Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity - PubMed (original) (raw)

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity

David Ribé et al. Free Radic Biol Med. 2008.

Abstract

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 A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- 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 A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (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, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

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Figures

Fig. 1

Fig. 1

O2•− production in wild-type and A2AR knockout heart homogenates detected by lucigenin chemiluminescence. (A) Kinetic measurement of O2•− at every minute for a total of 30 min. NADPH was added after 15 min of measurement. (B) Difference in NADPH-dependent O2•− production between wild-type and A2AR knockout heart homogenates. MLU: mean light unit of 15 measurements. (C) Effects of enzyme inhibitors on the level of O2•− production by wild-type heart homogenates. The results were presented as percentage of the control (without inhibitor). *P<0.05 for indicated values versus the control value in the same figure. _n_=12.

Fig. 2

Fig. 2

Dihydroethidium (DHE) fluorescence detection of the ROS production in cardiac sections. Cardiac sections from wild-type and A2AR were incubated with DHE in the presence or absence of tiron (an O2•− scavenger). The DHE fluorescence was visualised under confocal microscopy and quantified. *P<0.05 for A2AR knockout versus wild-type controls. _n_=18 sections from 6 hearts/per group.

Fig. 3

Fig. 3

Immunoblotting for the protein expression of NADPH oxidase subunits in wild-type and A2AR knockout hearts. (A) A neutrophil membrane preparation was used as a positive control for the detection of Nox2. Cardiac troponin I was used as a loading control. (B) Protein bands were quantified densitometrically and normalised to the expression of cardiac troponin I in the same sample. The results were expressed as arbitrary units. *P<0.05 for A2AR knockout versus wild-type controls. _n_=12.

Fig. 4

Fig. 4

Differences in cardiac MAPK activation and heart/body weight ratio between wild-type and A2AR knockout mice. (A) Immunoblotting for the expression of total and phosphorylated ERK1/2, p38MAPK, and JNK detected by phospho-specific monoclonal antibodies. (B) Protein bands were quantified densitometrically and the levels of MAPK phosphorylation were normalised to the total protein levels of these molecules in the same samples and expressed as arbitrary units. _n_=12 hearts. (C) Body weights, heart weights, and heart/body weight ratio of wild-type and A2AR knockout mice. _n_=24 mice. *P<0.05 for A2AR knockout versus wild-type controls.

Fig. 5

Fig. 5

The effect of SCH58261 on cardiac ROS production. (A) Kinetic measurement of NADPH-dependent O2•− production detected by lucigenin chemiluminescence in cardiac homogenates from wild-type mice treated in vivo with vehicle or SCH58261 (3 mg/kg body weight). Tiron (an O2•− scavenger) and apocynin (an NADPH oxidase inhibitor) were used to confirm the specificity of the assay. (B) Difference in NADPH-dependent O2•− production between heart homogenates from vehicle-treated (0 mg of SCH58261) and SCH58261 (3 and 10 mg/kg body weight)-treated mice. _n_=12 animals/per group. (C) The effect of SCH58261 on ROS production by cultured H9C2 cardiac myocytes. The cell homogenate from 2×106 cells was used for each measurement. _n_=3 independent cell cultures. MLU: mean light unit of 15 measurements. *P<0.05 for indicated values versus control value (0 mg SCH58261).

Fig. 6

Fig. 6

The effect of SCH58261 treatment on cardiac p47phox phosphorylation and MAPK activation. (A) Top panel: p47phox was immunoprecipitated down and detected by a phospho-serine-specific monoclonal antibody. Total p47phox was detected in parallel as loading controls. (B) ERK1/2, p38MAPK, and JNK phosphorylation detected by phospho-specific monoclonal antibodies. (C) Protein bands were quantified densitometrically and the results were normalised to the total protein levels of these molecules in the same samples and expressed as arbitrary units. _n_=12 hearts. *P<0.05 for indicated value versus vehicle control.

Fig. 7

Fig. 7

Confocal microscopy for the cellular expression of p47phox and Nox2 in cardiac sections. Endothelial cells (left panel) were labeled by CD31 (red). Cardiac myocytes (right panel) were outlined by laminin expressed in the sarcolemmal membrane (green). The yellow fluorescence in the lower panels shows the expression of p47phox (green, left middle panel) in endothelial cells, and the expression of Nox2 (red, right middle panel) in cardiac myocytes.

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