NA-K-ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

Na，K-ATPase是一种主动转运Na+的质膜酶 和K+离子对其电化学梯度的影响，以及 三磷酸腺苷。它被认为是世界上最大的能源消费国 中枢神经系统，占ATP水解率的30-40%。三 Na，K-ATPase的不同同工酶在中枢神经系统中表达，在一些 实例在不同单元格类型中的局部化程度相当显著。 它们的个体属性和生理作用尚不为人所知。 现有的证据表明，同工酶可能在它们的 三磷酸腺苷和心脏糖苷的亲和力以及它们对 受到不明细胞内因素的调节。此外，缺血 据报道，受伤会导致一种选择性的快速失活。 心脏中有两种同工酶。这项提议的假设是， 中枢神经系统中的Na，K-ATPase同工酶在不同的细胞类型中表达， 他们对ATP的亲和力或对调节或降解的敏感性 可加重或改善缺血性损伤和水肿。这个 Na，K-ATPase的性质可能在解释不同的 不同类型神经元对缺血的易感性。 该提案有几个目标：确定哪些细胞类型表达 哪些同工酶；研究它们的功能特性并预测 在边缘灌流的组织中，哪些同工酶将保持活性； 研究它们的失活继发于细胞内钙离子的升高。 我们将利用免疫细胞化学和特定的单抗 Na，K-ATPase在细胞和亚细胞中的分布 大鼠中枢神经系统。我们将分离同工酶，并确定它们的个体 对ATP、Na和K+的亲和力，以及研究 以GTP为底物。最后，我们将对其机制进行探讨。 依赖于钙离子的蛋白水解酶对各同工酶的修饰或失活 并通过钙调素介导的钙抑制作用。这项基础研究应该奠定 为严格分析离子运动、能量 消耗，以及它们在缺血脑细胞死亡和水肿中的作用，以及 有助于详细了解离子泵的功能。

The Na,K-ATPase is the plasma membrane enzyme that actively transports Na + and K+ ions against their electrochemical gradients, with the hydrolysis of ATP. It is thought to be the single largest consumer of energy in the central nervous system, accounting for 30-40% of ATP hydrolysis. Three different isozymes of the Na,K-ATPase are expressed in the CNS, and in some instances are rather strikingly localized within different cell types. Their individual properties and physiological roles are not yet well known. Available evidence suggests that the isozymes may differ in their affinities for ATP and cardiac glycosides, and in their susceptibility to regulation by unidentified intracellular factors. In addition, ischemic injury has been reported to cause a selective and rapid inactivation of one of two isozymes in the heart. The hypothesis of this proposal is that the Na,K-ATPase isozymes in the CNS are expressed in different cell types, where their affinity for ATP or susceptibility to regulation or degradation may either exacerbate or ameliorate ischemic injury and edema. The properties of the Na,K-ATPases may play a role in explaining the different vulnerability to ischemia of different classes of neurons. The proposal has several objectives: to determine which cell types express which isozymes; to investigate their functional properties and predict which isozymes will remain active in marginally perfused tissue; and to investigate their inactivation secondary to a rise in intracellular Ca2+. We will utilize immunocytochemistry with specific monoclonal antibodies to determine the cellular and subcellular distribution of the Na,K-ATPases in the rat CNS. We will separate the isozymes, and determine their individual affinities for ATP, Na and K+, as well as investigate the possible use of GTP as a substrate. Finally we will investigate the mechanism of modification or inactivation of each isozyme by Ca 2+-dependent proteolysis and by calnaktin-mediated Ca 2+ inhibition. This basic research should lay the groundwork for a rigorous analysis of ion movements, energy consumption, and their role in cell death and edema in ischemic brain, and contribute to a detailed understanding of ion pump function.