THE INHIBITION OF MITOCHONDRIAL RESPIRATION BY INHALATIONAL ANAESTHETIC AGENTS

Brit. J. Anaesth. (1973), 45,1005

THE INHIBITION OF MTTOCHONDRIAL RESPIRATION BY INHALATIONAL ANAESTHETIC AGENTS G. M. HALL, S. J. KISTLAND AND H. BAOM SUMMARY

The majority of cellular respiration is a reflection of the oxidation of reduced nicotinamide adenine dinudeotide (NADH) generated in the dehydrogenation reactions of the tricarboxylic add cycle. Flavoprotein-linked substrates, such as succinate, make but a small contribution to the total mitochondrial oxygen consumption, in vivo. The inner mitochondrial membrane is impermeable to NADH and to free diffusion of substrate molecules. Instead, specific carrier systems exist to transfer tricarboxylic add-cycle intermediates into the matrix of the mitochondria where the enzymes of the cyde are located. The choice of substrate for in-vitro work is thus determined by the characteristics of the specific membrane carrier system as well as by the enzyme specifidties of the cyde itself. Thus, when studying nicotinamide adenine dinudeotide (NAD)linked respiration it is common to use as added substrates pyruvate plus malate. Pyruvate gains ready access to the pyruvate dehydrogenase in the matrix, yielding acetyl coenzyme A, while malate enters the matrix giving rise (via the activity of malate dehydrogenase) to the oxaloacetate necessary to condense with acetyl coenzyme A to initiate the further reactions of the cycle. Mitochondrial respiratory states. Chance and Williams (1955) identified five states of respiration, each of which was defined by the rdative availabilities of substrate, oxygen and adenosine diphosphate (ADP) in the presence of excess phosphate. Only states LTI and IV are applicable to the present study and may be described as follows. A state TV respiratory rate is observed

in the presence of oxygen, phosphate and excess substrate but in the absence of ADP. In other words, state IV reflects a situation where respiration is limited by the rate of utilization of the conserved energy of electron transfer. A slow rate of respiration is found which is typical of mitochondria in the resting state. If ADP is added to the system a rapid increase in oxygen consumption occurs (state HI rate) until all the ADP is phosphorylated whereupon the mitochondria return to a state TV respiratory rate. The phosphorylation of ADP thus constitutes a mode of rapid utilization of the energy conserved in the respiratory chain. Normally mitochondrial respiration is tightly coupled to phosphorylation so that one might reasonably expect the state IV rate to be virtually zero. However, unknown "energy leaks" from the mitochondria produce a basal state IV respiratory rate. If these "energy leaks" are excessive then an increased state IV rate is found and the mitochondria are said to show "loss of respiratory control". The ability of halothane to inhibit reversibly the oxidation of NADH by the mitochondrial electron transfer is well documented (Cohen and Marshall, 1968; Miller and Hunter, 1970; Harris et aL, 1971). There is little information about the inhibition of NADH oxidation by other inhalational agents in intact mitochondria. The present study investigated G. M. HALL, M A , B.S., F.F.A.R.CS., Department of Anaesthetics, Hammersmith Hospital, London, W12 0HS; S. J. KlRTLAND*, B.SC, PHJX, H . BAUM, B.SC., PHJ)., F.R.I.C;

Department of Biochemistry, Chelsea College, Manresa Road, London S.W.3. •Present address: Strangeways Laboratory, Wort's Causeway, Cambridge.

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The effects of halothane, methoxyflurane, trichloroethylene, and chloroform on the oxidation of nicotinamide adenine dinudeotide (NAD)-linked substrates by isolated rat liver mitochondria were investigated. All four agents inhibited NADH oxidation at concentrations that did not affect respiration with succinate as the substrate. The mean inhibitory concentrations ( ± SD) for the abolition of stimulation of state IV respiration by ADP were: halothane 0.84 mM (±0.10 mM), methoxyflurane 1.05 mM (±0.25 mM), trichloroethylene 1.11 mM ( ± 0 2 6 mM), and chloroform 2.62 mM (±0.45 mM). The clinical relevance of this inhibition of electron transfer is discussed.

