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

Abstract

In this study, we looked into possible compensatory changes of other adenosine receptors (AR) in A 2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A 2A ARmediated vasodilation. Gene expression of ARs from coronary arteries of A 2A AR wild type mice (A2AWT) and A2AKO were 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 nonspecific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 µM in A 2A WT (76.32 ± 11.35% from baseline, n=5). In A 2A KO, 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 A 2A WT but not in A 2A KO (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 A 2A WT (346±22.85 to 277±31.39, n=6). No change in CF to CGS-21680 was noted in A 2A AKO. 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.

Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts

“…With difficulties in collecting large amount of tissues for Western blot, one of our recent studies has confirmed the absence of A 2A AR protein level in A 2A AR KO mice in isolated coronary arteries using immunohistochemistry [40]. Our functional data also confirmed the lack of CGS-21680 (a selective A 2A AR agonist)-induced coronary flow response in isolated hearts of A 2A AR KO mice and the lack of BAY 60-6583 (a selective A 2B AR agonist)-induced coronary flow response in isolated hearts of A 2B AR KO mice [16,20].…”

“…Mice were kept in cages with 12:12-h light-dark cycles and maintained on a standard laboratory diet with access to water ad libitum. The absence of A 2A and A 2B ARs at mRNA level in A 2A AR KO and A 2B AR KO mice has been confirmed by our previous studies using PCR in isolated aortas, mesenteric arteries, and coronary arteries [16,20,[39][40][41]. With difficulties in collecting large amount of tissues for Western blot, one of our recent studies has confirmed the absence of A 2A AR protein level in A 2A AR KO mice in isolated coronary arteries using immunohistochemistry [40].…”

“…Mice were weighed before hearts were rapidly removed into heparinized (5 U ml −1 ) ice-cold Krebs-Henseleit buffer containing (in mM) 119 NaCl, 11 glucose, 22 NaHCO 3 , 4.7 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 2.5 CaCl 2 , 2 pyruvate, and 0.5 EDTA. After removal of the surrounding tissue, the aorta was rapidly cannulated with a 20-gauge, blunt-ended needle; then, the heart was continuously perfused with 37°C buffer bubbled with 95 % O 2 /5 % CO 2 at a constant perfusion pressure of 80 mmHg [20,40]. Subsequently, through an opening in the left atrium, a fluid-filled balloon made of plastic wrap was inserted into the left ventricle across the mitral valve.…”

“…In separate experiments, the specific Nox2 inhibitor gp91 ds-tat (1 μM) was perfused for 20 min before acquiring CGS-21680 and BAY 60-658 concentration response curves (10 −10 -10 −6 M), respectively [16,37]. All compounds were infused at a rate of 1/100 ml min −1 of the coronary flow through an injection port directly proximal to the aortic cannula using a microinjection pump (Harvard Apparatus, Holliston, MA, USA) [16,20,37].…”

“…With an overall coronary vasodilation, A 2A AR predominantly and A 2B AR minimally contribute to dilating coronary vasculature [14][15][16], whereas A 1 AR and A 3 AR counteract the effects of A 2A /A 2B ARs resulting in a diminished coronary vasodilation [15,17,18]. At post-receptor levels, several effector pathways of adenosine-mediated coronary flow have been reported, such as activation of nitric oxide (NO) pathway [19,20], cyclic adenosine 5′-monophosphate (cAMP)-dependent pathway [21], activation of potassium channels [9,21], and the involvement of H 2 O 2 [19]. However, the downstream effectors linked to the activation of ARs in this process are not completely understood.…”