Up-regulation of A 2B adenosine receptor in A 2A adenosine receptor knockout mouse coronary artery

Abstract

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A 2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A 2A AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A 2A 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. A 2B AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A 2A AR expression was absent in A2AKO. There was no difference in A 1 and A 3 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 A 2B 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 A 2AAKO. Our data demonstrate, for the first time, that coronary A 2B AR was up-regulated in mice deficient in A 2A AR. We also provide direct evidence supporting a role for NO in A 2A AR-mediated coronary vasodilation. The data further support the role for A 2A AR in the regulation of basal coronary tone through the release of NO.