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the effect of halothane, methoxynurane, trichloroethylene and chloroform on NAD-linked substrates by isolated rat liver mitochondria. METHOD

Measurement of oxygen consumption. A Clark-type oxygen electrode (Yellow Springs Instrument Co.) was used to determine mitochondrial oxygen consumption. The 3.2-ml reaction cuvette was of perspex construction and machined so that the electrode was a tight fit A water jacket surrounded the reaction chamber and all estimations were carried out at 26 °C. The contents of the cuvette were continuously stirred by a teflon-coated magnetic stirrer to ensure that oxygen diffusion to the electrode did not limit the electrode response. Additions of ADP, substrate and anaesthetic agent were made from calibrated microlitre syringes through a narrowbore portal in the cuvette. The output of the electrode was linked to a pen recorder (Servoscribe). The electrode was polarized at 0.65 V except in the case of halothane when 0.5 V was used to minimize errors due to the polarographic reduction of halothane (Severinghaus et al., 1971). Preliminary calibration of the electrode showed that the plateau region of the current-voltage curve lay between 0.4 and 0.8 V. The apparatus was calibrated exactly as described by Robinson and Cooper (1970) in order to determine the oxygen concentration of the medium corresponding to 100% setting on the recorder—240 /iM. The chart reading was shown to have a linear response to varying oxygen concentrations over the range studied. The medium used for studying oxidative phosphory-

RESULTS

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Isolation of mitochondria. Starved Wistar rats were killed by cervical dislocation. The livers were rapidly removed and immersed in ice-cold medium. The medium used throughout the isolation of the mitochondria was 0.25 M sucrose + 1 mM EDTA (ethylene riiaminp tetraacetate), pH 7.4, and all subsequent procedures were carried out at 0-4°C. After homogenization the livers were centrifuged at 600 X g for 10 minutes to remove cellular debris. Mitochondria were then separated by centrifugation at 9,000 Xg for 10 minutes. Two further washes were given by resuspension in the isolation medium. After the final separation, the mitochondrial pellet was resuspended in a small volume of medium to provide a protein concentration of 20-40 mg/ml (Gornall, Bardawill and David, 1949), and stored on ice.

lation consisted of 0.25 M sucrose containing 5 mM magnesium chloride and 20 mM Tris chloride (buffer), pH 6.8. 1,0 /U of 1.0 M potassium phosphate buffer (pH 7.4) were added to provide excess phosphate. The substrate was either 20 /A sodium pyruvate (0.5 M)+10 /d. sodium malate (0.5 M) or 20 fd sodium sucdnate (0.5 M). The inhalational agents halothane, methoxyflurane, trichloroethylene, and chloroform were diluted to 0.6-1.0 M in absolute ethanol before addition to the cuvette. In a typical experiment 3.0 ml of medium were placed in the cuvette and 0.2 ml of mitochondria added. In the presence of succinate (as substrate) and excess phosphate, the state IH respiratory rate produced by incremental additions of 6 p\. ADP (40 mM) was noted. After this control experiment the procedure was repeated using pyruvate+malate as substrate, again with the addition of excess phosphate. Increments of the anaesthetic agent were added until the state III respiratory rate produced by the 6 jA additions of ADP was totally or almost totally inhibited. 20 [A of sodium succinate were then added to the cuvette and the state HI respiratory rate with this substrate determined. This value was compared with that found in the control experiment. An inhibitory concentration for each of the four anaesthetic agents studied was determined from one mitochondrial preparation. A total of six mitochondrial preparations were used.

Figure 1 shows a typical polarographic trace obtained using halothane with sodium pyruvate+malate as substrate. Increments of halothane to a final concentration of 0.93 M were required before ADP stimulation of the state IV respiratory rate was abolished, i.e. before respiration, rather than the rate of dissipation of the energized state, became rate limiting. Further additions of halothane inhibited even the state IV respiratory rate under these conditions but the point at which stimulation by ADP was just eliminated was a convenient one for titration purposes. The addition of sodium succinate caused a stimulation of respiration to a rate equal to the state HI respiratory rate with succinate as determined in the control experiment in the absence of halothane. This indicated that the site of inhibition by halothane was located between NADH and ubiquinone along the electron transfer chain (fig. 2). The re-establishment of state HI respiration by succinate indicated that the previous inhibition was of electron transfer

