Up-regulation of A2B adenosine receptor in A2A adenosine receptor knockout mouse coronary artery

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A2A AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A2A AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A2B AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A2A AR expression was absent in A2AKO. There was no difference in A1 and A3 ARs between WT and KO animals. The concentration–relaxation curve for adenosine-5′-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration–response curve for A2B selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 µM in A2AWT (76.32 ± 11.35% from baseline, n = 5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 µM, 93.32 ± 5.8% from baseline, n = 5). L-NMA (1 µM, n = 4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66 ± 3.23% from baseline in A2AWT, while 81.76 ± 8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 µM NECA was significantly blunted with 10 µM L-NAME (377.08 ± 25.23% to 305.41 ± 30.73%, n = 9) in A2AWT but not in A2AKO (153.66 ± 22.7% to 143.88 ± 36.65%, n = 5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346 ± 22.85 to 277 ± 31.39, n = 6). No change in CF to CGS-21680 was noted in A2AAKO. Our data demonstrate, for the first time, that coronary A2B AR was up-regulated in mice deficient in A2A AR. We also provide direct evidence supporting a role for NO in A2A AR-mediated coronary vasodilation. The data further support the role for A2A AR in the regulation of basal coronary tone through the release of NO.