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

我们已经报道，在化学预适应(IP)中，心脏保护归因于激活ECTO-5‘-核苷酸酶(ECTO-5’NT)，ECTO-5‘NT是犬心肌产生腺苷的关键酶，而ECTO-5’NT是由蛋白激酶C(PKC)激活的。开胸犬冠脉结扎4次，每次5分钟，间隔5分钟。心肌细胞胞外5‘NT活性和腺苷释放增加，但两者均被胞外5’NT抑制剂(AMP-CP)钝化。此外，短暂给予甲氧胺(α1-肾上腺素受体激动剂)和PMA(蛋白激酶C激活剂)(甲氧胺，α1-肾上腺素能受体激动剂)和PMA(蛋白激酶C激活剂)，可模拟缺血预适应(IP)后ECTO-5‘NT活性的增加和心肌梗死面积的限制作用。其次，我们验证了缺血预适应(IP)是由于α-PKC激活而导致缺血预适应(IP)激活…膜上2+和gt；依赖的PKC的假设更多派系。免疫印迹法(IB)显示，与对照组相比，心肌组织中α-PKC的含量增加，而β-、β-和epsilon-PKC的含量没有变化。在相同的膜组分中，外向-5‘-NT活性增加。然后，我们检测了在犬的预适应心肌中，PKC是否磷酸化了ecto-5‘T。免疫沉淀的ecto-5‘NT是带有抗磷酸化抗体的IB。我们观察了免疫沉淀物ecto-5‘NT的丝氨酸-茶氨酸磷酸化。此外，蛋白激酶C特异性抑制剂GY109203X可钝化IP引起的PKC和ECTO-5‘NT活性的升高，并且不能检测到免疫沉淀的ECTO-5’NT的丝氨酸-苏氨酸磷酸化。我们的结论是，心肌中的ECTO-5‘NT被IP中的PKC磷酸化并激活，从而增加了腺苷释放酶。通过磷酸化激活ECTO-5‘NT是IP心脏保护的重要机制。较少

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