Metabolism and Transport of Nitrate, Nitrite, and Nitric Oxide

硝酸盐、亚硝酸盐和一氧化氮的代谢和运输

• 批准号: 10248123
• 负责人: Alan Schechter
• 金额: $ 59.42万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10248123
• 关键词: Active Biological Transport; Affect; Animal Model; Area; Biological; Biological Assay; Blood; Blood Circulation; Blood Platelets; Blood Vessels; Blood coagulation; Blood flow; Carrier Proteins; Cell Culture Techniques; Cell Differentiation process; Cells; Chlorides; Complex; Confusion; Cyclic GMP; Development; Diet; Dietary intake; Disease; Endocrine; Enzymes; Erythrocytes; Exercise; Functional disorder; Gases; Generations; Genetically Modified Animals; Hemoglobin; Human; Hypoxia; Inhalation; Ions; Journals; Kinetics; Knock-out; Literature; Liver; Measurement; Measures; Metabolism; Methodology; Molecular; Muscle; Muscle Cells; Muscle Fibers; Myoblasts; Myocardium; Myoglobin; Nitrates; Nitric Oxide; Nitric Oxide Signaling Pathway; Nitric Oxide Synthase; Nitrites; Organ; Oxygen; Pathway interactions; Patients; Pharmacology; Phosphorylation; Physiological; Plasma; Platelet aggregation; Process; Property; Proteins; Proteomics; Publishing; Reaction; Reporting; Research; Rodent; Rodent Model; Role; Saliva; Sickle Cell Anemia; Signal Transduction; Skeletal Muscle; Smooth Muscle; Sodium Nitrite; Source; Specific qualifier value; Thailand; Therapeutic Intervention; Tissues; Transport Process; Venous; Work; XDH gene; clinical application; dietary manipulation; genetic manipulation; human disease; interest; targeted treatment; uptake; vasodilator-stimulated phosphoprotein

We have now largely completed our work on platelet and blood clotting inhibition by nitrite by studying the effects of changes in ambient oxygen levels on the processes in rodent models using the TEG methodologies. We have also measured the effects of red cell reduction of plasma nitrite on platelet signaling because of some confusion in the literature and have confirmed that VASP phosphorylation occurs with the generation of NO but that this signal is very dependent on the kinetics of reaction and the exact experimental conditions, especially red cell concentrations. The VASP phosphorylation appears to be an order of magnitude more sensitive than the direct measurements of cGMP that we have used in the past. These measurements have been very valuable for our collaborators in Bangkok, Thailand who have found such changes in circulating platelets in certain patients after sodium nitrite inhalation (see DK 025104). Because of the recent interest in the results of VASP phosphorylation studies we have published further on this process and produced a "Journal of Visualized Research" tutorial so others may better use this assay, as well as other methodological information. Our studies of skeletal muscle nitrate began during our studies of NO metabolism in rodents in which we discovered that nitrate levels in skeletal muscle were much higher than in blood or any other organ, indeed muscle appears to be the main reservoir for nitrate in the body. We then showed that during exercise the nitrate could be reduced to nitrite and then NO which we believe are the main pathway controlling the massive increase in blood flow with exercise, known for 150 years but without a good explanation until now. This work has also focused on better understanding of the mechanisms of these reductive processes and how muscle obtains such high levels of nitrate. So far our results are still most compatible with xanthine oxido-reductase as the major enzyme involved in the reductive processes. Dietary and genetic manipulations of rodents has shown that NOS 1 (nNOS) and myoglobin knockouts have markedly reduced levels of skeletal muscle nitrate suggesting that, as we predicted, both proteins are involved in these high levels. However, we also find that dietary limitations of nitrate and nitrite lower these levels greatly (more than in blood or liver) and that return of these ions to the diet results in rapid accumulation and, indeed, in some cases an "overshoot" of the levels. These result raise the possibility of some active transport mechanisms in the muscle, perhaps by the protein sialin which transports nitrate into the saliva from blood. We are also currently studying these processes in muscle cells, primary and continuous lines, in culture, including in cells which can be caused to differentiate from myoblasts to myocytes and myotubes. Our results show uptake of nitrate and nitrite in all developmental stages of muscle cell but that differentiation of muscle cells is required for enhancing nNOSitrate reduction to nitrite. We are now quantitating the roles of n-nitric oxide synthase, myoglobin, sialin, a chloride transporter protein, and other proteins in affecting the levels of nitrate in muscle tissues. We are planning to do proteomic analyses of these cells to identify new molecules that may be involved in these processes and we are also studying how hypoxia affects the relative contributions of nNOS, reductive processes and transport processes in determining levels of nitrate, nitrite and NO in muscle under specified conditions and if smooth and cardiac muscle have similar mechanisms.

