IN VIVO CEREBRAL OXIDATIVE METABOLISM IN HYPOXIC NEWBORNS

本研究将探讨低氧时钙/钙调素依赖的蛋白激酶IV(CaM Kinase IV)激活的机制，该激活导致环状AMP反应元件结合(CREB)蛋白的激活，并触发凋亡基因的转录，从而导致缺氧性神经元死亡。我们将验证这一假设，即低氧时CaM激酶IV的激活增加的机制是由于CaM蛋白的酪氨酸磷酸化和CaM激酶IV的酪氨酸磷酸化，即酪氨酸激酶、Src酪氨酸激酶和表皮生长因子受体(EGFR)酪氨酸激酶。我们认为，脑缺氧诱导的钙调蛋白酪氨酸磷酸化增加(Tyr99)导致其与CaM激酶IV酶的钙调蛋白结合域结合的亲和力增加，导致CaM激酶IV的激活增加。CaM激酶IV(Tyr154和Tyr172)的酪氨酸磷酸化增加导致酶的活性部位区域的修饰，导致对其底物的亲和力增加，导致CaM激酶IV的激活增加。将在新生仔猪上开展以下实验方案：(1)低氧对神经元核CaM激酶IV的CaM蛋白99的Tyr99磷酸化、CaM激酶IV的Tyr154和Tyr172磷酸化以及CaM激酶IV的活性的影响(2)利用特异性抗Tyr416-p-Src和抗Tyr1173-p-EGFR的抗体，研究低氧对小鼠大脑皮层中Src和EGFR活性的影响，以及作为酶活性指标的Src和EGFR活性部位的酪氨酸磷酸化。

The proposed studies will investigate the mechanism of activation of Ca++/calmodulin-dependent protein kinase IV (CaM kinase IV) during hypoxia that leads to activation of cyclic AMP-response element binding (CREB) protein and triggers the transcription of apoptotic genes resulting in hypoxic neuronal death. We will test the hypothesis that the mechanism of increased activation of CaM kinase IV during hypoxia is due to tyrosine phosphorylation of calmodulin and tyrosine phosphorylation of CaM kinase IV by the tyrosine kinases, Src tyrosine kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. We propose that cerebral hypoxia-induced increased tyrosine phosphorylation of calmodulin (at Tyr99) leads to increased affinity for its binding at the calmodulin binding domain of the CaM kinase IV enzyme and results in increased activation of CaM kinase IV. The increased tyrosine phosphorylation of CaM kinase IV (at Tyr154 and Tyr172) leading to modification of the active site domain of the enzyme leads to increased affinity for its substrate and results in increased activation of CaM kinase IV. Experimental protocols will be carried out on newborn piglets investigating: (1) the effect of hypoxia on Tyr99 phosphorylation of calmodulin, Tyr154 and Tyr172 phosphorylation of CaM kinase IV, and activity of CaM kinase IV in neuronal nuclei, (2) the effect of hypoxia on the activity of Src kinase and EGFR kinase, and tyrosine phosphorylation at the active site of Src kinase (Tyr416) and EGFR kinase (Tyr1173), an index of enzyme activation, using specific anti-Tyr416-p-Src kinase and anti-Tyr1173-p-EGFR kinase antibodies to demonstrate activation of Src and EGFR tyrosine kinases in the cerebral cortex of hypoxic piglets.