Complex I and F0F1-ATP Synthase Mediate Membrane Depolarization and Matrix Acidification by Isoflurane in Mitochondria

Abstract

Introduction: Short application of volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of heart and cardiac mitochondria against injury. Mild depolarization and acidification of mitochondrial matrix are involved in the protective mechanism, but the molecular basis for these changes is not known. In this study we investigated the electron transport chain, F0F1-ATP synthase and mitochondrial ion channels as potential targets of isoflurane in mitochondria.Methods: We have measured mitochondrial respiration, membrane potential, matrix pH, matrix swelling, and H2O2 release in isolated mitochondria in the presence and absence of isoflurane (0.5 mM). Pyruvate/malate, succinate/rotenone, or ascorbate/TMPD, were used as substrates for complex I, II and IV, respectively. Guanosine-diphosphate (GDP), oligomycin, paxilline and 5-hydroxydecanoic acid (5-HD) were used to probe involvement of uncoupling proteins, F0F1-ATP synthase, mitochondrial ATP- and Ca2+-sensitive K+ channel. Nigericin, a K+/H+ exchanger, was used to manipulate the matrix pH.Results: With pyruvate/malate as substrates, isoflurane inhibited mitochondrial respiration by 23±4%, depolarized membrane potential by 2.7±0.7% and decreased matrix pH by 11±3%. With complex II and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized ΔΨm. Depolarization and matrix acidification were only attenuated by oligomycin, but not GDP, paxilline, or 5-HD. Isoflurane did not induce matrix swelling, but decreased H2O2 release in the presence of succinate in an oligomycin and matrix pH sensitive manner.Conclusion: Our results indicate that isoflurane inhibited the electron transport chain at the site of complex I and also modified F0F1-ATP synthase. Both effects lead to an acidification of the mitochondrial matrix which is beneficial at the time of reperfusion. K+ channels and uncoupling proteins are likely not involved in these direct effects of isoflurane on isolated mitochondria.