Shvedova A A, Tyurina Y Y, Gorbunov N V, Tyurin V A, Castranova V, Kommineni C, Ojimba J, Gandley R, McLaughlin M K, Kagan V E
Health Effects Laboratory Division, Pathology and Physiology Research Branch, NIOSH, Morgantown, WV 26505, USA.
Biochem Pharmacol. 1999 May 1;57(9):989-1001. doi: 10.1016/s0006-2952(99)00007-6.
The goal of the present work was to determine whether nitric oxide (NO) released from different donors (NONOates and nitrosothiols) can act as a protective antioxidant against oxidative stress and cytotoxicity induced by extracellular hemoglobin/tert-butyl hydroperoxide (Hb/tert-BuOOH) in vascular smooth muscle cells (VSMCs). No changes in phospholipid composition were found in VSMCs incubated with oxyhemoglobin (oxyHb)/tert-BuOOH. Using our newly developed HPLC-fluorescence technique for measurement of site-specific oxidative stress in membrane phospholipids, we produced VSMCs in which endogenous phospholipids were metabolically labeled with an oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. In these cells, we were able to reliably quantitate oxidative stress in major phospholipid classes-phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol-induced by tert-BuOOH in the presence of oxyHb or methemoglobin (metHb). The oxidative stress was accompanied by cytotoxic effects of oxyHb/tert-BuOOH and metHb/tert-BuOOH on VSMCs. We further found that an NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen 1-ium-1,2-diolate (PAPANONO), but not nitrosothiols, protected VSMCs against oxidative stress and cytotoxicity induced by Hb/tert-BuOOH. The protective effect of PAPANONO was most likely due to its ability to form NO-heme Hb (detectable by low temperature EPR spectroscopy and visible spectrophotometry). These findings are important for further understanding the physiological antioxidant role of NO against oxidative stress induced by hemoproteins as well as for pathological hypertensive events induced by extracellular Hb via NO depletion.
本研究的目的是确定不同供体（硝普钠和亚硝基硫醇）释放的一氧化氮（NO）是否可以作为一种保护性抗氧化剂，抵抗血管平滑肌细胞（VSMC）中细胞外血红蛋白/叔丁基过氧化氢（Hb/tert-BuOOH）诱导的氧化应激和细胞毒性。用氧合血红蛋白（oxyHb）/叔丁基过氧化氢孵育的VSMC中未发现磷脂组成有变化。我们使用新开发的HPLC-荧光技术测量膜磷脂中位点特异性氧化应激，制备了内源性磷脂用氧化敏感荧光脂肪酸顺式十八碳四烯酸进行代谢标记的VSMC。在这些细胞中，我们能够可靠地定量在氧合血红蛋白或高铁血红蛋白（metHb）存在下叔丁基过氧化氢诱导的主要磷脂类——磷脂酰乙醇胺、磷脂酰胆碱、磷脂酰丝氨酸和磷脂酰肌醇中的氧化应激。氧化应激伴随着oxyHb/叔丁基过氧化氢和metHb/叔丁基过氧化氢对VSMC的细胞毒性作用。我们进一步发现，一种NO供体，（Z）-1-[N-（3-氨丙基）-N-（正丙基）氨基]重氮-1,2-二醇盐（PAPANONO），而不是亚硝基硫醇，可保护VSMC免受Hb/叔丁基过氧化氢诱导的氧化应激和细胞毒性。PAPANONO的保护作用很可能是由于其形成NO-血红素Hb的能力（可通过低温电子顺磁共振光谱和可见分光光度法检测）。这些发现对于进一步理解NO对血红素蛋白诱导的氧化应激的生理抗氧化作用以及细胞外Hb通过NO消耗诱导的病理性高血压事件具有重要意义。