现在，我们通过研究了使用TEG方法论的环境氧水平变化对啮齿动物模型中的过程的变化，从而在很大程度上完成了对亚硝酸盐抑制血小板和血液凝结抑制作用的工作。 我们还测量了血浆亚硝酸盐对血小板信号传导的降低的影响，因为文献中有些混乱，并证实VASP磷酸化是随着NO的产生而发生的，但该信号非常依赖于反应的动力学和精确的实验条件，尤其是红细胞浓度。 VASP磷酸化似乎比我们过去使用过的CGMP的直接测量值更敏感。对于我们在泰国曼谷的合作者，这些测量值非常有价值，他们在亚硝酸钠吸入后发现某些患者的循环血小板发生了这种变化（请参阅DK 025104）。由于最近对VASP磷酸化研究结果的兴趣，我们在此过程中发表了进一步发表，并产生了“可视化研究杂志”教程，因此其他人可以更好地使用该测定法以及其他方法论信息。 我们对骨骼肌硝酸盐的研究始于我们对啮齿动物无代谢的研究，其中我们发现骨骼肌的硝酸盐水平比血液或任何其他器官高得多，实际上，肌肉似乎是体内硝酸盐的主要储层。然后，我们表明，在运动过程中，硝酸盐可以减少到亚硝酸盐，然后我们认为这是控制血液流量大量增加的主要途径，该运动通过运动已知150年，但直到现在尚无良好的解释。 这项工作还集中在更好地理解这些还原过程的机制以及肌肉如何获得如此高的硝酸盐。 到目前为止，我们的结果仍然与黄嘌呤氧化还原酶是涉及还原过程的主要酶。啮齿动物的饮食和遗传操作表明，NOS 1（NNOS）和肌红蛋白敲除骨骼肌硝酸盐的水平显着降低，这表明，正如我们预测的那样，这两种蛋白质都涉及这些高水平。 但是，我们还发现，硝酸盐和亚硝酸盐的饮食局限性降低了这些水平（比血液或肝脏中的饮食限制），并且这些离子回到饮食中的饮食限制会导致迅速积累，实际上，在某些情况下，这些离子的水平“过时”。这些结果提高了肌肉中某些主动运输机制的可能性，也许是蛋白质酸粒蛋白从血液中运输到唾液中的蛋白质。 我们目前还在培养肌肉细胞，主要和连续线的肌肉细胞中研究这些过程，包括可能导致从肌细胞到成肌细胞和肌管的细胞中。我们的结果表明，在肌肉细胞的所有发育阶段，硝酸盐和亚硝酸盐的摄取量是肌肉细胞的分化以增强非西硝酸盐还原为亚硝酸盐。现在，我们正在量化N氮氧化物合酶，肌红蛋白，sialin，氯化物转运蛋白蛋白和其他蛋白质在影响肌肉组织中硝酸盐水平中的作用。我们计划对这些细胞进行蛋白质组学分析，以鉴定可能参与这些过程的新分子，我们还在研究缺氧如何影响NNOS，还原过程和运输过程在确定硝酸盐，硝酸盐和肌肉在特定条件下以及在特定条件下的肌肉和非平滑性肌肉的相对水平中的相对贡献。