Adenosine Prevents Activation of Transcription Factor NF-κB and Enhances Activator Protein-1 Binding Activity in Ischemic Rat Heart

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Background. Adenosine prevents myocardial TNF-α production induced by ischemia/reperfusion, but the mechanisms are poorly understood. Transcription factors NF-κB and AP-1 have been implicated in the regulation of a variety of inducible gene expressions in response to oxidative stress and cellular defense. The effects of adenosine on NF-κB and AP-1 activation have not been clearly defined. This study demonstrated differential effects of adenosine on NF-κB and AP-1 nuclear binding activity in ischemic myocardium. Methods. Isolated working rat hearts were subjected to 0, 1, 2, 3, 4, 5, 7.5, 10, 15, and 30 minutes of ischemia, with 4 to 6 hearts for each time point with and without adenosine (100 μmol/L). NF-κB and AP-1 binding activity in the nucleus were analyzed by electrophoretic mobility shift assay (EMSA). IκBα levels in the cytoplasm were measured by Western blot analysis. TNF-α mRNA levels were determined by RT-PCR. Results. NF-κB binding activity in the nucleus significantly increased after 4 minutes of ischemia and remained to 30 minutes. The levels of IκBα protein in the cytoplasm markedly decreased after 4, 5, 7.5, and 10 minutes of ischemia. TNF-α mRNA levels peaked after 10 minutes of ischemia. AP-1 DNA binding activity was induced and persisted during all ischemic periods. Adenosine significantly inhibited NF-κB binding activity in the nucleus, markedly prevented the loss of IκBα proteins from the cytoplasm, and concomitantly down-regulated TNF-α mRNA expression, but enhanced AP-1 binding activity in the nucleus of ischemic myocardium. Conclusions. Adenosine modulation of NF-κB activation may be the cellular molecular mechanism of down-regulation of TNF-α mRNA expression. The cardioprotective properties of adenosine may be involved in the differential modulation of NF-κB and AP-1 activation during myocardial ischemia.