Nitric Oxide Regulation of Smooth Muscle Tone

描述（由申请人提供）： 活性氧化剂（ROS），如一氧化氮（NO）和过氧化氢（H2 O2），在活组织中起重要的生理作用，并且在疾病中起病理生理作用，特别是那些涉及炎症的疾病。虽然环核苷酸是ROS诱导的细胞过程的关键介质，但环核苷酸非依赖性机制也很重要。在前一个赠款周期中，我们获得的数据显示，ROS对平滑肌收缩的环核苷酸非依赖性抑制是由于抑制Ca 2+敏感性的新机制。这种效应在完整组织中自发可逆，这是由于抑制了肌球蛋白II的肌动球蛋白ATP酶活性以及肌球蛋白轻链激酶（MLCK）和异源三聚体G蛋白的活性。目前的建议的总体目标是阐明这些蛋白质的氧化还原调节ROS的生化机制。目的A将检验ROS通过抑制催化位点的核苷酸结合和稳定肌球蛋白结构从而阻止F-肌动蛋白结合来抑制肌动球蛋白ATP酶活性的假设。这两种机制都是由于肌球蛋白上半胱氨酸（Cys）残基的可逆氧化。目的B将检验与ROS诱导的肌球蛋白调节轻链（rMLC）磷酸化抑制相关的假设。我们的初步数据表明，MLCK活性和GDP-GTP交换在G α亚基的异源三聚体G蛋白的活性氧抑制，这两种影响将抑制rMLC磷酸化。透化制剂用于原位生物化学研究，从而证明所提出的机制的生理相关性。分离的肌球蛋白II的可溶性蛋白水解片段和肌球蛋白上的候选Cys的定点突变被用于探索ROS对肌动球蛋白ATP酶活性的影响的特定生化机制。纯化的MLCK全酶和MLCK催化结构域的组成活性、蛋白水解亚片段被用于研究MLCK的生化机制。最后，粗膜制备和重组G蛋白亚基用于阐明新的氧化还原作用GDP-GTP交换在Ga。阐明这些机制对于理解ROS作为健康和疾病中生理效应的关键介质的作用具有重要意义。

DESCRIPTION (provided by applicant): Reactive oxidant species (ROS), such as nitric oxide (NO) and hydrogen peroxide (H2O2), play an important physiologic role in living tissue, and a pathophysiologic role in diseases, particularly those involving inflammation. Although cyclic nucleotides are key mediators of ROS-induced cellular processes, cyclic nucleotide-independent mechanisms are also important. In the previous grant cycle, we obtained data showing that the cyclic nucleotide-independent inhibition of smooth muscle contraction by ROS is due to novel mechanisms that inhibit Ca2+ sensitivity. This effect is spontaneously reversible in intact tissue and due to inhibition of the actomyosin ATPase activity of myosin II and the activities of myosin light chain kinase (MLCK) and heterotrimeric G-proteins. The overall goal of the current proposal is elucidate the biochemical mechanisms for redox regulation of these proteins by ROS. Aim A will test the hypotheses that ROS inhibit actomyosin ATPase activity by inhibiting nucleotide binding at the catalytic site and by stabilizing the myosin structure, thereby preventing F-actin binding. Both of these mechanisms are due to reversible oxidation of cysteine (Cys) residues on myosin. Aim B will test hypotheses related to ROS-induced inhibition of phosphorylation of the regulatory light chain of myosin (rMLC). Our preliminary data indicate that MLCK activity and GDP-GTP exchange at the Galpha subunit of heterotrimeric G-proteins are inhibited by ROS; both of these effects would inhibit rMLC phosphorylation. A permeabilized preparation is used for in situ biochemical studies, thereby demonstrating the physiologic relevance of the proposed mechanisms. Soluble proteolytic fragments of isolated myosin II and site-directed mutagenesis of candidate Cys on myosin are used to explore specific biochemical mechanisms for ROS effects on actomyosin ATPase activity. Purified MLCK holoenzyme and a constituitively active, proteolytic subfragment of the catalytic domain of MLCK are used to investigate biochemical mechanisms on MLCK. Finally, a crude membrane preparation and recombinant Gprotein subunits are used to elucidate novel redox effects on GDP-GTP exchange at Galpha. Elucidation of these mechanisms is of importance in understanding the role of ROS as key mediators of physiologic effects in both health and disease.