Activation of Ecto-5′-nucleotidase by Protein Kinase C Attenuates Irreversible Cellular Injury due to Hypoxia and Reoxygenation in Rat Cardiomyocytes

Adenosine, synthesized by ecto-5′-nucleotidase, is cardioprotective against ischemia and reperfusion injury. We have previously reported that activation of protein kinase C increases ecto-5′-nucleotidase activity of the rat cardiomyocytes, raising the possibility that activation of protein kinase C protects cardiomyocytes from the irreversible cellular injury via activation of ecto-5′-nucleotidase. To test this hypothesis, cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode buffer solution. The cardiomyocytes were incubated with and without exposure to methoxamine (1×10−6mol/l) or phorbol 12-myristate 13-acetate (PMA, 1×10−8mol/l). Ecto-5′-nucleotidase activity increased 15 min after the onset of an exposure to either methoxamine or PMA. Adenosine release during hypoxia and reperfusion was augmented in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated cardiomyocytes, which was inhibited byα,β-methyleneadenosine 5′-diphosphate (AOPCP), an inhibitor of ecto-5′-nucleotidase. Irreversible cellular injury assessed by the extent of release of lactate dehydrogenase and the trypan blue exclusion test following 60 min of hypoxia and 60 min of reoxygenation was attenuated in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated group, which was also blunted by AOPCP and 8-sulfophenyltheophylline, an adenosine receptor antagonist. An adenosine A1receptor agonist, N6-cyclohexyladenosine, restored the cardioprotection under the treatment with PMA and AOPCP. We conclude that activation of ecto-5′-nucleotidase via protein kinase C contributes to the attenuation of the irreversible injury of the rat cardiomyocytes due to hypoxia and reoxygenation.