Complex I and ATP synthase mediate membrane depolarization and matrix acidification by isoflurane in mitochondria

Abstract

Short application of the volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of the heart against injury. Mild depolarization and acidification of the mitochondrial matrix are involved in the protective mechanisms of isoflurane, but the molecular basis for these changes is not clear. In this study, mitochondrial respiration, membrane potential, matrix pH, matrix swelling, ATP synthesis and -hydrolysis, and H2O2 release were assessed in isolated mitochondria. We hypothesized that isoflurane induces mitochondrial depolarization and matrix acidification through direct action on both complex I and ATP synthase. With complex I-linked substrates, isoflurane (0.5 mM) inhibited mitochondrial respiration by 28±10%, and slightly, but significantly depolarized membrane potential and decreased matrix pH. With complex II- and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized mitochondrial membrane potential. Depolarization and matrix acidification were attenuated by inhibition of ATP synthase with oligomycin, but not by inhibition of mitochondrial ATP- and Ca2+-sensitive K+ channels or uncoupling proteins. Isoflurane did not induce matrix swelling and did not affect ATP synthesis and hydrolysis, but decreased H2O2 release in the presence of succinate in an oligomycin- and matrix pH-sensitive manner. Isoflurane modulated H+ flux through ATP synthase in an oligomycin-sensitive manner. Our results indicate that isoflurane-induced mitochondrial depolarization and acidification occur due to inhibition of the electron transport chain at the site of complex I and increased proton flux through ATP synthase. K+ channels and uncoupling proteins appear not to be involved in the direct effects of isoflurane on mitochondria.

Isoflurane modulates cardiac mitochondrial bioenergetics by selectively attenuating respiratory complexes

“…Compared to ISO, these uncoupling agents caused a pronounced state 2 depolarization (data not shown). In addition, matrix acidification and ΔΨ m depolarization are hallmarks of proton leak [41, 46], but we did not observe any change in matrix pH measured by BCECF fluorescence (data not shown).…”

“…These results extend previous findings [8, 23] that suggested that the prolonged duration of state 3 ADP phosphorylation can be attributed to slowed rates of electron transfer and proton pumping by NADH-linked intermediates; this is consistent with the increased durations of state 3 NADH, ΔΨ m , and respiration with the complex I substrate PM. A decrease in ΔΨ m can be caused by any protonophoric effect that would allow ADP-independent influx of protons into the matrix (proton leak) [40, 41], which would increase respiration to restore ΔΨ m . We did not observe any ISO-mediated change in matrix pH with BCECF-AM incubated mitochondria (data not shown), nor any increase in state 2 respiration to support a proton leak (Fig.…”

Anaesthetic Postconditioning at the Initiation of CPR Improves Myocardial and Mitochondrial Function in a Pig Model of Prolonged Untreated Ventricular Fibrillation

“…The solution contained experimental buffer, 0.2 μM diadenosine pentaphosphate, 30 μM ADP, 10 μg ml −1 mitochondria, 0.1 mg ml −1 luciferin, and 1.25 μg ml −1 luciferase. 15 Addition of pyruvate/malate or succinate (5 mM each) initiated the reaction that was measured for 120 sec. Defined ATP concentrations were used to obtain the standard curve.…”

Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia

“…We and others have shown that isoflurane inhibits the ETC at the level of complex I, which potentially may lead to a small rise in ROS production (13,24). In fact, a small burst of ROS as signaling molecules, originating from the mitochondria, was reported to be essential for triggering the Fig.…”