Modulators of Nitric Oxide Synthase Activity in Sickle Cell Disease

镰状细胞病中一氧化氮合酶活性的调节剂

• 批准号: 7654865
• 负责人: Mary E Fabry
• 金额: $ 41.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654865
• 关键词: Amino Acids; Animals; Antioxidants; Arginine; Benchmarking; Biological Availability; Biological Markers; Biological Preservation; Biopterin; Breeding; Cells; Citrulline; Dehydration; Dose; Enzymes; Erythrocytes; Event; Failure; Functional disorder; GTP Cyclohydrolase I; Hemoglobin; Hemolysis; Human; Hypoxia; Inflammation; Intervention; Lead; Metabolism; Modeling; Multienzyme Complexes; Mus; Nitric Oxide; Nitric Oxide Synthase; Ornithine; Oxidative Stress; Oxygen; Pathology; Pathway interactions; Patients; Phosphorylation; Pilot Projects; Plasma; Play; Polymers; Production; Rattus; Reaction; Response Elements; Role; Series; Serine; Severities; Sickle Cell; Sickle Cell Anemia; Superoxides; Supplementation; Testing; Threonine; Tissue Extracts; Tissues; Transgenic Mice; Transgenic Organisms; Vascular Diseases; base; blood perfusion; drinking water; improved; inhibitor/antagonist; mouse model; polymerization; prevent; sickling; tetrahydrobiopterin; vascular bed

The primary features of sickle cell disease are events resulting in low flow, poor perfusion and blood and tissue hypoxia that are both pre-disposing to and the consequence of vaso-occlusion. We will test the overall hypothesis that BH4 (tetra-hydrobiopterin, an essential co-factor of nitric oxide synthase (NOS)) levels are decreased in sickle cell disease and in sickle transgenic mice and treatment protective of tissue BH4 levels can have greater efficacy than arginine supplementation alone to interrupt the cycle of polymerization, vaso-occlusion and inflammation that is the origin of pathology in sickle cell disease. Depletion of either arginine or BH4 results in loss of NOS NO generating capacity and production shifts to superoxide, resulting in a vicious cycle in which BH4 is oxidized and more superoxide is generated. We have demonstrated that BH4 is depleted in sickle transgenic mice due in part to oxidative stress. NOS is up regulated by hypoxia via a HIF-sensitive, hypoxia-response element. We further speculate that failure to simultaneously up regulate BH4 will increase oxidative stress. Phosphorylation at serine 1177 and threonine 495 modulate NOS activity. We have demonstrated that the percent phosphorylation at serine 1177 is reduced in mild sickle transgenic mice when they are exposed to hypoxia. We speculate that simultaneous reduction of BH4 and serine phosphorylation will severely impact NOS activity. Hemolysis is thought to be a major factor in reduction of NO bioavailability, both through reaction of cell free hemoglobin with NO and through release of argininase that depletes arginine, the substrate of NOS. We have demonstrated that arginine supplementation reduces hemolysis and oxidative stress in sickle transgenic mice, in part, via inhibition of the Gardos channel. Inhibition of the Gardos channel prevents red cell dehydration and inhibits polymer formation and hemolysis that further reduces NO bioavailability. However, arginine supplementation does not completely correct the vasculopathy observed in sickle transgenic mice. We hypothesize that some of the benefit of arginine supplementation may be due to preservation of BH4. To further test this hypothesis, we are breeding mice that over-express GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in BH4 synthesis into our sickle transgenic strains. These observations may be applicable to patient interventions.

镰状细胞病的主要特征是导致低血流、低血流灌注、血液和组织缺氧的事件，这些事件既是血管闭塞的预处理，也是血管闭塞的后果。我们将测试总体假设，即在镰状细胞疾病和镰刀转基因小鼠中，BH4(四氢生物蝶呤，一氧化氮合酶(NOS)的基本辅助因子)水平降低，治疗保护组织BH4水平可以比单独补充精氨酸更有效地中断聚合、血管闭塞和炎症的循环，这是镰状细胞疾病的病理起源。精氨酸或BH4的耗尽会导致一氧化氮合酶生成能力的丧失，生产转移到超氧化物，导致BH4被氧化而产生更多的超氧化物的恶性循环。我们已经证明，在镰刀形转基因小鼠中，BH4被耗尽，部分原因是氧化应激。缺氧通过对缺氧反应敏感的元件上调一氧化氮合酶的表达。我们进一步推测，未能同时上调BH4将增加氧化应激。丝氨酸1177和苏氨酸495的磷酸化调节一氧化氮合酶的活性。我们已经证明，当轻度镰刀转基因小鼠暴露在低氧环境中时，丝氨酸1177的磷酸化百分比降低。我们推测，BH4和丝氨酸磷酸化的同时减少将严重影响NOS活性。溶血被认为是降低NO生物利用度的主要因素，这既是通过细胞外血红蛋白与NO的反应，也是通过释放精氨酸酶来耗尽一氧化氮合酶的底物精氨酸。我们已经证明，补充精氨酸减少了镰刀转基因小鼠的溶血和氧化应激，部分是通过抑制Gardos通道。抑制Gardos通道可防止红细胞脱水，抑制聚合物形成和溶血，从而进一步降低NO的生物利用度。然而，补充精氨酸并不能完全纠正在镰刀转基因小鼠中观察到的血管病变。我们推测补充精氨酸的一些好处可能是由于保存了BH4。为了进一步验证这一假设，我们正在培育过表达GTP环水解酶I(GTPCH)的小鼠，GTPCH是BH4合成中的限速酶，进入我们的镰刀转基因品系。这些观察结果可能适用于患者干预。