Modulation of NADPH oxidase activity and redox signaling by the adenosine A2A receptor in mouse heart

David Ribé1, David Sawbridge2, Susanna Hourani1, Li Jian-Mei1
1University Of Surrey, Guildford, Surrey, United Kingdom, 2University Of Edinburgh, Edinburgh, United Kingdom

Reactive oxygen species (ROS) production in cardiac tissues is tightly regulated and plays an important role in cardiac development and cardiovascular function via redox signalling pathways. NADPH oxidase is constitutively expressed in cardiac tissue and is a major contributor of the ROS involved in redox signalling (Li, 2004). Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells (Jacobson, 2006). However, the relationship between A2AR signalling and cardiac ROS production under physiological conditions remains unknown. In this study, we investigated the role of A2AR in the regulation of NADPH oxidase expression and activity in cardiac tissues obtained from the wild-type and A2AR knockout mice (Ledent, 1997) on a CD1 background.

NADPH-dependent superoxide production, as measured by lucigenin chemiluminescence, was significantly decreased by 39±8% (p<0.01, n=9) in hearts from male A2AR knockout mice compared to age (10 weeks) matched wild-type controls. This ROS production was inhibited by DPI (a flavoprotein inhibitor) and apocynin (a specific NADPH oxidase inhibitor) but not by oxypurinol (a xanthine oxidase inhibitor), rotenone (a mitochondrial oxidase inhibitor) or L-NAME (a nitric oxide synthase inhibitor). Reduced ROS production 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 (p<0.05, n=6). The effect of A2AR deficiency on the regulation of cardiac ROS production by NADPH oxidase was further examined in vivo in wild-type male CD1 mice by intraperitoneal injection of the selective A2AR antagonist SCH58261 (10 mg/kg body weight or vehicle as control) (Monopoli, 1998). Cardiac tissues were harvested from these mice 90 min after treatment. We found that treatment with SCH58261 caused a 48 ± 8% reduction in NADPH-dependent ROS production compared to controls (p<0.05, n=6). Reduced ROS production was accompanied by a 54 ± 28% reduction in JNK and ERK1/2 activation compared to controls (p<0.05, n=6). SCH58261 treatment reduced the phosphorylation of p47phox (a major regulatory subunit of the NADPH oxidase) as detected by immunoprecipitation followed by immunoblotting using phospho-serine specific antibody (p<0.05, n=3).

In conclusion, our data indicates that A2AR is involved in the regulation of cardiac ROS generation by NADPH oxidase. Knockout of A2AR or using selective antagonists to A2AR reduces cardiac ROS production and inhibits redox-sensitive MAPK signalling pathways.

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