INHIBITION OF MTTOCHONDRIAL RESPIRATION

150nAO

observations) on NADH oxidation and ATP-driven, succinate-linked, NAD reduction using sub-mitochondrial particles. In these studies it was observed that halothane inhibited the oxidation of NADH added to a suspension of submitochondrial particles—a preparation in which the respiratory chain has direct access to added NADH and where respiration is not limited by the rate of energy transfer (i.e. the particles exhibit no respiratory control). Furthermore, the addition of adenosine triphosphate (ATP) to such particles permits NAD to be reduced by added succinate, the electrons being driven to traverse the span between NADH and ubiquinone in the reverse direction (see fig. 2). Halothane was found to inhibit this "reverse electron transfer", but not other ATPdriven processes. The concentrations of halothane used in these experiments were not closely controlled and therefore a direct quantitative comparison cannot be made. Investigation of methoxyflurane, trichloroethylene, and chloroform produced similar traces to that of halothane, i.e. inhibition of pyruvate + malate oxidation without inhibition of succinate oxidation. Six values were determined for each anaesthetic agent with mean inhibitory concentrations as shown in table I. In the presence of these concentrations of

SUCCINATE

5 MIN FIG. 1. Typical polarographic trace showing the inhibition of state III respiration (NAD-linked substrates) by halothane. Initial substrate 20/4 pyruvate (0.5 M) and 10 pi malate (0.5 M). Each ADP addition 6 ^1 (40 mM). Hi and H,, 2 jul of 0.6 M halothane; H,, 1 /U of 0.6 M halothane. Final halothane concentration of 0.93 mM. The ordinate, which is the output of the electrode system is a measure of oxygen concentration although calibrated in terms of oxygen content. The bar represents a decrease in oxygen content corresponding to the consumption of 150 ng atoms of oxygen.

TABLE I. The mean concentration of anaesthetic agent required to eliminate the stimulation by ADP of the oxidation of NAD-linked substrates.

and not of energy transfer. Inhibition of state HI respiration by energy transfer inhibitors such as oligomycin is not reversed by succinate. A direct indication that the inhibition is at the level of the respiratory chain and is not of energy transfer, substrate transport, or substrate dehydrogenation, came from confirmatory studies (E. Grist, unpublished

(FERfilCYAN IDE)

Agent

(NAD-LINKED)

0.84 ±0.10 1.05 ±0.25 l.ll±0.26 2.62 ±0.45

Halothane Methoxyflurane Trichloroethylene Chloroform

SUCCIHATE

FATTY ACYL COENZYfE A

FLAVOPROTEIN

FATTY ACYL CoA DEHYDROGEIIASE

(SUCCINATE L I N K E D )

•FLAVOPROTEIN

Mean inhibitory concentration ( ± SD) (mM)

-»JiOH-HAB1 IRON PROTEINS

(FLAVOPROTEIN)

UBIOUIfKWE

CYT.B,C,

'CYT.c-

CYT.A.A3-

INHIBITORS ACTING AT THE SITE INDICATED - ANYTAL. ROTENONE. PIERICIDIII A- HALOTHAIIE. METHOXYFLURAHE. TRICHLOROETHYLENE AND CHLOROFORM

FIG. 2. Abbreviated representation of the electron transfer chain showing the site of action of the inhibitors of NADH oxidation.

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I

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anaesthetic agents the state HI respiratory rate with succinate was the same as in the control experiments. Absolute ethanol was used as the solvent for the anaesthetic agents and was added in these experiments in concentrations up to 20 mM. We therefore investigated the effect of ethanol on mitochondrial oxygen consumption and found that it had no effect at concentrations less than 108 mM. The concentrations of anaesthetic agents which inhibited NADH oxidation were independent of the mitochondrial protein concentration in the cuvette which varied from 1 to 3 mg/ml. DISCUSSION

