Regulation of Vascular Smooth Muscle Calcium by NADPH Redox

证据表明磷酸戊糖途径(PPP)/葡萄糖-6-磷酸 脱氢酶(G6PD)和NADPH氧化还原参与冠状动脉收缩功能的调节 (CA)动脉。然而，G6PD和NADPH调节血管收缩功能的机制(S) CA是默默无闻的。因此，这一提议的主要焦点将是阐明信号通路 G6PD和NADPH氧化还原对血管平滑肌细胞L钙电流的影响 和CA功能。为了实现这些目标，我们将在目标1中确定G6PD是否在 静息和收缩CA的细胞部分，通过估计葡萄糖氧化速率和G6PD 生物化学和放射性同位素示踪分析的活性水平。进一步，我们将确定机制(S) 通过研究PKC和代谢的作用，参与了收缩剂诱导的G6PD激活 小路。在目标2中，我们将确定G6PD是否介导L型钙通道活动， 静息和收缩CA时细胞内钙离子和血管舒缩张力，通过检测L型钙离子功能， 药物抑制G6PD后细胞内钙变化及血管舒缩功能的测定 试剂和siRNA转染法，以及在G6PD缺陷的小鼠主动脉中。在目标3中，我们将确定 葡萄糖-6-磷酸脱氢酶是否调节细胞内L型钙通道功能 CA中通过与离子通道蛋白的直接物理相互作用引起的Ca~(2+)收缩和氧化还原变化 (CaV1.2的Alpha亚基)，通过免疫共沉淀、共定位和体外结合试验。 此外，我们还将确定NADP+或NADPH是否直接与L类型的钙通道结合 蛋白质使通道失活以及还原型/氧化型谷胱甘肽(GSH)水平是否发生变化 或过氧化氢(H_2O_2)，由NADPH水平降低(由于G6PD抑制)诱导 活性)，调节冠状动脉和冠状动脉平滑肌细胞的L型钙通道功能 G6PD缺陷小鼠的主动脉。PPP/G6PD和NADPH氧化还原在糖尿病中上调， 肺动脉高压和心力衰竭，从而提示G6PD和NADPH的潜在作用 氧化还原在这些疾病中严重损害血管的收缩功能。这项研究，关于 如预期的那样完成，将被证明在开发新的治疗方法方面是有用的 肺动脉高压、糖尿病和心力衰竭时的血管功能障碍。 项目说明第6页

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. Project Description Page 6