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.

通过使用 TEG 方法研究环境氧气水平变化对啮齿动物模型过程的影响，我们现已基本完成了亚硝酸盐抑制血小板和血液凝固的工作。 由于文献中的一些混乱，我们还测量了红细胞减少血浆亚硝酸盐对血小板信号传导的影响，并证实 VASP 磷酸化随着 NO 的产生而发生，但该信号非常依赖于反应动力学和确切的实验条件，尤其是红细胞浓度。 VASP 磷酸化似乎比我们过去使用的 cGMP 直接测量敏感一个数量级。这些测量结果对于我们在泰国曼谷的合作者来说非常有价值，他们发现某些患者吸入亚硝酸钠后循环血小板发生了这种变化（参见 DK 025104）。由于最近对 VASP 磷酸化研究结果的兴趣，我们进一步发表了有关此过程的文章，并制作了“可视化研究杂志”教程，以便其他人可以更好地使用此测定以及其他方法学信息。 我们对骨骼肌硝酸盐的研究始于对啮齿动物一氧化氮代谢的研究，其中我们发现骨骼肌中的硝酸盐水平远高于血液或任何其他器官中的硝酸盐水平，事实上，肌肉似乎是体内硝酸盐的主要储存库。然后我们证明，在运动过程中，硝酸盐可以还原为亚硝酸盐，然后还原为一氧化氮，我们相信这是控制运动时血流量大量增加的主要途径，这一点已有 150 年的历史，但迄今为止还没有一个很好的解释。 这项工作还侧重于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水平的硝酸盐。 到目前为止，我们的结果仍然与黄嘌呤氧化还原酶最为一致，黄嘌呤氧化还原酶是参与还原过程的主要酶。对啮齿动物的饮食和基因操作表明，一氧化氮合酶 1 (nNOS) 和肌红蛋白敲除显着降低了骨骼肌硝酸盐的水平，这表明，正如我们预测的那样，这两种蛋白质都参与了这些高水平的过程。 然而，我们还发现，饮食中硝酸盐和亚硝酸盐的限制会大大降低这些水平（比血液或肝脏中的水平更低），并且这些离子返回饮食会导致快速积累，实际上，在某些情况下会导致水平“超调”。这些结果提出了肌肉中某些主动转运机制的可能性，可能是通过唾液酸蛋白将硝酸盐从血液转运到唾液中。 我们目前也在研究肌肉细胞、原代和连续系、培养物中的这些过程，包括可导致从成肌细胞分化为肌细胞和肌管的细胞。我们的结果表明，肌细胞的所有发育阶段都会吸收硝酸盐和亚硝酸盐，但肌肉细胞的分化是增强 nNOSitrate 还原为亚硝酸盐所必需的。我们现在正在定量一氧化氮合酶、肌红蛋白、唾液酸蛋白、氯转运蛋白和其他蛋白质在影响肌肉组织中硝酸盐水平方面的作用。我们计划对这些细胞进行蛋白质组学分析，以确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、还原过程和转运过程的相对贡献，以确定特定条件下肌肉中硝酸盐、亚硝酸盐和一氧化氮的水平，以及平滑肌和心肌是否具有相似的机制。