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. 2002 Sep 6;544(Pt 3):687–693. doi: 10.1113/jphysiol.2002.025015

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

Peter J Hanley *, John Ray *, Ulrich Brandt *, Jürgen Daut *
PMCID: PMC2290615  PMID: 12411515

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 ≈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.

Depression of cardiac function is the most important side-effect of commonly used volatile anaesthetics such as halothane, isoflurane and sevoflurane. In an elaborate review, Rusy & Komai (1987) discussed three major mechanisms which could be responsible for the negative inotropic action of volatile anaesthetics: (i) a reduction in Ca2+ availability, (ii) a decrease in responsiveness of the contractile proteins to Ca2+, and (iii) inhibition of mitochondrial function. Since that time, studies using intact cardiac muscle have convincingly shown that the volatile anaesthetics halothane, isoflurane and sevoflurane depress contractility by decreasing both Ca2+ availability and the responsiveness of the contractile proteins to Ca2+ (Hanley & Loiselle, 1998; Jiang & Julian, 1998a, b; Harrison et al. 1999; Davies et al. 2000; Housmans et al. 2000; Hannon et al. 2001). These inhibitory actions decrease the energy expenditure of the heart via the accompanying reduction in the activity of the major cytosolic energy consumers actomyosin-ATPase and Ca2+-ATPase (Schramm et al. 1994). Whether volatile anaesthetics also decrease energy supply by inhibiting mitochondrial ATP synthesis remains controversial and only modest progress has been made in elucidating this question.

From previous work with isolated mitochondrial preparations, halothane has been deduced to inhibit the electron transport chain at complex I (Hall et al. 1973; Merin et al. 1973; Rusy & Komai, 1987). Consistent with inhibition of complex I, an increase in NADH fluorescence evoked by halothane, as well as by isoflurane, has been observed in isolated ventricular trabeculae (Hanley & Loiselle, 1998) and isolated, perfused hearts (Kissin et al. 1983). It is therefore tempting to speculate that NADH: ubiquinone oxidoreductase (complex I) may be a common target of volatile anaesthetics such as halothane, isoflurane and sevoflurane. We tested this hypothesis using intact isolated cardiomyocytes and submitochondrial particles.

Methods

Isolation of cardiac ventricular myocytes

Myocytes were isolated as previously described (Ray et al. 2002), and the experiments were performed in accordance with the animal welfare guidelines at the Regierungspräsidium Giessen. In brief, guinea-pigs, weighing 300-350 g, were anaesthetized with 3-4 % isoflurane in oxygen and decapitated. Isolated hearts were attached to a cannula via the aorta and perfused for 5 min with physiological salt solution (PSS) containing (mm): 115 NaCl, 5.4 KCl, 1.5 MgCl2, 0.5 NaH2PO4, 5 Hepes, 16 taurine, 5 sodium pyruvate, 15 NaHCO3, 1 CaCl2 and 5 glucose (pH 7.4). Subsequently, the heart was perfused for 4-5 min with nominally Ca2+-free solution, followed by low Ca2+ solution containing 0.6 mg ml−1 (≈180 U ml−1) collagenase type I (Sigma), 0.1 % bovine serum albumin and 40-60 μm Ca2+. After enzymatic digestion (5-7 min), ventricular myocytes were dissociated by trituration with a wide-bore pipette in a ‘recovery’ solution containing (mm): 45 KCl, 70 potassium glutamate, 3 MgSO4, 15 KH2PO4, 16 taurine, 10 Hepes, 0.5 EGTA and 10 glucose (pH 7.4). After 60 min incubation in the recovery solution, myocytes were resuspended in Dulbecco's Modified Eagle's Medium (Gibco BRL).

NADH fluorescence

Myocytes were placed in a Perspex bath (volume, 100 μl) located on the stage of an inverted microscope (Diaphot 300, Nikon) and superfused via gravity flow (≈1 ml min−1), or, during application of volatile anaesthetics, via a syringe pump (1 ml min−1). The volatile anaesthetics were prepared at final concentrations in PSS. The syringe pump (with a glass barrel) was connected to the bath via stainless-steel tubing. The time constant of solution washout, determined by measuring the decay of tetramethylrhodamine ethyl ester (TMRE) fluorescence, was 7.2 s. The bath was placed on an electrically heated aluminium plate, which, in turn, was attached to a Perspex microscope stage insert. The temperature of the metal plate was monitored via an embedded thermistor and was maintained at 37 °C using a feedback circuit (TC-324A heater controller, Warner Instrument Corp., Hamden, CT, USA). Bath temperature was continuously monitored via a second thermistor. Immediately prior to entering the bath, solutions passed through a heat exchanger. The oil-immersion objective was heated to 37.5-38 °C with a brass water jacket.

