Nuszkowski A, Gräbner R, Marsche G, Unbehaun A, Malle E, Heller R
Center of Vascular Biology and Medicine, Friedrich-Schiller-University of Jena, 99089 Erfurt, Germany.
J Biol Chem. 2001 Apr 27;276(17):14212-21. doi: 10.1074/jbc.M007659200. Epub 2001 Jan 22.
Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.
活化吞噬细胞的髓过氧化物酶/H₂O₂/卤化物系统产生的次氯酸/次氯酸盐，已被证明在体外可氧化/修饰低密度脂蛋白（LDL），并可能参与体内致动脉粥样硬化脂蛋白的形成。因此，在人类动脉粥样硬化病变中已检测到次氯酸盐修饰的（脂蛋白），它们与巨噬细胞和内皮细胞共定位。本研究调查了次氯酸盐修饰的LDL对内皮细胞合成一氧化氮（NO）的影响，通过在凝血酶或离子霉素刺激细胞后测量瓜氨酸（NO的副产物）和cGMP（NO激活的可溶性鸟苷酸环化酶的产物）的形成来评估。与用天然LDL预孵育细胞相比，用次氯酸盐修饰的LDL预处理人脐静脉内皮细胞导致激动剂诱导的瓜氨酸和cGMP合成出现时间和浓度依赖性抑制。这种抑制既不是由于内皮型一氧化氮合酶（eNOS）表达降低，也不是由于其辅因子四氢生物蝶呤缺乏。同样，eNOS的底物L-精氨酸进入细胞的过程也未受影响。次氯酸盐修饰的LDL引起细胞内eNOS分布的显著变化，包括从质膜易位和高尔基体定位的解体，而不改变该酶的肉豆蔻酰化或棕榈酰化。相比之下，已知可耗尽质膜胆固醇并破坏小窝的环糊精仅导致eNOS从质膜消失，这与激动剂诱导的瓜氨酸和cGMP形成减少无关。本研究结果表明，NOS的错误定位是次氯酸盐修饰的LDL处理的人脐静脉内皮细胞中NO生成减少的原因，并指出高尔基体定位的NOS在这些过程中的重要作用。我们得出结论，次氯酸盐修饰的LDL对NO合成的抑制可能是内皮功能障碍发展和动脉粥样硬化早期发病机制中的一个重要机制。
