NMR STUDIES OF CYTOSOLIC METAL IONS AND OXIDATIVE STRESS

描述（由申请人提供）：我们的长期目标是阐明细胞内矿物质离子（Na+, K+，游离Mg2+和游离Ca2+）和氧化应激在原发性高血压和2型糖尿病病理生理中的作用。我们试图了解细胞内必需金属离子和膜脂浓度的调节如何在健康疾病，特别是高血压和糖尿病中误入歧途。我们的主要研究工具是核磁共振光谱学。我们的实验室在开发用于测量各种细胞内阳离子的核磁共振方法方面发挥了关键作用，并且我们在使用1H核磁共振分析膜磷脂组成和膜脂肪酸不饱和程度（双键）和平均链长方面也具有相当的专业知识。氧化应激可能在糖尿病和高血压的发病机制中发挥重要作用，它引起膜脂的过氧化降解，导致脂肪酸双键的损失，这可以通过1H NMR来定量。本研究的具体目的如下：(1)探讨盐敏感性高血压患者肾钠稳态改变的机制；(2)证明氧化应激导致活性氧（ROS）的过量产生，可导致膜磷脂脂酰基链不饱和状态的丧失（通过H NMR测量），并验证氧化应激期间通过膜脂肪酸不饱和状态的丧失测量的脂质过氧化降解导致细胞内离子变化的假设，类似于原发性高血压；(3)探讨镁对氧化应激的可能保护作用，以及低镁环境下组织的抗氧化能力（以谷胱甘肽（GSH）水平衡量）是否降低；(4)验证膜脂肪酸不饱和缺陷与人原发性高血压相关的假说，探讨高血压中鞘磷脂-神经酰胺通路是否发生改变；(5)研究抑制一氧化氮合酶导致正常大鼠高血压的一氧化氮合成障碍是否引起与原发性高血压相似的膜脂变化；(6)验证高血糖引起膜脂过氧化降解的假设，如1H NMR测量，特别是在血管组织中，如果是这样，是否高血糖相关的脂肪酸不饱和损失和细胞内离子改变可以通过膳食抗氧化剂如维生素C和E来逆转；(7)探讨高血压和高血糖肾和心肌对缺血性损伤的易感性增加及其与膜脂过氧化降解增加的关系；(8)设计、开发和测试一种使用三量子过滤（TQ） 23na NMR非侵入性测量“细胞内钠”的方法。提出的细胞内离子和氧化应激在高血压和高血糖状况的核磁共振研究可能最终导致这些健康疾病的管理更好的策略。

DESCRIPTION (provided by applicant): Our long-range goal is to elucidate the role of intracellular mineral ions (Na+, K+, free Mg2+ & free Ca2+) and oxidative stress in the pathophysiology of essential hypertension and type 2 diabetes. We seek to understand how the regulation of intracellular concentrations of essential metal ions and membrane lipids goes astray in health disorders, especially hypertension and diabetes. Our main research tool is NMR spectroscopy. Our laboratory played a key role in the development of NMR methods for measuring various intracellular cations and we also have considerable expertise in the use of 1H NMR for analyzing membrane phospholipid composition and the degree of membrane fatty acid unsaturation (double bonds) and average chain length. Oxidative stress, which may play a contributory role in the pathogenesis of diabetes as well as hypertension, causes peroxidative degradation of membrane lipids resulting in a loss of fatty acid double bonds that can be quantitated by 1H NMR. The following specific aims will be pursued: (1) To investigate the mechanism of altered renal sodium homeostasis in salt-sensitive hypertension; (2) To demonstrate that oxidative stress, which results in overproduction of reactive oxygen species (ROS), can cause loss of unsaturation in fatty acyl chains of membrane phospholipids as measured by 'H NMR, and to test the hypothesis that peroxidative degradation of lipids as measured by loss of membrane fatty acid unsaturation during oxidative stress results in intracellular ionic changes similar to those seen in essential hypertension; (3) To investigate a possible protective role of magnesium against oxidative stress and whether there is a decrease in antioxidant capacity, as measured by glutathione (GSH) levels, in tissues exposed to low Mg environment; (4) To test the hypothesis that a deficit in membrane fatty acid unsaturation is associated with human essential hypertension and to investigate if there is an alteration in sphingomyelin-ceramide pathway in hypertension; (5) To find out if impairment of nitric oxide synthesis by inhibition of nitric oxide synthase, which causes hypertension in a normal rat, produces membrane lipid changes similar to those seen in essential hypertension; (6) To test the hypothesis that hyperglycemia causes peroxidative degradation,of membrane lipids as measured by 1H NMR, especially in vascular tissue, and, if so, whether hyperglycemia associated loss of fatty acid unsaturation and intracellular ionic alterations can be reversed by dietary antioxidants such as vitamin C and E; (7) To investigate increased vulnerability of hypertensive as well as hyperglycemic kidney and myocardium to ischemic damage and its relationship to increased perexidative degradation of membrane lipids; and (8) To design, develop and test a method for non-invasive measurement of "intracellular sodium using triple-quantum filtered (TQ)23 Na NMR. The proposed NMR investigations of intracellular ions and oxidative stress in hypertension and hyperglycemia condition may eventually lead to better strategies for the management of these health disorders.