Regulation of Vascular Smooth Muscle Calcium by NADPH Redox

NADPH 氧化还原对血管平滑肌钙的调节

• 批准号: 7372575
• 负责人: SACHIN A GUPTE
• 金额: $ 23.12万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7372575
• 关键词: 6-Aminonicotinamide; 6-phosphogluconate; Angiotensin II; Aorta; Arts; Binding; Biochemical; Biochemical Reaction; Bioinformatics; Biological Assay; Blood Vessels; Calcium; Calcium Channel; Cardiovascular system; Cell Survival; Cell membrane; Cells; Co-Immunoprecipitations; Commit; Complex; Contracts; Coronary; Coronary artery; Coupled; Culture Techniques; Cysteine; Cytosol; DNA; Data; Development; Diabetes Mellitus; Disease; Drug Delivery Systems; Electrophoresis; Environment; Enzymes; Epiandrosterone; Figs - dietary; Functional disorder; Future; Gene Expression Regulation; Genes; Glucose; Glucose-6-Phosphate; Glucosephosphate Dehydrogenase; Glutathione; Glutathione Disulfide; Glycolysis; Goals; Heart; Heart failure; Human; Hydrogen Peroxide; Hypertension; In Vitro; Influentials; Ion Channel; Ion Channel Protein; L-Type Calcium Channels; Label; Link; Localized; Lung; Measures; Mediating; Membrane Potentials; Metabolic; Metabolic Diseases; Metabolic Pathway; Methods; Molecular Biology Techniques; Morbidity - disease rate; Mus; Muscle function; Myocardial; NADP; Organoids; Oxidation-Reduction; Oxidoreductase; PMCA1 protein; Pathway interactions; Pentosephosphate Pathway; Perfusion; Personal Satisfaction; Phosphorylation; Play; Process; Protein Kinase C; Proteins; Pulmonary Hypertension; Pulmonary artery structure; RNA chemical synthesis; Radio; Radioisotopes; Rate; Regulation; Relaxation; Research; Research Personnel; Resource Sharing; Rest; Role; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Sodium-Calcium Exchanger; Testing; Thromboxane A2; Tracer; Transfection; U46619; Vascular Smooth Muscle; Vasomotor; analog; attenuation; base; cellular imaging; channel blockers; dehydroepiandrosterone; glucose metabolism; inhibitor/antagonist; innovation; inorganic phosphate; knock-down; mortality; mutant; mutant mouse model; novel; novel therapeutics; oxidation; research study; ribose-5-phosphate; thioredoxin glutathione reductase; voltage

DESCRIPTION (provided by applicant): We have previously provided evidence that the pentose phosphate pathway (PPP)/glucose-6-phosphate dehydrogenase (G6PD) and NADPH redox is involved in modulating contractile function of the coronary (CA) artery. However, the machanism(s) by which G6PD and NADPH modulates contractile function of CA are obscure. Therefore, the primary focus of this proposal will be, to elucidate the signaling pathways involved in mediating the effects of G6PD and NADPH redox on smooth muscle cell L-type Ca2+ currents and CA function. To achieve these goals, we will, in Aim #1 determine if G6PD is active in the sub- cellular fractions of resting and contracting CA, by estimating the rate of glucose oxidation, and the G6PD activity levels by biochemical and radioisotope tracer assays. Furthermore, we will identify mechanism(s) involved in contractile agents-induced-G6PD activation, by investigating the role of PKC and metabolic pathways. In Aim #2, we will determine whether G6PD mediates L-type Ca2+ channel activity, intracellular Ca2+, and vasomotor tone in resting and contracting CA, by examining L-type Ca2+ function, measure intracellular Ca2+ changes and vasomotor function after inhibiting G6PD with pharmacological agents and siRNA transfection, and in G6PD deficient mouse aorta. In Aim #3, we will determine whether glucose-6-phosphate dehydrogenase modulates the L-type Ca2+ channel function, intracellular Ca2+, contraction and redox changes, in CA via direct physical interaction with the ion channel proteins (alpha subunit of CaV1.2), by co-immunoprecipitation, co-localization and in-vitro binding assays. Additionally, we will determine whether direct binding of NADP+ or NADPH to the L-type Ca2+ channel protein inactivates the channel and whether changes in the levels of reduced/oxidized glutathione (GSH) or hydrogen peroxide (H2O2), induced by decrease in NADPH levels (due to the inhibition of G6PD activity), modulates L-type Ca2+ channel function, in smooth muscle cells isolated from coronary and aorta of G6PD deficient mouse. The PPP/G6PD and NADPH redox is up-regulated in diabetes, pulmonary hypertension and heart failure, thereby suggesting a potential role for G6PD and NADPH redox in profoundly impairing the contractile function of blood vessels in these diseases. This study, on completion as anticipated, will prove to be useful in developing novel therapies for the treatment of vascular dysfunction in pulmonary hypertension, diabetes, and heart failure. In the current project, we have undertaken a task to determine whether metabolic changes play a role in the development of circulatory system malfunction, which is a major cause of morbidity and mortality in the USA. This study, therefore, on completion as anticipated, will prove to be useful in developing novel therapies to treat vascular dysfunction in pulmonary hypertension, diabetes, and heart failure.

