NMR STUDIES OF SODIUM HOMEOSTASIS IN ISCHEMIC MYOCARDIUM

先前的研究表明，细胞内的CS2+ 超负荷在心肌缺血性损伤中起重要作用， 钙离子稳态的一个重要决定因素是对钙离子的控制 细胞内Na+(Na+)。然而，Na+的动态平衡在 对缺血仍知之甚少。拟议的项目将 采用最先进的核磁共振技术的独特组合 获得对Na+i如何控制的全面评估 心肌，在缺血和缺氧期间。使用完好无损的 大鼠心脏灌流模型，交错~(23)Na和~(31)P核磁共振波谱 将监测Na+i和细胞能量的变化。在相同的 制剂，87Rb核磁共振波谱将用于监测 Rb+摄取和Na+/K+ATPase活性。使用7Li核磁共振 光谱，Li+摄取量将被测量以监测电压- 门控Na+通道活动。这种方法不仅衡量了 净Na+积累，但也被设计为同时提供 关于单向Na+通量如何变化的信息 诱导它。将解决以下具体目标： 1.肌膜Na~+/K~+-ATPase活性测定 控制常氧灌流、低氧、低流量缺血。这个 实验将确定Na+的排出量是减少还是 在后两种情况下增加。另外，为了检查 Na+/K+ATP活性在不同代谢背景下的变化 钠离子、细胞能量状态、pH等调节剂的顺序 评估功能改变如何发生在 缺血/缺氧。 2.确定单向Na+内流的速率，在 控制常氧灌注、低氧和低流量缺血，通过 使用Na+和Na+挤出速率测量。这些 测量将确定单向Na+内流是否发生改变 在各种情况下，特别是如果它是 与对照组相比，低流量缺血时基因表达下调 常氧和低氧。此外，为了比较这些Na+内流 与通过测量Li+摄取获得的变化， 其对Na+通道活性具有特异性。 3.测定Na~+/H~+的内流速率 交换，在控制期间，常氧灌流，低氧，和低- 血流缺血，并评估这一机制在 在不同缺血/缺氧状态下导致Na+积聚 条件。 4.测量发生单向Na+内流的速率 通过Na+-HCO3共转运，在对照常氧灌流期间， 低氧和低流量缺血，并评估其重要性 这一机制在不同的条件下促进了Na+的积累 缺血/缺氧状态。此外，为了评估可能的 这种pH调节机制与Na~+/H~+的相互作用 交换(抑制一方是否刺激另一方)。 5.测量Na+内流的速率。 失活Na+通道电流，在对照期间，常氧， 低氧和低流量缺血，并评估其重要性 这一机制在不同的条件下促进了Na+的积累 缺血/缺氧状态。

Previous studies have demonstrated that intracellular Cs2+ overload plays an important role in myocardial ischemic injury, and that an important determinant of Ca2+ homeostasis is the control of intracellular Na+ (Na+). However, Na+ homeostasis during ischemia is still poorly understood. The proposed project will employ a unique combination of state-of-the-art NMR techniques to obtain a comprehensive assessment of how Na+I is controlled in the myocardium, during ischemia and hypoxia. Using the intact perfused rat heart model, interleaved 23Na and 31P NMR spectra will monitor changes in Na+I and cellular energy. In the same preparations, 87Rb NMR spectroscopy will be used to monitor Rb+ uptake and Na+/K+ ATPase activity. Using 7Li NMR spectroscopy, Li+ uptake will be measured to monitor voltage- gated Na+ channel activity. This methodology not only measures net Na+ accumulation, but is designed to also provide simultaneous information concerning how changes in unidirectional Na+ fluxes induce it. The following specific aims will be addresses: 1. To measure sarcolemmal Na+/K+ ATPase activity during control normoxic perfusion, hypoxia, and low-flow ischemia. The experiments will determine whether Na+ extrusion decreases or increases under the latter two conditions. Also, to examine Na+/K+ ATP activity in the context of the various metabolic modulators such as Na+, cellular energy status, pH, etc., in order to assess how functional alteration may occur during ischemia/hypoxia. 2. To determine the rate of unidirectional Na+ influx, during control normoxic perfusion, hypoxia, and low-flow ischemia, by using the Na+ and Na+ extrusion rate measurements. These measurements will determine if unidirectional Na+ influx is altered under the various conditions, and in particular, if it is downregulated during low-flow ischemia, as compared to control normoxia and hypoxia. Also, to compare these Na+ influx alterations with those obtained from measurement of Li+ uptake, which has specificity for Na+ channel activity. 3. To measure the rates of Na+ influx which occur via Na+/H+ exchange, during control normoxic perfusion, hypoxia, and low- flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions. 4. To measure the rates of unidirectional Na+ influx which occur via Na+-HCO3- cotransport, during control normoxic perfusion, hypoxia, and low-flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions. Also, to assess the possible interaction of this pH regulatory mechanism with Na+/H+ exchange (whether inhibition of one stimulates the other). 5. To measure the rate of Na+ influx which occur via non- inactivating Na+ channel current, during control, normoxia, hypoxia, and low-flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions.