amino-3-iodophenhyl)ethylamino]adenosine (125I-APE), was found to bind to adenosine receptors in porcine coronary artery smooth muscle membranes. Specific 125I-APE binding is temperature sensitive with maximal binding detected at 4 degrees C. 125I-APE binds to a high affinity low density site with a KD of 0.59 +/- 0.11 nM and a Bmax 7 +/- 0.8 fmoles/mg protein. A high abundance lower affinity site is suggested by the fact that APE competes for 125I-APE binding with a concentration that inhibits 50% (IC50) of 0.96 microM. Competition with various other adenosine receptor agonists results in a potency order of (IC50, microM): 2-phenylaminoadenosine (CV 1808, 0.34) > APE (0.96) > CGS 22988 (5.2) > 2-chloroadenosine (30) > CGS 21680 and NECA (> 100). Agonist binding is not affected by GppNHp (10(-7)-10(-3) M). Among antagonists the potency order is (microM): CGS 15943 (1.1) > 8-(3-chlorostyryl)-caffeine (CSC, 5.3) > 8-sulfophenyltheophylline (SPT, 86) > theophylline (> 100). These binding characteristics are similar to the properties of a putative A4 binding site characteristic of A2a receptors assayed at a low temperature. Photoaffinity labeling of porcine coronary artery membrane proteins with the azide derivative of 125I-APE revealed a 45,000-Da binding site. Photolabeling is prevented by coincubation of membranes at 4 degrees C with various adenosine receptor antagonists (1 microM CSC, 1 microM CGS 15943 or 100 microM theophylline). In conclusion, adenosine receptors of coronary arteries have been detected for the first time by radioligand binding and photoaffinity labeling. This ligand appears to label porcine A4 binding sites that may correspond to A2a receptors assayed at 4 degrees C.