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Mitochondria may be exposed to an anaesthetic agent in eidier the gaseous or aqueous phase. Most previous investigators (Cohen and Marshall, 1968; Miller and Hunter, 1970; Cohen and Mclntyre, 1972) have equilibrated a mitochondrial suspension with the anaesthetic vapour at 0°C and subsequently studied its respiration at 25-26°C. This method, which involves the higher solubility of an anaesthetic at the lower temperature, will expose the mitochondria to higher concentrations than would occur in vivo at the same partial pressure of anaesthetic. Harris and associates (1971) utilized aqueous solutions of halo thane prepared by equilibration at 30 °Q In the present investigation the anaesthetic agents were diluted in absolute ethanol to concentrations sufficient to produce inhibition on the addition of a few microlitres. This technique involved minimal disturbance of the polarographic trace. The mean inhibitory concentration of halothane (0.84 mM) obtained in this study is similar to that found by Miller and Hunter (1970). They observed inhibition of NADH oxidation at about 1 mM in the aqueous phase at 25 °C. Harris and associates (1971) showed that there was a 75% inhibition of the state HI respiratory rate by 1.3 mM halodiane. The effect of other anaesthetic agents on intact mitochondria is poorly documented. Cohen and Mclntyre (1972) expressed their results as the concentration of anaesthetic required to produce 50% inhibition of the state HI respiratory rate. After equilibration of the mitochondria with anaesthetic vapour at 0°C, they found values for halothane of 1.35%, methoxyflurane 0.59%, and diediyl ether 7.88%. Britt, Kalow and Endrenyi (1972) exposed rat muscle mitochondria at 25 °C to halothane and methoxyflurane vapour; 5% halothane produced 85% inhibition of the ADP-stimulated rate while 2% methoxyflurane gave 78% inhibition.

Amytal, rotenone, and piericidin A are known to inhibit electron transfer between NADH and ubiquinone. Gutman and associates (1970) investigated the exact site of action of these compounds and concluded that they probably block electron transfer between non-haem iron and ubiquinone (fig. 2). Harris and associates (1971) suggested that halothane had a similar site of action because of its ability to inhibit NADH oxidation, its lack of inhibition of succinate oxidation and its poor inhibition of NADH-ferricyanide reductase. The results of this study, which show that methoxyflurane, trichloroethylene, and chloroform also inhibit NADH oxidation wimout effect on the oxidation of succinate, indicate that these agents act at a similar point on the electron transfer rhain The non-specific nature of this inhibition is illustrated by the lack of effect of varying the mitochondrial protein concentration and by die high concentration of anaesthetic required. Inhibitors such as rotenone, which binds stoichiometrically at a specific site in die NADH-ubiquinone reductase complex, block electron transfer at a concentration of 25-30 nM/g protein (Ernster, Dallner and Azzone, 1963). Inhibition of NADH oxidation by anaesthetic agents would be expected to cause a decreased rate of formation of adenosine triphosphate (ATP). Nilsson and Siesjo (1970), however, were unable to show a fall in rat cerebral ATP levels following a variety of anaesthetic agents. It is generally concluded that any reduction in ATP formation is matched by the decreased requirement for this high energy compound under anaesdiesia. Biebuyck, Lund and Krebs (1972a) examined the effect of perfusing an isolated rat liver with 2.5% halothane on various metabolic functions. In fed animals they found a fall in oxygen uptake associated with a raised lactate level together with decreased urea synthesis and gluconeogenesis. The inhibition of these metabolic processes was accompanied by a fall in ATP production. These effects were much less in rats starved for 48 hours and could be minimized in fed animals by the simultaneous infusion of oleate (Biebuyck, Lund and Krebs, 1972b). The f3 oxidation of fats is partially dependent on flavoprotein-linked enzymes which bypass the anaesthetic-induced inhibition of electron transfer (fig. 2). This work indicates that the ability of anaesthetic agents to inhibit NADH oxidation may have clinical relevance to biosynthetic processes. Cohen and Marshall (1968) demonstrated that the action of halodiane on mitochondrial oxygen con-

INHIBITION OF MTTOCHONDRIAL RESPIRATION

ACKNOWLEDGEMENT

We should like to thank Mrs R. Mason for valuable secretarial assistance. REFERENCES

