Regulation of Vascular Smooth Muscle Calcium by NADPH Redox

NADPH 氧化还原对血管平滑肌钙的调节

基本信息

批准号：
7743739
负责人：
SACHIN A GUPTE
金额：
$ 36.75万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7743739
关键词：
6-Aminonicotinamide 6-phosphogluconate Address Angiotensin II Aorta Arts Binding Biochemical Biochemical Reaction Biological Assay Blood Vessels Calcium Calcium Channel Cardiovascular system Cell Survival Cell membrane Cells Co-Immunoprecipitations Commit Complex Confusion Contracts Coronary Coronary artery Critiques Cysteine Cytosol DNA Data Deletion Mutation Development Diabetes Mellitus Disease Drug Delivery Systems Electrophoresis Enzymes Epiandrosterone Figs - dietary Functional disorder Future Genes Glucose Glucose-6-Phosphate Glucosephosphate Dehydrogenase Glutathione Glutathione Disulfide Glycolysis Goals Grant Heart Heart failure Human Hydrogen Peroxide Hypertension In Vitro Influentials Ion Channel Ion Channel Protein L-Type Calcium Channels Label Laboratories Link Lung Measures Mediating Membrane Potentials Metabolic Metabolic Diseases Metabolic Pathway Methods Modification Morbidity - disease rate Mus Muscle function Myocardial NADP Oxidation-Reduction Oxidoreductase PMCA1 protein Pathway interactions Pentosephosphate Pathway Perfusion Phosphorylation Play Principal Investigator Process Protein Kinase C Proteins Protocols documentation Publishing Pulmonary Hypertension Pulmonary artery structure RNA chemical synthesis Radio Radioisotopes Regulation Relaxation Rest Role Sarcoplasmic Reticulum Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Small Interfering RNA Smooth Muscle Smooth Muscle Myocytes Sodium-Calcium Exchanger Techniques Testing Thromboxane A2 Tracer Transfection Vascular Smooth Muscle Vasomotor analog attenuation base channel blockers dehydroepiandrosterone glucose metabolism inhibitor/antagonist innovation inorganic phosphate mortality mutant mutant mouse model novel novel therapeutic intervention oxidation patch clamp programs research study response ribose-5-phosphate thioredoxin glutathione reductase voltage

项目摘要

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磷酸戊糖途径（PPP）/6-磷酸葡萄糖脱氢酶（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 功能影响的信号通路。为了实现这些目标，在目标#1中，我们将通过估计葡萄糖氧化速率来确定G6PD在静息和收缩CA的亚细胞部分中是否具有活性，并通过生化和放射性同位素示踪测定来确定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参与收缩剂诱导的 G6PD 激活的机制。在目标#2中，我们将通过检查L型Ca2+功能，测量用药理学抑制G6PD后的细胞内Ca2+变化和血管舒缩功能，确定G6PD是否介导静息和收缩CA中的L型Ca2+通道活性、细胞内Ca2+和血管舒缩张力。试剂和 siRNA 转染，以及 G6PD 缺陷的小鼠主动脉。在目标#3中，我们将确定葡萄糖-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来调节CA中的L型Ca2+通道功能、细胞内Ca2+、收缩和氧化还原变化。），通过免疫共沉淀、共定位和体外结合测定。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会使通道失活，以及 NADPH 水平降低是否会引起还原型/氧化型谷胱甘肽 (GSH) 或过氧化氢 (H2O2) 水平的变化（由于抑制 G6PD 活性），调节从 G6PD 缺陷小鼠的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，从而表明 G6PD 和 NADPH 氧化还原在严重损害这些疾病中血管收缩功能方面具有潜在作用。这项研究如预期完成，将有助于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。在当前的项目中，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，循环系统功能障碍是美国发病和死亡的主要原因。因此，这项研究如预期完成，将有助于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。