Argininosuccinate lyase is an essential regulator of systemic nitric oxide produc

描述（由申请人提供）：本更新申请的重点是研究补充一氧化氮（NO）作为精氨酸琥珀酸解酶（ASL）缺乏引起的精氨酸琥珀酸尿（ASA）血管并发症的治疗效果，ASL是第二常见的尿素循环紊乱和一氧化氮（NO）缺乏的人体模型。ASL具有独特的催化功能和结构功能，这为理解ASA患者无视精氨酸悖论（即，尽管补充精氨酸治疗，他们仍无法有效产生NO）提供了结构模型。在一项原则性案例研究中，我们成功地治疗了一名ASA合并严重高血压的患者，该患者对多种抗高血压药物难治性补充NO。我们的数据表明，组织水平一氧化氮缺乏会导致ASA并发症，仅预防高氨血症可能无法预防长期发病。为了将这一发现转化为一种新的治疗策略，我们建议回答以下问题：补充NO是否可以用于治疗ASA患者的内皮功能障碍？我们将对ASA患者进行双盲、安慰剂对照、交叉研究。我们假设，一部分ASA患者由于血管内皮缺乏一氧化氮依赖的一氧化氮生成而表现出内皮功能障碍，而通过营养药物Neo40(R)补充低剂量亚硝酸钠等一氧化氮不依赖的一氧化氮来源可以纠正这一缺陷。目标2。我们能否开发出体外试验来预测体内对NO补充的临床反应和临床变异性？我们将通过亚硝酸盐和亚硝基硫醇测定ASA试验患者成纤维细胞产生NO的能力，并将Aim 1中的临床反应和表型与精氨酸补充后体外NO的产生联系起来。目标3。ASL/NOS复合物调节NO的机制基础是什么？一氧化氮的合成可能在一定程度上受到一氧化氮参与正调控复合物和负调控复合物的调节。为了探索调节这些结构复合体的机制，我们将检验一个假设，即NO的产生是由涉及NOS和小窝蛋白的负调控复合体与涉及NOS和ASL的正合成复合体的平衡控制的。这些研究的见解可能为研究NO在其他疾病背景下的多种生物学效应提供一个范例。虽然ASA的发病机制很复杂，可能延伸到其他精氨酸依赖机制，但我们的临床前和临床研究支持NO缺乏在本研究中测试的终点中占主导地位。此外，本文提出的方法和机制可能广泛应用于其他疾病状态下NO的失调。

DESCRIPTION (provided by applicant): This renewal application focuses on studying the efficacy of nitric oxide (NO) supplementation as a treatment for the vascular complications in argininosuccinic aciduria (ASA) caused by deficiency of argininosuccinate lyase (ASL), the second most common urea cycle disorder and a human model of nitric oxide (NO) deficiency. ASL serves distinct catalytic vs. structural functions that provide a structural model for understanding why ASA patients defy the arginine paradox, i.e., they are unable to generate NO efficiently in spite of supplemental arginine therapy. In a proof-of-principle case study, we successfully treated an ASA patient with severe hypertension refractory to multiple antihypertensive medications with NO supplementation. Our data imply that NO deficiency at the tissue level contributes to the complications of ASA and preventing hyperammonemia alone may not prevent long-term morbidity. To translate this discovery into a new treatment strategy, we propose to answer the following questions: Aim 1. Can NO supplementation be used to treat endothelial dysfunction in patients with ASA? We will perform a double-blind, placebo-controlled, cross over study in patients with ASA. We hypothesize that a subset of ASA patients will demonstrate endothelial dysfunction due to lack of NOS-dependent NO production from the vascular endothelium and that supplementation with an NOS-independent NO source such as low dose sodium nitrite via the nutriceutical Neo40(R) would correct this defect. Aim 2. Can we develop in vitro assays that predict in vivo clinical response to NO supplementation and clinical variability? We will determine the ability of ASA trial patient fibroblasts to produce NO as measured by nitrite and nitrosothiols and correlate the clinical response and phenotype in Aim 1 with in vitro production of NO in response to arginine supplementation. Aim 3. What is the mechanistic basis of NO regulation by the ASL/NOS complex? NO synthesis may be regulated in part by the participation of NOS in both positive and negative regulatory complexes. To explore the mechanisms that regulate these structural complexes, we will test the hypothesis that NO production is controlled by a balance of negative regulatory complex involving NOS and caveolin vs. a positive, synthetic complex involving NOS and ASL. The insights from these studies may offer a paradigm for studying the diverse biological effects of NO in other disease contexts. While the pathogenesis of ASA is complex and may extend to other arginine- dependent mechanisms, our preclinical and clinical studies support the dominant contribution of NO deficiency on the endpoints tested in this study. Moreover, the approaches and mechanisms proposed here may be broadly application to dysregulation of NO in other disease states.