Halothane, isoflurane and sevoflurane inhibit NADH:ubiquinone oxidoreductase (complex I) of cardiac mitochondria

Abstract

We have investigated the effects of volatile anaesthetics on electron transport chain activity in the mammalian heart. Halothane, isoflurane and sevoflurane reversibly increased NADH fluorescence (autofluorescence) in intact ventricular myocytes of guinea-pig, suggesting that NADH oxidation was impaired. Using pig heart submitochondrial particles we found that the anaesthetics dose-dependently inhibited NADH oxidation in the order: halothane > isoflurane = sevoflurane. Succinate oxidation was unaffected by either isoflurane or sevoflurane, indicating that these agents selectively inhibit complex I (NADH:ubiquinone oxidoreductase). In addition to inhibiting NADH oxidation, halothane also inhibited succinate oxidation (and succinate dehydrogenase), albeit to a lesser extent. To test the hypothesis that complex I is a target of volatile anaesthetics, we examined the effects of these agents on NADH:ubiquinone oxidoreductase (EC 1.6.99.3) activity using the ubiquinone analogue DBQ (decylubiquinone) as substrate. Halothane, isoflurane and sevoflurane dose-dependently inhibited NADH:DBQ oxidoreductase activity. Unlike the classical inhibitor rotenone, none of the anaesthetics completely inhibited enzyme activity at high concentration, suggesting that these agents bind weakly to the 'hydrophobic inhibitory site' of complex I. In conclusion, halothane, isoflurane and sevoflurane inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. At concentrations of approximately 2 MAC (minimal alveolar concentration), the activity of NADH:ubiquinone oxidoreductase was reduced by about 20 % in the presence of halothane or isoflurane, and by about 10 % in the presence of sevoflurane. These inhibitory effects are unlikely to compromise cardiac performance at usual clinical concentrations, but may contribute to the mechanism by which volatile anaesthetics induce pharmacological preconditioning.

Figure 1. Volatile anaesthetics increase NADH fluorescence of ventricular myocytes

A, effects of 1.5 mm halothane, B, 1.5 mm isoflurane or C, 1.5 mm sevoflurane on NADH fluorescence, measured in three different isolated ventricular myocytes. Rotenone (10 μm), a potent inhibitor of NADH:ubiquinone oxidoreductase, was used to obtain maximal NADH fluorescence.

Figure 2. Inhibition of NADH oxidation by volatile anaesthetics

Effects of halothane (•), isoflurane (□) and sevoflurane (▵) on NADH oxidation by pig heart submitochondrial particles.

Figure 3. Inhibition of NADH:DBQ oxidoreductase (complex I) activity

Effects of halothane (A), isoflurane (B) and sevoflurane (C) on NADH:DBQ oxidoreductase activity (NADH:DBQ activity) of bovine heart submitochondrial particles. DMSO, used to solubilise the anaesthetics, had no effect (not shown). Data were fitted by a first order exponential decay function:where y (%) is the enzyme activity in the absence of the drug, y₀ (%) is the enzyme activity in the presence of high concentrations of the drug, C (mm) is the drug concentration, and C_D (mm) is a constant describing the concentration dependence of the inhibitory effects of the drug. Although only the effects of anaesthetics in the range 0 to 2 mm are shown, all data, 11-15 measurements for each anaesthetic titration (0-10 mm), were used in the fitting procedure. The estimated concentrations of the anaesthetics corresponding to 2 MAC are indicated by arrows.

Figure 4. Electron transport chain inhibition by volatile anaesthetics

Schematic diagram of the mitochondrial sites of action of volatile anaesthetics. Halothane inhibits both NADH:ubiquinone (Q) oxidoreductase and succinate dehydrogenase. Isoflurane and sevoflurane selectively inhibit NADH:Q oxidoreductase. Nitrous oxide (N₂O) inhibits cytochrome c oxidase (complex IV).