Evidence for the involvement of NADPH oxidase in adenosine receptor-mediated control of coronary flow using A₁and A₃knockout mice

Abstract

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox) and 4 are the major sources for reactive oxygen species (ROS) in cardiovascular system. In conditions such as ischemia-reperfusion injury and hypoxia, both ROS and adenosine are released suggesting a possible interaction. We hypothesized that ROS generated through Nox is involved in adenosine-induced coronary flow (CF) responses. Adenosine (10−8-10−5.5 M) increased CF in isolated hearts from wild type (WT; C57/BL6), A1 adenosine receptor (AR) knockout (A1KO), A3AR KO (A3KO) and A1 and A3AR double KO (A1/A3DKO) mice. The Nox inhibitors apocynin (10−5 M) and gp91 ds-tat (10−6 M) or the SOD and catalase-mimicking agent EUK134 (50 μM) decreased the adenosine-enhanced CF in the WT and all the KOs. Additionally, adenosine increased phosphorylation of p47-phox subunit and ERK 1/2 without changing protein expression of Nox isoforms in WT. Moreover, intracellular superoxide production was increased by adenosine and CGS-21680 (a selective A2A agonist), but not BAY 60-6583 (a selective A2B agonist), in mouse coronary artery smooth muscle cells (CASMCs) and endothelial cells (CAECs). This superoxide increase was inhibited by the gp91 ds-tat and ERK 1/2 inhibitor (PD98059). In conclusion, adenosine-induced increase in CF in isolated heart involves Nox2-generated superoxide, possibly through ERK 1/2 phosphorylation with subsequent p47-phox subunit phosphorylation. This adenosine/Nox/ROS interaction occurs in both CASMCs and CAECs, and involves neither A1 nor A3 ARs, but possibly A2A ARs in mouse.

Adenosine Production by Biomaterial‐Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury

“…Many adenine nucleotides including ATP and AMP are potent vasodilators, yet they serve no benefit in regulating MI/R injury 16. Furthermore, the vasodilation seen in coronary blood flow with adenosine has been previously reported to be dependent on upregulation of NADPH‐oxidase–mediated ROS formation in coronary endothelial cells 57, 58. In light of our findings that MSCs are capable of increasing adenosine levels while simultaneously preventing significant increases in ROS formation, it seems unlikely that vasodilation is the primary therapeutic mechanism at play.…”

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

“…However, little is known about the involvement of Nox2-derived ROS in adenosine-mediated flow response in the coronary circulation. Although activation of A 1 and A 3 AR has been shown to have vascular effects through ROS [33,36], our recent studies have already excluded the involvement of A 1 and A 3 ARs in ROS-mediated regulation of coronary flow [37]. Therefore, with the focus on the adenosine signaling, the first aim of the present study was to further investigate whether A 2A and/or A 2B AR are involved in Nox2-derived ROS-mediated coronary vasodilation.…”

“…Adenosine concentration response curves (10 −8 -10 −5 M) were acquired in perfused hearts from WT, A 2A AR KO, and A 2B AR KO mice. Each concentration of adenosine was infused for 5 min followed by a minimum of 5 min of perfusion for drug washout [16,37]. In separate experiments, the specific Nox2 inhibitor gp91 ds-tat (1 μM) [42] or the superoxide dismutase and catalase-mimicking drug EUK134 (50 μM) was perfused for 20 min before acquiring adenosine concentration response curves (10 −8 -10 −5 M) in perfused hearts from those mice [37].…”

“…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].…”

“…After 5 min of adenosine incubation, additional images were taken. Hydrogen peroxide (200 μM) served as the positive control at the end of experiments [37,40]. Only the bottom half of vessels (more proximal to the scanner) were scanned by xyz sectioning, in order to avoid the quenching as much as possible.…”