Britt, B. A., Kalow, W., and Endrenyi, L. (1972). Effects of halothane and methoxyflurane on rat skeletal muscle mitochondria. Biochem. Pharmacol., 21, 1159. Chance, B., and Williams, G. R. (1955). Respiratory enzymes in oxidative phosphorylation. I l l : The steady state. J. biol. Chenu, 217, 409. Cohen, P. J., and Marshall, B. E. (1968). Toxicity of Anesthetics (ed. B. R. Fink), p. 24. Baltimore: Williams & Wilkins. Mclnryre, R. (1972). Cellular Biology and Toxicity of Anesthetics (ed. B. R. Fink), p. 109. Baltimore: Williams & Wilkins. Emster, L., Dallner, G., and Azzone, G. F. (1963). Differential effects of rotenone and amytal on mitochondrial electron and energy transfer. J. biol. Chem., 238, 1124. Gomall, A. G., Bardawill, C. H., and David, M. M. (1949). Determination of serum proteins by means of the Biuret reaction. J. biol. Chem., 177, 751. Gutman, M., Singer, T. P., Beinhert, H., and Casida, J. E. (1970). Reaction sites of rotenone, piericidin A, and amytal in relation to the nonheme iron components or NADH dehydrogenase. Proc. not. Acad. Sci. (.Wash.), 65, 763. Harris, R. A., Munroe, J., Farmer, B., Kim, K. C , and Jenkins, P. (1971). Action of halothane upon mitochondrial respiratioa. Arch. Biochem. Biophys., 142, 435. Levy, L., and Featherstone, R. M. (1954). The effect of xenon and nitrous oxide on in vitro guinea-pig brain respiration and oxidative phosphorylation. J. Pharmacol, exp. Ther., 110, 221. Miller, R. N., and Hunter, F. E. (1970). The effect of halothane on electron transport, oxidative phosphorylarion, and swelling in rat liver mitochondria. Molec. Pharmacol., 6, 67. Nilsson, L., and Siesjo, B. K. (1970). The effect of anaesthetics upon labile phosphates and upon extraand intracellular lactate, pyruvate, and bicarbonate concentrations in the rat brain. Acta physiol. scand., 80, 235. Robinson, J., and Cooper, J. M. (1970). Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Arm. Biochem., 33, 390.

Biebuyck, J. F., Lund, P., and Krebs, H. A. (1972a). The effects of halothane (2-bromo-2-chloro-l,l,l,-mfluoroethane) on glycolysis and biosynthetic processes of the isolated perfused rat liver. Biochem. J., 128, 711. Severinghaus, J. W., Weiskopt, R. B., Nishimura, M., and Bradley, A. F. (1971). Oxygen electrode errors due (1972b). The protective effect of oleate to the polarographic reduction of halothane. J. appL on metabolic changes produced by halothane in. rat Physiol, 31, 640. liver. Biochem. J., 12«, 721.

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sumption was easily reversible within the clinical concentration range. This inhibition could be regarded as an example of an anaesthetic-protein interaction that by altering mitochondrial membrane configuration interferes with enzymic action. Why one particular segment of the electron transfer chain is more susceptible than others is not known. However, it is templing to speculate that inhibition of electron transfer at This point is an essential property of an anaesthetic agent. Levy and Featherstone (1954) were unable to show any effect of nitrous oxide and xenon on guineapig brain mitochondria although the concentrations used may have been too low (80% gas/20% oxygen). Specific biochemical inhibitors for this site, such as rotenone and piericidin A, act essentially irreversibly and are not suitable models to invoke in this discussion. In conclusion, the inhibitory effect of riinirai levels of inhalational agents on electron transfer might be regarded as a biochemical curiosity reflecting no more than the sensitivity of one particular lipoprotein complex to a perturbation analogous to that which at a different locus is truly responsible for anaesthesia. On the other hand, inhibition of NADH oxidation at specific sites in the central nervous system might (e.g. by causing local depression of ATP synthesis or local inhibition of the metabolism of a neurotransmitter) actually underlie anaesthesia. Even in the former case the phenomenon may have clinical relevance in terms of the interference by anaesthetics with specific metabolic pathways in sensitive tissues.

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