I-125 APE BINDING TO ADENOSINE RECEPTORS IN CORONARY-ARTERY - PHOTOAFFINITY-LABELING WITH I-125 AZIDOAPE

Coronary arteries are known to contain adenosine receptors that elicit vasodilation. Past attempts to characterize these receptors by radiol igand binding have been unsuccessful. in the present study, a newly sy nthesized iodinated adenosine analogue, 25]2-[2-(4-amino-3-iodophenhyl ethylamino]adenosine (I-125-APE), was found to bind to adenosine recep iors in porcine coronary artery smooth muscle membranes. Specific I-12 5-APE binding is temperature sensitive with maximal binding detected a t 4 degrees C. I-125-APE binds to a high affinity tow density site wit h a K-D 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 c ompetes for I-125-ApE binding with a concentration that inhibits 50% ( IC50) of 0.96 mu M. Competition with various other adenosine receptor agonists results in a potency order of (IC50 mu M): 2-phenylaminoadeno sine (CV 1808, 0.34) > APE (0.961 > CGS 22988 (5.2) > 2-chloroadenosin e (30) > CGS 21680 and NECA (>100). Agonist binding is not affected by GppNHp (10(-7)-10(-3) M). Among antagonists the potency order is luRn ): CGS 15943 (1.1) > 8-(3-chlorostyryl)-caffeine (CSC, 5.3) > 8-sulfop henyltheophyIline (SPT, 86) > theophylline (>100). These binding chara cteristics are similar to the properties of a putative A(4) binding si te characteristic of A(2a) receptors assayed at a low temperature. Pho toaffinity labeling of porcine coronary artery membrane proteins with the azide derivative of I-125-APE revealed a 45,000-Da binding site. P hotolabeling is prevented by coincubation of membranes at 4 degrees C with various adenosine receptor antagonists (1 mu M CSC, 1 mu M CGS 15 943 or 100 mu M theophylline). In conclusion, adenosine receptors of c oronary arteries have been detected for the first time by radioligand binding and photoaffinity labeling. This ligand appears to label porci ne A(4) binding sites that may correspond to A(2a) receptors assayed a t 4 degrees C.