ATP DEPENDENCE OF ION GRADIANTS IN NORMOXIC HEARTS

这一提议的假设是，稳态心肌钙离子和 Na+离子梯度由肌浆的平衡状态设定 网状(SR)Ca~(2+)-ATPase和肌膜(SL)Na+/K+-ATPase 因此，钙离子和钠离子的梯度分别取决于 ATP水解自由能，DeltaGATP。 特定目标1将开发两个降低DeltaGATP的模型系统 充氧灌流的大鼠心脏。提供三磷酸腺苷的底物通量 合成定义了这两个模型：模型1 ATP合成将是 糖酵解；模拟的ATP合成将是氧化的。模型1约束 可用于三元酸循环的乙酰辅酶A通量 代谢抑制剂。因此，模型1中的能源需求和增量GATP为 设定由底物水平的糖酵解磷酸化。模式2将 耗尽心脏的糖原和底物的氧化将受到 非糖酵解底物的可用性。因此，能源需求和 模型2中的DeltaGATP是由氧化磷酸化设定的，即 这是由底物可用性控制的。在这两个模型中，都是deltaGATP 将因工作需求增加而进一步减少。31P核磁共振波谱 将测量计算所需的磷酸化代谢物 增量GATP。此外，氧气消耗、底物氧化和 乳酸盐的产量将被确定。《特定目标2》使用了这些模型 来定义deltaGATP和[Ca2+]i之间的关系。这将是 用荷有木犀草素的心脏来测量钙瞬变， 收缩期[Ca~(2+)]i峰值和舒张期[Ca~(2+)]i以增量GATP表示 细胞内钙离子的内流和外流均受到调节。具体目标3 使用这些模型来定义deltaGATP和 SL Na+梯度。这将使用23Na核磁共振波谱来完成 测量[Na+]I、39K核磁共振波谱测量[K+]I和87Rb核磁共振 用分光光度法测定模型1和模型2的Na+/K+ATPase活性。 钙离子和钠离子梯度的改变是由于 心肌缺血。这些变化构成了很大一部分 在缺血过程中发生的损伤。这些调查将 模拟缺血的能量后果，而不是一些 使效果复杂化。理解对世界经济的积极贡献 控制正常心脏的离子动态平衡可能会改善 脑缺血综合征的治疗。

The hypothesis of this proposal is that steady state myocardial Ca2+ and Na+ ion gradients are set by an equilibrium state of the sarcoplasmic reticulum (SR) Ca2+ ATPase and the sarcolemmal (SL) Na+/K+ ATPase reactions, respectively Thus, the Ca2+ and Na+ gradients depend on the free energy of ATP hydrolysis, deltaGATP. SPECIFIC AIM 1 will develop two model systems with reduced deltaGATP in the oxygenated perfused rat heart. The substrate flux that provides ATP synthesis defines these two models: MODEL 1 ATP synthesis will be glycolytic; MODEL ATP synthesis will be oxidative. MODEL 1 restrains the flux of acetyl-CoA available to the tricarboxylic acid cycle using metabolic inhibitors. Hence, energy demand and deltaGATP in MODEL 1 is set by substrate level phosphorylation of glycolysis. MODEL 2 will deplete hearts of glycogen and substrate oxidation will be limited by the availability of non-glycolytic substrates. Hence, energy demand and deltaGATP in MODEL 2 is set by oxidative phosphorylation, the rate of which is controlled by substrate availability. In both MODELS deltaGATP will be further reduced by increased work demand. 31P NMR spectroscopy will measure the phosphorylated metabolites necessary to calculate deltaGATP. In addition, oxygen consumption, substrate oxidation, and lactate production will be determined. SPECIFIC Aim 2 uses these MODELS to define the relationship between deltaGATP and [Ca2+]i. This will be done using aequorin-loaded hearts to measure the Ca2+ transient, the peak systolic [Ca2+]i and the diastolic [Ca2+]i as deltaGATP is decreased and the influx and efflux of Ca2+ modulated. SPECIFIC Aim 3 uses these MODELS to define the relationship between deltaGATP and the SL Na+ gradient. This will be done using 23Na NMR spectroscopy to measure [Na+]i, 39K NMR spectroscopy to measure [K+]i and 87Rb NMR spectroscopy to measure Na+/K+ ATPase activity in MODELS 1 and 2. Alterations in the Ca2+ and Na+ gradients occur as a result of myocardial ischemia. These alterations underlie a significant portion of the damage that occurs during ischemia. These investigations will mimic the energetic consequence of ischemia without some of its complicating effects. Understanding the energetic contribution to the control of ion homeostasis in normal hearts may lead to improved therapies for ischemic syndromes.