Heinecke J W, Rosen H, Suzuki L A, Chait A
J Biol Chem. 1987 Jul 25;262(21):10098-103.
Extracellular superoxide (O2-.) was detected in cultures of monkey arterial smooth muscle cells as measured by the superoxide dismutase-inhibitable reduction of cytochrome c and acetylated cytochrome c. Reduction of cytochrome c by these cells required L-cystine in the incubation medium. A variety of other sulfur-containing amino acids, including D-cystine, L-cystathionine, L-methionine, and djenkolic acid did not support O2-. generation when present at concentrations equimolar to L-cystine. At millimolar concentrations, the chelators EDTA and diethylene triamine penta-acetic acid inhibited O2-. production by smooth muscle cells. This effect was maximal when the chelator was present at the same concentration as the sum of the Ca2+ and Mg2+ in the medium, suggesting a role for these cations in O2-. generation by cells. Modification of low density lipoprotein (LDL) by arterial smooth muscle cells, as assessed by changes in lipid peroxide content, mobility on agarose gel electrophoresis, and apoprotein B fragmentation, was also L-cystine-dependent. LDL modification also required micromolar concentrations of the transition metal ion Cu(II) or Fe(III) and was inhibited by superoxide dismutase. LDL modified by smooth muscle cells in the presence of L-cystine and Cu(II) was taken up and degraded less well than native LDL by human skin fibroblasts, suggesting that recognition by the LDL receptor was lost. In contrast, LDL modified by smooth muscle cells was taken up and degraded to a greater degree than native LDL by mouse peritoneal macrophages, consistent with recognition by the scavenger receptor. These results indicate that monkey arterial smooth muscle cells produce O2-. and modify LDL by an L-cystine-dependent process. This may involve reduction of cystine to a thiol, possibly cysteine or a cysteine-containing peptide such as glutathione. Sulfur-containing amino acids may play a role in atherogenesis by supporting cell-mediated generation of reactive oxygen species and modification of lipoprotein to a form recognized by the scavenger receptor.
通过超氧化物歧化酶抑制的细胞色素c和乙酰化细胞色素c的还原作用，在猴动脉平滑肌细胞培养物中检测到细胞外超氧化物（O2-.）。这些细胞对细胞色素c的还原作用需要在孵育培养基中加入L-胱氨酸。当以与L-胱氨酸等摩尔浓度存在时，包括D-胱氨酸、L-胱硫醚、L-甲硫氨酸和豆薯酸在内的多种其他含硫氨基酸均不能支持O2-.的产生。在毫摩尔浓度下，螯合剂乙二胺四乙酸（EDTA）和二乙烯三胺五乙酸抑制平滑肌细胞产生O2-.。当螯合剂的浓度与培养基中Ca2+和Mg2+的总和相同时，这种作用最大，表明这些阳离子在细胞产生O2-.过程中起作用。通过脂质过氧化物含量、琼脂糖凝胶电泳迁移率和载脂蛋白B片段化的变化评估，动脉平滑肌细胞对低密度脂蛋白（LDL）的修饰作用也依赖于L-胱氨酸。LDL修饰还需要微摩尔浓度的过渡金属离子铜（II）或铁（III），并被超氧化物歧化酶抑制。在L-胱氨酸和铜（II）存在的情况下，经平滑肌细胞修饰的LDL被人皮肤成纤维细胞摄取和降解的能力不如天然LDL，这表明LDL受体识别能力丧失。相反，经平滑肌细胞修饰的LDL被小鼠腹腔巨噬细胞摄取和降解的程度比天然LDL更大，这与清道夫受体的识别作用一致。这些结果表明，猴动脉平滑肌细胞通过依赖L-胱氨酸的过程产生O2-.并修饰LDL。这可能涉及将胱氨酸还原为硫醇，可能是半胱氨酸或含半胱氨酸的肽如谷胱甘肽。含硫氨基酸可能通过支持细胞介导的活性氧生成以及将脂蛋白修饰为清道夫受体识别的形式，在动脉粥样硬化形成中发挥作用。
