Kaczara Patrycja, Motterlini Roberto, Rosen Gerald M, Augustynek Bartlomiej, Bednarczyk Piotr, Szewczyk Adam, Foresti Roberta, Chlopicki Stefan
Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow 30-348, Poland.
INSERM U955, Equipe 12, Créteil, 94000, France; University Paris-Est, Faculty of Medicine, Créteil, 94000, France.
Biochim Biophys Acta. 2015 Oct;1847(10):1297-309. doi: 10.1016/j.bbabio.2015.07.004. Epub 2015 Jul 14.
Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.
一氧化碳（CO）是血红素加氧酶降解血红素的产物，在血管稳态中起重要作用。最近的证据表明，线粒体是介导CO细胞作用的众多分子靶点之一。在本研究中，我们使用电子顺磁共振（EPR）血氧测定法和海马XF技术，表征了CORM - 401释放的CO对完整人内皮细胞线粒体呼吸和糖酵解的影响。我们发现，CORM - 401（10 - 100μM）可导致氧消耗率（OCR）持续增加，同时伴有糖酵解抑制（细胞外酸化率，ECAR）和ATP周转率降低。此外，CORM - 401增加了质子泄漏，降低了线粒体储备能力，并增强了非线粒体呼吸。无活性的CORM - 401（iCORM - 401）既不诱导线粒体解偶联，也不抑制糖酵解，这支持了CO在CORM - 401诱导的内皮代谢反应中的直接作用。有趣的是，用派迷西林阻断线粒体大电导钙调节钾离子通道（mitoBKCa）可消除CORM - 401促进的OCR增加，而不影响ECAR；膜片钳实验证实，CORM - 401衍生的CO激活了线粒体中存在的mitoBKCa通道。相反，MG132对糖酵解的稳定作用可防止CORM - 401介导的ECAR降低，但不改变OCR反应。总之，我们在完整的内皮细胞中证明，CO诱导了一种双组分代谢反应：依赖于mitoBKCa通道激活的线粒体呼吸解偶联和独立于mitoBKCa通道的糖酵解抑制。
