Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle.
Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle.
复制标题
DOI：
10.1152/ajplung.1994.267.6.l815
复制
发表时间：
1994-12
期刊：
影响因子：
--
通讯作者：
K. Mohazzab;M. Wolin
中科院分区：
文献类型：
--
作者：
K. Mohazzab;M. Wolin
本研究通过测量由O2-反应产生的化学发光，研究了犊牛肺动脉平滑肌匀浆和亚细胞组分中超氧阴离子（O2-）产生的来源。因为最近的证据表明内源性产生的活性氧似乎介导了某些血管反应。在匀浆组分中，NADH （0.1 mM）依赖性氧化还原酶活性是化学发光的主要检测来源。NADPH （0.1 mM）仅产生3%的O2-。NADH观察。O2-的某些其他潜在来源的定量。（优化条件下），包括黄嘌呤氧化酶（0.1 mM次黄嘌呤），线粒体（5 mM琥珀酸+ 30微米抗霉素），环加氧酶/脂加氧酶（1微米花生四烯酸+ 0.1 mM NADPH），或自氧化（0.1 mg/ml超氧化物歧化酶），导致检测到微量的化学发光（< 3%的NADH）。线粒体O2-的测定。从组织呼吸速率的生产表明，lucigenin是一个很差的检测线粒体内O2-。这些观察结果通过检查亚细胞组分产生的化学发光得到证实，其中检测到的主要活性是NADH氧化还原酶，其分离方式与微粒体标记酶鱼藤酮不敏感NADH细胞色素c还原酶的活性密切匹配。因为这种NADH氧化还原酶似乎是血管平滑肌来源的O2-的主要来源。因此，该系统有可能成为生成血管活性活性氧的重要内源。
Sources of superoxide anion (O2-.) production in calf pulmonary artery smooth muscle homogenate and subcellular fractions were examined in this study by measurement of the chemiluminescence produced by the reaction of O2-. with 50 microM lucigenin, because recent evidence suggests that endogenously produced reactive O2 species appear to mediate certain vascular responses. In the homogenate fraction, an NADH (0.1 mM)-dependent oxidoreductase activity was the major detected source of chemiluminescence. NADPH (0.1 mM) produced only 3% of the O2-. observed with NADH. Quantitation of certain other potential sources of O2-. (under optimized conditions), including xanthine oxidase (0.1 mM hypoxanthine), mitochondria (5 mM succinate + 30 microM antimycin), cyclooxygenase/lipoxygenase (1 microM arachidonic acid + 0.1 mM NADPH), or autooxidation (0.1 mg/ml superoxide dismutase), resulted in the detection of minimal amounts (< 3% of NADH) of chemiluminescence. Estimation of mitochondrial O2-. production from tissue respiration rates suggests that lucigenin is a poor detector of intramitochondrial O2-.. These observations were confirmed by examination of chemiluminescence produced by subcellular fractions, where the major activity detected was an NADH oxidoreductase, which fractionated in a manner closely matching the activity of the microsomal marker enzyme rotenone-insensitive NADH-cytochrome c reductase. Because this NADH oxidoreductase appears to be a major vascular smooth muscle-derived source of O2-. production, this system has the potential to be an important endogenous source for the generation of vasoactive reactive O2 species.