Myocytes were excited at 350 nm and NADH fluorescence detected at 450 nm (bandwidth, 65 nm) using a Deltascan 4000 fluorescence system (Photon Technology International, Photomed, Seefeld, Germany). Note that the reduced form (NADH), but not the oxidised form (NAD+), of the redox couple NAD+/NADH is fluorescent. At the end of experiments, maximal NADH fluorescence was obtained by addition of the selective NADH: ubiquinone oxidoreductase inhibitor rotenone (10 μm).

Electron transport chain activity

Submitochondrial particles (SMP; disrupted mitochondria) were isolated from pig hearts (Clark & Switzer, 1977), freshly obtained from an abattoir. In brief, minced ventricular tissue (including remnants of the atria) was washed twice in a solution containing 10 mm potassium phosphate buffer and 1 mm EDTA (pH 7.4), drained via cheesecloth and then homogenised in a cold room (4 °C) in a solution containing 30 mm potassium phosphate and 1 mm EDTA (pH 7.4). The homogenate was then centrifuged at 1076 g (3000 r.p.m.) for 15 min using a cooled (4 °C) Sorvall centrifuge with a SS34 rotor. The supernatant (first supernatant) was stored on ice and the precipitate was homogenised and centrifuged as previously, yielding a second supernatant. The first and second supernatants were combined and the pH was reduced to 6.6 by adding acetic acid. The combined supernatants were centrifuged for 15 min at 7649 g (8000 r.p.m.). The precipitate was washed once in ice water and then centrifuged for 5 min at 2988 g (5000 r.p.m.). The pellet was resuspended in a solution containing 30 mm potassium phosphate buffer and 0.1 mm EDTA (pH 7.4), and aliquots were stored at -25 °C. Protein concentration was determined by the Lowry-Folin method.

An aliquot of 100 μg of the submitochondrial particles was suspended in phosphate buffer solution, containing (mm): 138 NaCl, 2.7 KCl and 10 phosphate buffer (pH 7.4). The rate of NADH oxidation was assessed by adding 800 μm NADH (substrate) and using oxygen as an electron acceptor. A custom-made Clark-type oxygen electrode was used to measure the rate of oxygen consumption. The electrode protruded into a temperature-controlled (25 °C) glass chamber (volume ≈0.6 ml) which had a neck of 1 mm diameter and contained a magnetic stirrer. Succinate oxidation was determined after adding 5 mm succinate. In selected experiments, the succinate dehydrogenase activity of complex II was measured by monitoring 2,6-dichlorophenol-indophenol (DCIP) reduction at 578 nm in the presence of 5 mm sodium cyanide and 1 μg antimycin A (mg protein)−1.

Volatile anaesthetics were added from near saturating concentrations in physiological NaCl solution (McKenzie et al. 1995): halothane (13.6 mm), isoflurane (14.2 mm) and sevoflurane (12.7 mm). The stock solutions were prepared by injecting the anaesthetic into a gas-tight syringe (Aesculap, Germany) containing 10 ml of a solution composed of (mm) 138 NaCl, 2.7 KCl and 10 phosphate buffer (pH 7.4). The solution in the sealed syringe was mixed for 15-20 min via a magnetic stirrer.

NADH:ubiquinone oxidoreductase activity

NADH:ubiquinone oxidoreductase activity was determined essentially as previously described (Okun et al. 1999). In brief, NADH:ubiquinone oxidoreductase activity of bovine heart submitochondrial particles, isolated as previously described (Smith, 1967; Thierbach & Reichenbach, 1981; Okun et al. 1999), was determined using the ubiquinone analogue decylubiquinone (DBQ) as substrate (Lenaz, 1998). Steady-state enzyme activity was measured by monitoring NADH oxidation at 340-400 nm (ɛ340-400 nm = 6.10 mm−1 ×cm−1) using a Shimadzu UV-300 spectrophotometer. NADH (100 μm) and 50 μg of SMP were added to buffer solution containing 50 mm Tris HCl (pH 7.4), 5 μm Kresoxim-Methyl Brio and 2 mm KCN. Before addition of DBQ, anaesthetics (dissolved in DMSO) were added via a Hamilton gas-tight (1700-series) syringe combined with a PB-600 mechanical dispenser. DMSO did not inhibit NADH:ubiquinone oxidoreductase activity. The reaction was initiated by addition of 60 μm DBQ. The final volume of the reaction mixture was 1 ml. Experiments were performed at 30 °C.

