Argininosuccinate lyase is an essential regulator of systemic nitric oxide produc

描述（由申请人提供）：本再注册申请重点研究一氧化氮（NO）补充剂作为治疗由乙酰氨基琥珀酸裂解酶（ASL）缺乏引起的乙酰氨基琥珀酸尿症（阿萨）血管并发症的有效性，乙酰氨基琥珀酸裂解酶（ASL）是第二种最常见的尿素循环障碍，是一氧化氮（NO）缺乏的人类模型。ASL具有不同的催化功能和结构功能，为理解阿萨患者为何无视精氨酸悖论提供了结构模型，即，尽管补充精氨酸治疗，但它们不能有效地产生NO。在一项原理验证病例研究中，我们成功地治疗了一名阿萨患者，该患者患有多种抗高血压药物难治性重度高血压，并补充了NO。我们的数据表明，NO缺乏在组织水平有助于阿萨的并发症和预防高血氨单独可能无法防止长期的发病率。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：目的1。补充NO可以用于治疗阿萨患者的内皮功能障碍吗？我们将在阿萨患者中进行一项双盲、安慰剂对照、交叉研究。我们假设一部分阿萨患者由于血管内皮缺乏NOS依赖性NO产生而表现出内皮功能障碍，通过营养品Neo 40（R）补充NOS非依赖性NO来源（如低剂量亚硝酸钠）将纠正这种缺陷。目标二。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阿萨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响应于精氨酸补充的体外NO产生相关联。目标3。ASL/NOS复合物调节NO的机制基础是什么？NO的合成可能部分受NOS参与正、负调节复合物的调节。为了探索调节这些结构复合物的机制，我们将测试一氧化氮的产生是由涉及NOS和小窝蛋白的负调节复合物与涉及NOS和ASL的正合成复合物的平衡控制的假设。这些研究的见解可能为研究NO在其他疾病背景下的不同生物学效应提供了一个范例。虽然阿萨的发病机制是复杂的，并可能扩展到其他精氨酸依赖性机制，我们的临床前和临床研究支持NO缺乏对本研究中测试的终点的主要贡献。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的NO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.