title: 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.
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow 30-348, Poland. Electronic address: patrycja.kaczara@jcet.eu.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kaczara
authors.affiliation: INSERM U955, Equipe 12, Créteil, 94000, France; University Paris-Est, Faculty of Medicine, Créteil, 94000, France. Electronic address: roberto.motterlini@inserm.fr.
authors.forename: Roberto
authors.initials: R
authors.lastname: Motterlini
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA. Electronic address: grosen@umaryland.edu.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Rosen
authors.affiliation: Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland. Electronic address: b.augustynek@nencki.gov.pl.
authors.forename: Bartlomiej
authors.initials: B
authors.lastname: Augustynek
authors.affiliation: Department of Biophysics, Warsaw University of Life Sciences - SGGW, Warsaw 02-776, Poland. Electronic address: p.bednarczyk@nencki.gov.pl.
authors.forename: Piotr
authors.initials: P
authors.lastname: Bednarczyk
authors.affiliation: Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland. Electronic address: a.szewczyk@nencki.gov.pl.
authors.forename: Adam
authors.initials: A
authors.lastname: Szewczyk
authors.affiliation: INSERM U955, Equipe 12, Créteil, 94000, France; University Paris-Est, Faculty of Medicine, Créteil, 94000, France. Electronic address: roberta.foresti@inserm.fr.
authors.forename: Roberta
authors.initials: R
authors.lastname: Foresti
authors.affiliation: Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow 30-348, Poland. Electronic address: stefan.chlopicki@jcet.eu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Chlopicki
keywords: CO-RM; Carbon monoxide; Endothelium; Glycolysis; Mitochondrial BKCa channels; Oxidative phosphorylation; Respiration