Binding of the Novel Nonxanthine A _2A Adenosine Receptor Antagonist [ ³ H]SCH58261 to Coronary Artery Membranes

Abstract

This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-epsilon]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900 +/- 61, 892 +/- 35, and 959 +/- 76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoaden osine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261 > HE-NECA = CSC = CGS 21680 = XAC > NECA = R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radio-ligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.

SCH 58261: A selective A2A adenosine receptor antagonists

“…Thus, in human platelets, lympho- Varani et al, 1997a,b]. Likewise, in cell cultures such as PC12 or CHO human A2A cells it blocks NECA-induced cyclic AMP formation in the low nM range (Table 4) and behaves as a competitive antagonist [Belardinelli et al, 1996;Dionisotti et al, 1997]. The best known adenosine receptor antagonists are the naturally-occurring xanthines caffeine and theophylline, which also interact with phosphodiesterases [Fredholm, 1995].…”

“…Use of specific tissues such as platelets containing A 2A receptors only or vascular districts whose relaxation is modulated by A 2A receptors [Belardinelli et al, 1996] has made it possible to examine both potency and the type of antagonism of SCH 58261. In the rabbit platelet aggregation assay, SCH 58261 antagonizes the effects induced by CGS 21680 with a pA 2 value of 7.9 [Zocchi et al, 1996a].…”

Role of A2A‐adenosine receptor activation for ATP‐mediated coronary vasodilation in guinea‐pig isolated heart

“…These actions of adenosine result in an increase in the ratio of oxygen supply to oxygen demand in the myocardium (Shryock & Belardinelli, 1997). The presence of A 2A -adenosine receptors (A 2A -AdoRs) in coronary vessels has been demonstrated by radioligand binding (Belardinelli et al, 1996). Activation of these receptors mediates coronary vasodilation by adenosine in guinea-pig isolated hearts .…”

“…Until now the lack of selective and potent antagonists for either P 2Y or A 2A -adenosine receptors has been an obstacle to the identi®cation of the type receptor(s) that mediate ATP-induced coronary vasodilation. Recently, a potent and selective antagonist at the A 2A -AdoR, 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-E]-1,2,4-triazolo [1,5-c]pyrimidine (SCH58261) has been characterized (Zocchi et al, 1996;Belardinelli et al, 1996;1998). In this study, the A 2A -AdoR antagonist SCH58261 was used to test the hypothesis that exogenous ATP causes coronary vasodilation via stimulation of A 2A -AdoRs in the guinea-pig isolated perfused heart.…”

Regadenoson in the detection of coronary artery disease

“…There is however a large A2A receptor "reserve" in the coronary artery, so that an ideal A2A agonist may well have a low affi nity (Belardinelli et al 1996). Since the discovery of the prototypical A2A agonist CGS-21680 in 1989, several potent, selective A2A adenosine receptor agonists have been synthesized, and preliminary experimental studies have been promising.…”