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）：我们先前提供了证据表明，五肽磷酸盐途径（PPP）/6-磷酸盐脱氢酶（G6PD）和NADPH氧化还原参与调节冠状动脉（CA）动脉的收缩功能。但是，G6PD和NADPH调节CA的收缩功能的MACHANISM是晦涩的。因此，该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+ CA2+电流和CA功能的影响所涉及的信号通路。为了实现这些目标，我们将通过AIM＃1确定G6PD是否通过估计葡萄糖氧化的速率以及通过生化和放射性异位素示踪剂测定的G6PD活性水平来确定静息和收缩CA的亚细胞分数中的活性。此外，我们将通过研究PKC和代谢途径的作用来确定与收缩剂诱导的G6PD激活有关的机制。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+和静止和收缩CA中的血管肌张力张力，通过检查L型Ca2+功能，测量抑制药理学生产者和Sirna Transfection和Sirna Transfection和Sirna Transfection和Insirna Transfection和InsirNA Transfection和Insirna Transfection和Insirna transfection and insirna transfection和insirna g6pd，并测量L型Ca2+ CA2+ CA2+变化和加血管的功能。 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从G6PD缺乏小鼠的冠状动脉和主动脉中分离出来。 PPP/G6PD和NADPH氧化还原在糖尿病，肺动脉高压和心力衰竭中被上调，从而表明G6PD和NADPH Redox在严重损害了这些疾病中血管的收缩功能方面具有潜在的作用。这项研究按预期完成后，将被证明可用于开发用于治疗肺动脉高压，糖尿病和心力衰竭血管功能障碍的新型疗法。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故障的发展中起作用，这是美国发病率和死亡率的主要原因。因此，在预期的完成后，这项研究将被证明可用于开发新的疗法，以治疗肺动脉高压，糖尿病和心力衰竭的血管功能障碍。