Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels

Abstract

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.