Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A 2A receptor (A 2A R) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A 2A R in the regulation of cardiac ROS production remains unknown. We found that knockout of A 2A R significantly decreased (39±8%) NADPHdependent O 2•− production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A 2A R antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47 phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48±8%), and decreased JNK and ERK1/2 activation by 54±28% (all P<0.05). In conclusion, A 2A R through MAPK signaling regulates p47 phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A 2A R activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

The translocation of glucose transporter-4 (GLUT4) to the cell surface is a complex multistep process that involves movement of GLUT4 vesicles from a reservoir compartment, and docking and fusion of the vesicles with the plasma membrane. It has recently been proposed that a p38 mitogen-activated protein kinase (MAPK)-dependent step may lead to intrinsic activation of the transporters exposed at the cell surface. In contrast to data obtained in muscle and adipocyte cell lines, we found that no insulin activation of p38 MAPK occurred in rat adipose cells. However, the p38 MAPK inhibitor SB203580 consistently inhibited transport activity after preincubation with the adipose cells. These apparently contradictory findings led us to hypothesize that the inhibitor may have a direct effect on the transport catalytic activity of GLUT4 that was independent of inhibition of the kinase. Kinetic analysis of 3-O-methyl-d-glucose transport activity revealed that SB203580 was a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but was a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport. This pattern of inhibition of GLUT4 was also observed with cytochalasin B. The pattern of inhibition is consistent with interaction at the endofacial surface, but not the exofacial surface of the transporter. Occupation of the endofacial substrate site reduces maximum velocity under zero-trans conditions, because return of the substrate site to the outside is blocked, and no substrate is present inside to displace the inhibitor. Under equilibrium-exchange conditions, internal substrate competitively displaces the inhibitor, and the transport K(m) is increased.

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