Abstract：
Persistent pulmonary hypertension of the newborn (PPHN) is a hypoxic disorder of pulmonary vascular relaxation, mediated in part by adenylyl cyclase (AC). Neonatal pulmonary arteries (PA) express mainly AC6 isoform, followed by AC3, 7 and 9. AC6 expression is upregulated in hypoxia. We reported AC enzyme inhibition due to S-nitrosylation in PPHN PA, and in PA myocytes exposed to hypoxia. We hypothesize that hypoxia promotes cysteine thiol nitrosylation of AC6, impairing cAMP production. HEK293T cells stably expressing AC isoforms (AC3, 5, 6, 7, 9), or cysteine-to-alanine mutants AC6_C1004A, AC6_C1145A or AC6_C447A were cultured in normoxia (21% O2) or hypoxia (10% O2) for 72 hours, or challenged with nitroso donor S-nitrosocysteine (CysNO). AC activity was determined by real-time live-cell cAMP measurement (cADDis assay) or terbium-norfloxacin AC catalytic assay, with or without challenge by allosteric agonist forskolin; protein S-nitrosylation detected by biotin switch method and quantified by affinity precipitation. Only AC6 catalytic activity is inhibited in hypoxia or by S-nitrosylating agent, in presence or absence of forskolin; impaired cAMP production in hypoxia correlates with increased cysteine nitrosylation of AC6. Selective AC6 inhibition in pulmonary artery myocytes extinguishes AC sensitivity to inhibition by hypoxia. Alanine substitution of C1004, but not of other cysteines, decreases S-nitrosylation of AC6. AC activity is diminished in AC6_C1004A compared to AC6 wild type. Substitution of C1004 also extinguishes the inhibition of AC6 by hypoxia. We conclude AC6 is uniquely S-nitrosylated in hypoxia, inhibiting its activity and cAMP generation. We speculate that S-nitrosylation at C1004 may inhibit AC6 interaction with Gαs, playing a role in PPHN pathophysiology.
摘要：
新生儿持续性肺动脉高压(PPHN)是一种肺血管舒张的缺氧性疾病,部分由腺苷酸环化酶（AC）介导。新生儿肺动脉（PA）主要表达AC6亚型，其次是AC3、7和9。AC6表达在缺氧中上调。我们报道了由于PPHNPA中S-亚硝基化引起的AC酶抑制，和暴露于缺氧的PA心肌细胞。我们假设缺氧会促进AC6的半胱氨酸硫醇亚硝基化，从而损害cAMP的产生。HEK293T细胞稳定表达AC亚型（AC3，5，6，7，9），或半胱氨酸到丙氨酸突变体AC6_C1004A，AC6_C1145A或AC6_C447A在常氧(21%O2)或低氧(10%O2)条件下培养72小时,或用亚硝基供体S-亚硝基半胱氨酸（CysNO）攻击。通过实时活细胞cAMP测量（cADDis测定）或tr-诺氟沙星AC催化测定确定AC活性，有或没有变构激动剂福司可林的攻击;蛋白质S-亚硝基化通过生物素转换方法检测并通过亲和沉淀定量。只有AC6的催化活性在缺氧或S-亚硝基化剂抑制，在存在或不存在毛喉素的情况下;缺氧时cAMP产生受损与AC6的半胱氨酸亚硝基化增加相关。肺动脉心肌细胞中的选择性AC6抑制消除了AC对缺氧抑制的敏感性。丙氨酸取代C1004，但不取代其他半胱氨酸，降低AC6的S-亚硝基化。与AC6野生型相比,AC6_C1004A中的AC活性降低。C1004的取代也消除了缺氧对AC6的抑制。我们得出结论，AC6在缺氧时具有独特的S-亚硝基化，抑制其活性和cAMP生成。我们推测，在C1004的S-亚硝基化可能抑制AC6与Gαs的相互作用，在PPHN病理生理学中发挥作用。