Volatile anaesthetics and minimal alveolar concentration

Anaesthetic concentrations are expressed in millimoles per litre and where appropriate, equivalent MAC (minimal alveolar concentration) values were estimated. MAC expressed as volume percentage (or percentage atmosphere) varies considerably with temperature whereas the equivalent liquid-phase concentration, expressed in millimoles per litre, changes little (Franks & Lieb, 1996). The equivalent MAC values for our experiments were calculated from the concentration of the anaesthetic and the conversion factors reported for halothane (1 MAC, 0.27 mm), isoflurane (1 MAC, 0.31 mm) and sevoflurane (1 MAC, 0.35 mm) in rat at 37 °C (Franks & Lieb, 1996).

Gas chromatography

Anaesthetic concentrations were determined using a gas chromatograph (Carlo Erba, Milan, Italy) equipped with a flame ionisation detector (FID). The carrier gas was hydrogen (60 kPa column head pressure) and the fused silica capillary column, coated with polysiloxane SE-30, was 25 m × 0.25 mm. Injector temperature was 250 °C, FID temperature was 300 °C and the oven was maintained at 90 °C. A mixture of halothane, isoflurane and sevoflurane in DMSO was used as internal standard.

Statistics

Results were analysed by two-way analysis of variance. Differences among means were tested for statistical significance (P < 0.05). Data are expressed as means ± s.e.m.

Results

NADH fluorescence measurements in cardiac myocytes

We tested whether inhibition of complex I can be observed in intact cardiac ventricular myocytes. Inhibition of complex I would be expected to cause a decrease in the NAD+/NADH ratio. Consistent with inhibition of complex I, Fig. 1 shows that the volatile anaesthetics induced a reversible increase in NADH fluorescence. To scale these effects, myocytes were exposed to the specific complex I inhibitor rotenone, which elicited maximal NADH fluorescence. The inhibitory action of rotenone was not reversible. On average, halothane (1.5 mm, ≈5.6 MAC), isoflurane (1.5 mm, ≈4.8 MAC) and sevoflurane (1.5 mm, ≈4.3 MAC) increased NADH fluorescence to 31.7 ± 7.8 % (n = 4), 25.1 ± 6.0 % (n = 4) and 14.3 ± 2.3 % (n = 4) of maximum, respectively. Following washout of each volatile anaesthetic, a small transient increase in NADH fluorescence was observed. The cause of this secondary effect is unclear.

Figure 1. Volatile anaesthetics increase NADH fluorescence of ventricular myocytes.

Figure 1

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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.

Oxidation of NADH and succinate by submitochondrial particles

The increase in NADH fluorescence seen in myocytes suggests that volatile anaesthetics impair mitochondrial NADH oxidation. Using pig heart submitochondrial particles, we tested the effects of the volatile anaesthetics on both NADH and succinate oxidation. Halothane, isoflurane and sevoflurane inhibited the rate of NADH oxidation in a concentration-dependent fashion (Fig. 2). Halothane was the most potent inhibitor. At a concentration of 0.9 mm (≈3 MAC), halothane decreased the rate of NADH oxidation to 67.5 ± 1.0 % (n = 5) of control. At the highest concentration tested (1.8 mm), halothane decreased the rate of NADH oxidation to 34.7 ± 1.8 % (n = 5) of control. Isoflurane and sevoflurane had qualitatively similar, but smaller effects on complex I (Fig. 2). Isoflurane (1 mm, ≈3.2 MAC) decreased complex I activity to 81.1 ± 3.8 % (n = 5) of control; sevoflurane (0.9 mm, ≈2.6 MAC) decreased enzyme activity to 84.8 ± 5.8 % (n = 5) of control. The effects of isoflurane and sevoflurane were not significantly different.

Figure 2. Inhibition of NADH oxidation by volatile anaesthetics.