描述（由申请人提供）：我们之前提供的证据表明戊糖磷酸途径(PPP)/葡萄糖-6-磷酸脱氢酶（G6PD）和NADPH氧化还原参与调节冠状动脉（CA）的收缩功能。然而，G6PD和NADPH调节CA收缩功能的机制尚不清楚。因此，本提案的主要重点将是阐明介导G6PD和NADPH氧化还原对平滑肌细胞l型Ca2+电流和CA功能的影响的信号通路。为了实现这些目标，我们将在目标1中确定G6PD在静止和收缩CA的亚细胞部分是否有活性，方法是估计葡萄糖氧化速率，并通过生化和放射性同位素示踪剂测定G6PD活性水平。此外，我们将通过研究PKC和代谢途径的作用来确定收缩剂诱导g6pd激活的机制。在Aim #2中，我们将确定G6PD是否介导l型Ca2+通道活性、细胞内Ca2+和静息和收缩CA的血管舒张性，通过检查l型Ca2+功能，测量细胞内Ca2+变化和血管舒张功能后，用药物和siRNA转染抑制G6PD，并在G6PD缺陷小鼠主动脉。在Aim #3中，我们将通过共免疫沉淀、共定位和体外结合试验，确定葡萄糖-6-磷酸脱氢酶是否通过与离子通道蛋白（CaV1.2的α亚基）的直接物理相互作用，调节CA中的l型Ca2+通道功能、细胞内Ca2+、收缩和氧化还原变化。此外，我们将确定NADP+或NADPH与l型Ca2+通道蛋白的直接结合是否使通道失活，以及NADPH水平降低（由于抑制G6PD活性）引起的还原性/氧化性谷胱甘肽（GSH）或过氧化氢（H2O2）水平的变化是否调节从G6PD缺陷小鼠冠状动脉和主动脉分离的平滑肌细胞中的l型Ca2+通道功能。PPP/G6PD和NADPH氧化还原在糖尿病、肺动脉高压和心力衰竭中上调，从而提示G6PD和NADPH氧化还原在这些疾病中深刻损害血管收缩功能的潜在作用。这项研究如预期的那样完成，将证明对开发治疗肺动脉高压、糖尿病和心力衰竭血管功能障碍的新疗法是有用的。在目前的项目中，我们承担了一项任务，以确定代谢变化是否在循环系统功能障碍的发展中发挥作用，这是美国发病率和死亡率的主要原因。因此，这项研究如预期的那样完成，将证明对开发治疗肺动脉高压、糖尿病和心力衰竭血管功能障碍的新疗法是有用的。