Regulation of Vascular Smooth Muscle Calcium by NADPH Redox

描述(申请人提供)：我们先前提供的证据表明，磷酸戊糖途径(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(S)尚不清楚。因此，本研究的重点将是阐明G6PD和NADPH氧化还原对平滑肌细胞L钙电流和CA功能影响的信号转导途径。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以及通过生化和放射性同位素示踪分析确定G6PD活性水平，从而确定G6PD在静息和收缩CA的亚细胞部分是否活跃。此外，我们将通过研究蛋白激酶C和代谢途径的作用，确定参与收缩药物诱导的G6PD激活的机制(S)。目的#2通过检测G6PD对静息和收缩CA的作用，测定G6PD抑制G6PD后和G6PD缺乏的小鼠主动脉细胞内钙的变化和血管收缩功能，以确定G6PD是否介导了G6PD在静息和收缩CA过程中的钙通道活性、细胞内钙离子和血管收缩张力。在目标3中，我们将通过免疫共沉淀、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)直接物理相互作用来调节CA的L型钙通道功能，细胞内钙、收缩和氧化还原的变化。此外，我们还将确定NADP+或NADPH与L型钙通道蛋白的直接结合是否使该通道失活，以及由于NADPH水平降低(由于抑制G6PD活性)而引起的还原型/氧化型谷胱甘肽(GSH)或过氧化氢(H_2O_2)水平的变化是否调节G6PD缺乏小鼠冠状动脉和主动脉的L型钙通道功能。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严重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后，将被证明对开发治疗肺动脉高压、糖尿病和心力衰竭的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，即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率和死亡率的主要原因。因此，这项研究一旦如预期完成，将被证明对开发治疗肺动脉高压、糖尿病和心力衰竭的血管功能障碍的新疗法是有用的。

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.