Figure 2

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

When succinate was used as substrate, neither isoflurane (1.9 mm) nor sevoflurane (1.7 mm) decreased the rate of oxygen consumption of the submitochondrial particles (not shown). Hence, we can infer that isoflurane and sevoflurane do not inhibit complex II, III or IV. However, halothane (1.8 mm) decreased the rate of oxygen consumption to 77.3 ± 3.1 % of control (n = 4) when succinate was used as substrate (not shown). In order to differentiate between inhibition at complex II versus complexes III and IV, we examined the effect of halothane (1.8 mm) on the activity of succinate dehydrogenase, the catalytic component of complex II. Halothane inhibited the activity of succinate dehydrogenase, assessed using DCIP, by a comparable extent (79.7 ± 4.0 % of control; n = 4; P > 0.05). Thus, with succinate as substrate, the inhibitory effect of halothane on oxygen consumption rate can be accounted for by succinate dehydrogenase (complex II) inhibition.

Measurement of NADH:ubiquinone oxidoreductase (complex I) activity

The ability of volatile anaesthetics to reduce the rate of NADH oxidation by submitochondrial particles suggested that NADH:ubiquinone oxidoreductase is inhibited. We therefore examined the effects of the volatile anaesthetics on NADH:ubiquinone oxidoreductase activity using DBQ as substrate. After making exploratory measurements over a wide range of concentrations to estimate the range in which the anaesthetics affected complex I activity, an incremental titration of 11-15 measurements was performed in the range 0 to 10 mm for each anaesthetic. Figure 3 shows the effects of the anaesthetics in the range of concentrations used clinically (0-2 mm). Halothane decreased NADH:DBQ oxidoreductase activity in a dose-dependent fashion (Fig. 3A). At much higher halothane concentrations enzyme activity was not completely inhibited. For example, at the highest concentration tested (10 mm), activity was reduced to ≈34 % of control (not shown).

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

Figure 3

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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:
graphic file with name tjp0544-0687-mu1.jpg
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 CD (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.

Similar to halothane, isoflurane (Fig. 3B) and sevoflurane (Fig. 3C) also inhibited NADH:DBQ oxidoreductase activity, albeit less potently. At very high concentrations (10 mm), isoflurane and sevoflurane decreased enzyme activity to, respectively, ≈56 and ≈75 % of control (not shown). Hence, none of the anaesthetics completely inhibited enzyme activity at very high doses.

Discussion

The mechanism underlying the anaesthetic-induced increase in NADH fluorescence

We have examined the effects of halothane, isoflurane and sevoflurane on electron transport chain activity in the heart and tested the hypothesis that these agents inhibit complex I (NADH:ubiquinone oxidoreductase). As far as we are aware, there are no previous studies examining the effects of isoflurane and sevoflurane on either NADH oxidation or NADH:ubiquinone oxidoreductase activity. Consistent with complex I inhibition, we found that all three volatile anaesthetics reversibly increase NADH fluorescence in cardiomyocytes. These results corroborate and extend previous reports showing that inhalational anaesthetics increase NADH fluorescence (alternatively referred to as NADH autofluorescence) in isolated perfused hearts or in cardiac ventricular trabeculae (Kissin et al. 1983; Jiang & Julian, 1997; Hanley & Loiselle, 1998; Riess et al. 2002). We have elucidated the underlying cause of the increase in NADH fluorescence using two approaches. First, we found that halothane, isoflurane and sevoflurane inhibit NADH oxidation in pig heart submitochondrial particles. Second, we demonstrated that volatile anaesthetics inhibit NADH:DBQ oxidoreductase activity of bovine submitochondrial particles.

At clinical concentrations (<1 mm), NADH:DBQ oxidoreductase activity was more sensitive to inhibition by the volatile anaesthetics than NADH oxidation. This difference suggests that inhibition of complex I activity may be underestimated by measurements of oxygen consumption of submitochondrial particles. Since DMSO did not inhibit NADH:DBQ oxidoreductase activity, we were able to examine the effects of high concentrations of volatile anaesthetics. At concentrations as high as 10 mm, complex I activity was reduced to 34, 56 and 75 % of control by, respectively, halothane, isoflurane and sevoflurane. These results suggest that volatile anaesthetics bind weakly to the ‘hydrophobic inhibitory site’ (Degli Esposti, 1998; Okun et al. 1999) of complex I. Succinate oxidation was not affected by isoflurane or sevoflurane suggesting that complexes II, III and IV were not inhibited. However, halothane was found to inhibit both complex I and succinate dehydrogenase, the catalytic component of complex II, as shown schematically in Fig. 4.

Figure 4. Electron transport chain inhibition by volatile anaesthetics.

Figure 4

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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).

