Mitochondrial Mechanism Responsible For Adenosine A2 Receptor Activation-induced Protection Against Myocardial Reperfusion Injury

Objective This study was aimed to test the effect of adenosine A2 receptors activation on mitochondrial oxidative injury at reperfusion and underlying mitochondrial signaling mechanism.Methods Isolated rats hearts and cardiac H9c2 cells were subjected to ischemia followed by reperfusion.The mitochondrial morphological and functional changes were determined by the transmission election microscopy(TEM)and high resolution respirometry,respectively.Mitochondrial fluorescence indicator JC-1,DCFH-DA and Mitosox Red were used to determine mitochondrial membrane potential(??m),reactive oxygen species(ROS)and mitochondrial superoxide.Amplex red hydrogen peroxide/peroxidase assay kit was used to detect the level of H2O2 and catalase activity.The mitochondrial permeability transition pore(m PTP)opening was evaluated by measuring mitochondrial swelling.The protein carbonylation test was used to measure the oxidative stress of mitochondria.The mitochondrial complex I activity was determined using the complex I enzyme activity dipstick assay kit.Mitochondrial complex I was collected by immunoprecipitation to determine tyrosine-phosphorylation and to identify phosphorylated(tyrosine)subunits of complex I.The molecular targets and phosphorylation sites of complex I by tyrosine kinase were identified using mass spectrometry(MS)and Net Phos K 1.0.Results 1.Compared with the sham group,the respiratory control ratio(RCR),??m and the susceptibility of mitochondria to calcium chloride-induced mitochondrial swelling were decreased in I/R group(P < 0.05,respectively),which were prevented by NECA.The adenosine A2 A receptor antagonist SCH58261(SCH)and the adenosine A2 B receptor antagonist MRS1706(MRS)inhibited the protective effects of NECA(P < 0.05,respectively).These data indicate that both the adenosine A2 A and adenosine A2 B receptors are involved in the protective effect of NECA on mitochondrial damage caused by I/R.2.I/R increased mitochondrial proteins carbonylation,ROS and H2O2 generation at reperfusion,implying that ROS were increased upon reperfusion(P < 0.05,respectively).NECA decreased the oxidative stress caused by I/R,which was inhibited by both SCH and MRS(P < 0.05,respectively).3.NECA and Peg-SOD can prevent I/R injury induced ? ?m loss and ROS generation in H9c2 cells(P < 0.05,respectively).All of these data indicate that the adenosine A2 A and adenosine A2 B receptors activation could prevent oxidative injury by inhibiting oxidative stress upon reperfusion.Compared to the sham group,the activity of superoxide dismutase(SOD)was decreased by I/R,which was not altered by NECA(P < 0.05,respectively).However,NECA increased the catalase activity comparing to the I/R group,which was reversed by both SCH and MRS(P < 0.05,respectively).This data indicate that NECA can scavenge ROS by increasing the catalase activity but not by increasing SOD activity.4.Comparing to the sham group,the complex I activity was increased in I/R group,which was reversed by NECA(P < 0.05,respectively).The selective adenosine A2 A receptor agonist CGS21680(CGS)and the selective adenosine A2 B receptor agonist Bay60-6583(BAY)could mimic NECA's effect(P < 0.05,respectively),implying that A2 receptors are involved in the action of NECA to inhibit ROS generation.5.NECA increased Src tyrosine kinase activity compared to the sham group,and this effect was blocked by MRS(P < 0.05,respectively).In addition,the selective Src tyrosine kinase inhibitor PP2 blocked the effect of NECA on ? ?m,the susceptibility of mitochondria to calcium chloride-induced mitochondrial swelling,ROS generation and complex I activity(P < 0.05,respectively),pointing to that the adenosine A2 receptors activation may prevent I/R induced mitochondrial injury by reducing ROS generation via Src.6.Tyr-118,191 and 192 of NDUFV2,tyr-146,245 of NDUFS3 and tyr-151 of NDUFS2 seem to be the potential sites of tyrosine phosphorylation.Conclusions 1.The adenosine A2 A or A2 B receptors activation may prevent I/R induced mitochondrial damage by inhibiting m PTP opening by increasing ROS scavenging via catalase and by decreasing ROS generation via the inhibition of complex I activity.2.The adenosine A2 receptors activation may prevent ROS generation by inhibiting complex I activity via Src tyrosine kinase.3.The adenosine A2 receptors activation may inhibit complex I activity by increasing tyr-118,191 and 192 of NDUFV2,tyr-146,245 of NDUFS3 and tyr-151 of NDUFS2 phosphorylation via Src tyrosine kinase.