The pathophysiological significanse and mechanism of activation of key enzyme responsible for adenosine production in ischemic preconditioning.

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We have reported that cardioprotection in is chemic preconditining (IP) is attributable to activation of ecto-5'-nucleotidase (ecto-5'NT), akey enzyme responsible for adenosine production in the canine myocadium, and that ecto-5'NT is activated by protein kinase C (PKC). In the open chest dogs, IP was produced by 4 times of 5-min coronary occlusion with a 5-min interval. Myocardial ecto-5'NT activity and adenosine release were increased after IP,but both were blunted by an inhibitor of ecto-5'NT (AMP-CP). The infarct size-limiting effect was also blunted by AMP-CP.Futhermore, transient administration (4 times of 5-min coronary infusion with a 5-min interval) of methoxamine (an stimulator of alpha 1-adrenoceptor) and PMA (an activator of PKC) mimicked the increase in ecto-5'NT activity and the infarct size-limiting effect after IP.Next, we tested the hypothesis that ischemic preconditining (IP) is attributable to activation of alpha-PKC.IP activated Ca^<2+>-dependent PKC of the membrane … More faction. Immunoblotting (IB) of the membrane fraction revealed the increases in the contents of alpha-PKC without changing the contents of beta-, delta-, and epsilon-PKC compared with the control myocardium. In the same membrane fractions, ecto-5'NT activity increased. And then, we examined whether PKC phospholylates ecto-5' T in the canine preconditioned myocardium. Immunoprecipitated ecto-5'NT was IB with antiphosphorylation antibodies. We observed the serine-theonine phosphorylation of immunoprecipitated ecto-5'NT due to the IP procedure. Futhermore, the increases in the activities of PKC and ecto-5'NT due to IP were blunted and the serine-threonine phosphorylation of immunoprecipitated ecto-5'NT was not detected by GY109203X (a specific inhibitor of protein kinase C). We conclude that ecto-5'NT in the myocardium is phosphorylated and activated by PKC in the IP to increase the adenosine relase. Activation of ecto-5'NT by phosphorylation is an important mechanism for cadioprotection in IP. Less