Haddad John J
Molecular Signaling Research Group, Division of Biological Sciences, Department of Biology, Faculty of Arts and Sciences, Lebanese International University, Beirut, Lebanon.
Protein Pept Lett. 2007;14(4):361-71. doi: 10.2174/092986607780363925.
The molecular regulation of MAPKs and apoptosis was investigated in a model of hypoxia-tolerance. Survival of neurons in Chrysemys picta bellii, an anoxia-tolerant turtle, involves a reduction in energy metabolism. The biochemical/physiological mechanisms of anoxia tolerance have been examined at the level of ion transport and ATP turnover. However, changes in the phosphorylation state of key enzymes and kinases, mainly, MAPKs, may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis.
If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain was then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing brain in a buffer, followed by threonine and tyrosine phosphorylation determination of MAPKs, and caspase activity.
MAPK(p38) was decreased after reoxygenation following 1 day and 1 week hypoxia. The effect of hypoxia on the phosphorylation of MAPK(ERK) was biphasic: Enhancement at 5h and inhibition at 6 weeks. Pro-caspases 8/9 were unchanged by hypoxia until increasing at 6 weeks. Both pro-caspases were upregulated by reoxygenation at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation induced the cleavage of pro-caspases 8/9 into p20 and p10, respectively. Furthermore, hypoxia induced Bax at 3 days and 1 week, and reoxygenation increased Bax #8776; 4-fold at 1 day. Although the expression of Bcl-2 was slightly increased by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with Bax.
These results indicate that hypoxia up-regulates MAPK(ERK) but not MAPK(p38;) hypoxia/reperfusion increases the expression of caspases and pro-apoptotic cofactors. The patterns of MAPK regulation suggest the significance of these kinases in cellular adaptation to oxygen deprivation with biomedical correlations, and thereby identify novel natural responsive signaling cofactors in Chrysemys picta bellii with potential pharmacologic and clinical applications.
在耐缺氧模型中研究了丝裂原活化蛋白激酶（MAPKs）和细胞凋亡的分子调控。耐缺氧的乌龟——彩龟（Chrysemys picta bellii）中神经元的存活涉及能量代谢的降低。已经在离子转运和ATP周转水平上研究了耐缺氧的生化/生理机制。然而，在缺氧期间可能会发生关键酶和激酶（主要是MAPKs）磷酸化状态的变化，因此可逆的蛋白质磷酸化可能是持久厌氧代谢重组的关键因素和主要机制。
如果将乌龟置于类似于其原生栖息地所经历的缺氧环境中，将其放入一个装满水且水面距顶部半英寸的玻璃水族箱中。在乌龟麻醉后，通过长时间缺氧或麻醉，将动物断头处死。然后取出大脑并置于缺氧的人工脑脊液中。通过在缓冲液中匀浆大脑进行总蛋白提取，随后测定MAPKs的苏氨酸和酪氨酸磷酸化以及半胱天冬酶活性。
在缺氧1天和1周后复氧，MAPK（p38）降低。缺氧对MAPK（ERK）磷酸化的影响是双相的：5小时时增强，6周时抑制。缺氧直到6周时前半胱天冬酶8/9才发生变化。在缺氧1天或6周时复氧均使两种前半胱天冬酶上调。缺氧和复氧均未诱导前半胱天冬酶8/9分别裂解为p20和p10。此外，缺氧在3天和1周时诱导Bax表达，复氧在1天时使Bax增加约4倍。尽管缺氧使Bcl-2的表达略有增加，但与Bax相比，[Bcl-2]小3至4倍。
这些结果表明，缺氧上调MAPK（ERK）但不上调MAPK（p38）；缺氧/再灌注增加半胱天冬酶和促凋亡辅因子的表达。MAPK调控模式表明这些激酶在细胞适应缺氧过程中的重要性及其与生物医学的相关性，从而在彩龟中鉴定出具有潜在药理和临床应用的新型天然反应性信号辅因子。
