Abstract-Adenosine is a proangiogenic purine nucleoside released from ischemic and hypoxic tissues. Of the 4 adenosine receptor (AR) subtypes (A 1 , A 2A , A 2B , and A 3 ), the A 2 and A 3 have been previously linked to the modulation of angiogenesis. We used the chicken chorioallantoic membrane (CAM) model to determine whether A 1 AR activation affects angiogenesis. We cloned and pharmacologically characterized chicken AR subtypes to evaluate the selectivity of various agonists and antagonists. Application of the A 1 AR-selective agonist N 6 -cyclopentyladenosine (CPA; 100 nmol/L) to the CAM resulted in a 40% increase in blood vessel number (PϽ0.01), which was blocked by the A 1 AR-selective antagonist C 8 -(N-methylisopropyl)-amino-N 6 -(5Ј-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571; 1 mol/L). Selective A 2A AR agonists did not stimulate angiogenesis in the CAM. In an ex vivo rat aortic ring model of angiogenesis that includes cocultured endothelial cells, fibroblasts, and smooth muscle cells, 50 nmol/L CPA did not directly stimulate capillary formation; however, medium from human mononuclear cells pretreated with CPA, but not vehicle, increased capillary formation by 48% (PϽ0.05). This effect was blocked by WRC-0571 (1.5 mol/L) or anti-VEGF antibody (1 g/mL). CPA (5 nmol/L) stimulated a 1.7-fold increase in VEGF release from the mononuclear cells. This is the first study to show that A 1 AR activation induces angiogenesis. Stimulation of A 2 ARs on endothelial cells results in proliferation and tube formation, and A 2 and A 3 ARs on inflammatory cells modulate release of angiogenic factors. We conclude that adenosine promotes a coordinated angiogenic response through its interactions with multiple receptors on multiple cell types. (Circ Res.

PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G(i)-coupled A1 AR (adenosine receptor) that is without effect on G(s)-coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1/A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with G(s) and G(i), and PD81,723 increases the potency of N6-cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with G(i) or G(s). This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G(i)-coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G(i) or G(s).