Department of Biophysics, Redox Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Am J Physiol Heart Circ Physiol. 2011 Sep;301(3):H803-12. doi: 10.1152/ajpheart.00210.2011. Epub 2011 Jun 17.
S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, L-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas L-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.
巯基在细胞信号通路中关键蛋白的 S-亚硝基化被认为是一氧化氮（NO）依赖性控制血管（病理）生理学的重要因素。多种代谢酶既是 NO 的靶点，也是 S-亚硝基化的靶点，包括参与糖酵解和氧化磷酸化的酶。因此，了解这些代谢途径如何被 NO 依赖性机制整合非常重要。在这里，我们使用细胞外通量技术比较了 NO 和 S-亚硝基化对牛主动脉内皮细胞糖酵解和氧化磷酸化的影响，以确定共同和独特的调节点。使用 S-亚硝基-L-半胱氨酸（L-CysNO）作为细胞内 S-亚硝基化的起始化合物，因为它可以被转运到细胞内，并在体外导致稳定的 S-亚硝基化。将其作用与 NO 供体 DetaNONOate（DetaNO）进行比较。DetaNO 处理仅导致储备呼吸能力下降；然而，L-CysNO 以浓度依赖性方式损害了这一参数和基础呼吸。此外，DetaNO 刺激细胞外酸化率（ECAR），这是糖酵解的替代标志物，而 L-CysNO 在低浓度下刺激 ECAR，在高浓度下抑制 ECAR。此外，还确定了 NO 和 S-亚硝基化对代谢影响之间的时间关系，即 NO 导致线粒体功能迅速受损，最终被 S-亚硝基化依赖的过程所克服。综上所述，这些结果表明，严重的药理学硝化应激可能通过细胞内 S-亚硝基化和 NO 依赖性机制对代谢途径进行差异调节。此外，这些数据提供了关于 NO 和相关化合物在血管（病理）生理学中的作用的见解。
