NMR STUDIES OF SODIUM HOMEOSTASIS IN ISCHEMIC MYOCARDIUM

以前的研究表明，细胞内Cs2+ 超负荷在心肌缺血损伤中起重要作用， Ca 2+稳态的一个重要决定因素是控制 细胞内Na+（Na+）。 然而，Na+稳态期间， 对局部缺血仍然知之甚少。 拟议项目将 采用最先进的NMR技术的独特组合， 获得关于Na+I如何控制的全面评估， 心肌缺血和缺氧时。 使用完整的 灌注大鼠心脏模型，23 Na和31 P交替NMR谱 将监测Na+I和细胞能量的变化。 在同一 制备，87 Rb NMR光谱将用于监测 Rb+摄取和Na+/K+ ATP酶活性。 使用7 Li NMR 在光谱学中，将测量Li+吸收以监测电压。 门控Na+通道活性。 这种方法不仅可以衡量 净Na+积累，但也旨在提供同时 关于单向Na+通量如何变化的信息 将致力于实现以下具体目标： 1.测定心肌细胞膜Na+/K+ ATP酶活性， 控制常氧灌注、缺氧和低流量缺血。 的 实验将确定Na+挤出是否减少或 在后两种情况下会增加。 此外，为了检查 Na+/K+ ATP活性在各种代谢的背景下 调节剂如Na+、细胞能量状态、pH等，为了 为了评估功能改变如何发生， 缺血/缺氧。 2.为了确定单向Na+内流的速率，在 控制常氧灌注、缺氧和低流量缺血， 使用Na+和Na+挤出速率测量。 这些 测量将确定单向Na+流入是否改变 在各种条件下，特别是，如果 与对照组相比，在低流量缺血期间下调 常氧和缺氧。 此外，为了比较这些Na+流入 与从Li+吸收的测量获得的那些改变， 其对Na+通道活性具有特异性。 3.测量通过Na+/H+发生的Na+内流速率 交换，在控制常氧灌注，缺氧，低- 血流缺血，并评估这一机制的重要性， 在各种缺血/缺氧条件下促进Na+积累 条件 4.为了测量发生的单向Na+流入速率， 通过Na+-HCO 3-共转运，在对照常氧灌注期间， 缺氧和低血流缺血，并评估 这种机制有助于Na+积累在各种条件下， 缺血/缺氧状况。 此外，为了评估可能的 这种pH调节机制与Na+/H+相互作用 交换（是否抑制一个刺激另一个）。 5.为了测量Na+内流率， 失活Na+通道电流，在对照、常氧期间， 缺氧和低流量缺血，并评估其重要性 这种机制有助于Na+积累在各种条件下， 缺血/缺氧状况。

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.