Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells
Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells
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DOI：
10.1152/ajpheart.00196.2002
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发表时间：
2002-11-01
影响因子：
4.8
通讯作者：
Goligorsky, MS
中科院分区：
文献类型：
--
作者：
Brodsky, SV;Gao, SJ;Goligorsky, MS
累积的超微结构和生化证据高度提示线粒体一氧化氮（NO）合酶（mtNOS）的存在，其中NO的局部产生调节电子传递沿着呼吸链。在这里，在活细胞中原位mtNOS的功能能力进行了检查，使用活体荧光NO指示剂，4，5-二氨基荧光素，采用一种新的程序，用于将其加载到线粒体中，以证明在未破坏的内皮细胞和分离的线粒体以及稳定表达内皮NOS的人胚肾细胞中的局部NO生成。使用这种方法，我们表明，在高浓度的D-葡萄糖（但不是L-葡萄糖）的存在下孵育的内皮细胞的特点是减少NO合成功能的线粒体，尽管酶的丰度不变。与此同时，在高浓度D-葡萄糖存在下培养的内皮细胞中，线粒体产生的超氧化物增加。通过用细胞可渗透的超氧化物歧化酶模拟物处理细胞，高血糖环境中NO的产生和超氧化物的产生都可以恢复到控制水平。此外，增强的线粒体超氧化物的产生可以抑制与NOS抑制剂在刺激的内皮细胞。总之，数据1）提供了内皮细胞中线粒体NO产生的直接证据，2）证明了其在高血糖环境中的抑制和增强的超氧化物产生，以及3）提供了“解偶联”mtNOS代表在含高葡萄糖的培养基中孵育的内皮细胞中超氧阴离子的重要来源的证据。
The accumulated ultrastructural and biochemical evidence is highly suggestive of the existence of mitochondrial nitric oxide (NO) synthase (mtNOS), where local production of NO regulates the electron transport along the respiratory chain. Here, the functional competence of mtNOS in situ in a living cell was examined using an intravital fluorescent NO indicator, 4,5-diaminofluorescein, employing a new procedure for loading it into the mitochondria to demonstrate local NO generation in undisrupted endothelial cells and in isolated mitochondria as well as in human embryonic kidney cells stably expressing endothelial NOS. With the use of this approach, we showed that endothelial cells incubated in the presence of high concentration of D-glucose (but not L-glucose) are characterized by the reduced NO synthetic function of mitochondria despite the unaltered abundance of the enzyme. In parallel, mitochondrial generation of superoxide was augmented in endothelial cells incubated in the presence of a high concentration of D-glucose. Both the NO generation and superoxide production in hyperglycemic environment could be restored to control levels by treating cells with a cell-permeable superoxide dismutase mimetic. In addition, enhanced mitochondrial superoxide production could be suppressed with an inhibitor of NOS in stimulated endothelial cells. In conclusion, the data 1) provide direct evidence of mitochondrial NO production in endothelial cells, 2) demonstrate its suppression and enhanced superoxide generation in hyperglycemic environment, and 3) provide evidence that "uncoupled" mtNOS represents an important source of superoxide anions in endothelial cells incubated in high glucose-containing medium.