Functional implications

The NADH:ubiquinone oxidoreductase is the first stage of proton and electron donation for NADH (Fig. 4) and thus is a potential source of electron leakage with the formation of free radicals (Turrens & Boveris, 1980). Recently, volatile anaesthetics have been suggested to induce pharmacological preconditioning (i.e. to protect the heart against subsequent ischaemic damage) via the production of reactive oxygen species (ROS; Müllenheim et al. 2002; Novalija et al. 2002; Tanaka et al. 2002). Rotenone (and other complex I inhibitors) has been shown to increase the rate of production of ROS by cardiac submitochondrial particles (Hasegawa et al. 1990; Ide et al. 1999). Furthermore, the K+ channel openers diazoxide and pinacidil, which have been widely used for pharmacological preconditioning, have also been shown to inhibit components of the respiratory chain (Hanley et al. 2002). Taken together, these findings suggest that partial inhibition of the electron transport chain may be a common mechanism by which various drugs, including volatile anaesthetics and diazoxide, induce preconditioning in mammalian heart.

We found that halothane, isoflurane and sevoflurane dose dependently inhibit NADH:DBQ oxidoreductase activity. At a concentration of ≈2 MAC (see Fig. 3), enzyme activity was reduced by about 20 % in the presence of halothane or isoflurane and by about 10 % in the presence of sevoflurane. Since volatile anaesthetics are usually employed in combination with ≈0.7 MAC nitrous oxide (N2O), a concentration of ≥ 2 MAC is seldom attained for a single agent; the majority of anaesthetics are administered using concentrations of < 1.5 MAC. Volatile anaesthetic concentrations ≥ 2 MAC are, nevertheless, delivered for brief periods during inhalational induction (Kwek & Ng, 1997; Epstein et al. 1998), a technique commonly employed for children.

Interestingly, nitrous oxide has been reported to decrease the rate of succinate and cytochrome c oxidation by bovine heart submitochondrial particles (Sowa et al. 1987), suggesting that it inhibits cytochrome c oxidase (complex IV of the respiratory chain; see Fig. 4). Indeed, N2O (≤ 100 %; equivalent to ≤ 1 MAC at 37 °C) was found to inhibit cytochrome c oxidase by up to 60 %, depending on the cytochrome c concentration (Einarsdóttir & Caughey, 1988; Chervin & Thibaud, 1992). Thus, we estimate that inhibition of the respiratory chain by 20-25 % could occur when inhalational agents (including N2O) at ≥ 2 MAC are used to induce or maintain surgical anaesthesia.

Despite its inhibitory effect on the electron transport chain, halothane (1.5 %, corresponding to ≈2 MAC) was found to produce no significant change in the steady-state concentrations of phosphocreatine or ATP in isolated perfused rabbit hearts, either paced (Murray et al. 1987) or spontaneously beating (McAuliffe & Hickey, 1987). However, under these in vitro experimental conditions, halothane decreased the rate pressure product by about 50 %, and this negative inotropic effect, which decreases energy demand, should have counterbalanced the partial inhibition of the respiratory chain. The situation is quite different in vivo, where negative inotropic effects are (at least partially) compensated by an increased activation of the sympathetic nervous system. During application of volatile anaesthetics at a total concentration of ≥ 2 MAC, the combination of negative inotropy and partial inhibition of the respiratory chain is expected to decrease cardiac reserve (the maximum percentage that cardiac output can increase above normal). This decrease in cardiac reserve is probably not critical for most patients undergoing general anaesthesia, and the major factor responsible for this reduction of maximum cardiac output is the negative inotropic effect of volatile anaesthetics. Nevertheless, inhibition of the respiratory chain (energy supply) by volatile anaesthetics may limit cardiac output under pathophysiological conditions, for example, during strong stimulation of the sympathetic nervous system (which decreases the efficiency of cardiac energy transduction; see Graham et al. 1968; Gibbs, 1982), during recovery of the myocardium from ischaemic episodes, or in patients with heart disease (Montgomery, 2000).

In conclusion, we have shown that halothane, isoflurane and sevoflurane inhibit NADH:ubiquinone oxidoreductase (complex I) in a dose-dependent fashion. Halothane, but not isoflurane or sevoflurane, also inhibits succinate oxidation and succinate dehydrogenase activity (complex II). Although this inhibition of the respiratory chain is unlikely to compromise cardiac performance at usual clinical concentrations, it may contribute to the mechanism by which volatile anaesthetics confer ischaemic-like preconditioning.

Acknowledgments

We thank R. Graf and K. Schneider for mechanical workshop support and Professor M. Löffler for preparing the submitochondrial particles. Gas chromatography was performed in the laboratory of Professor V. Schurig. This study was supported by the P. E. Kempkes Stiftung.

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