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Effect of halothane on adenylate kinase in porcine malignant hyperpyrexia
225
Clmica Chimica Aeta, 122 (1982) 225-230 Elsevier Biomedical Press
CCA 2167
Effect of halothane on adenylate kinase in porcine malignant hyperpyrexia Leo Department
A. Marjanen * and Michael A. ~enborough
of Medicine and Clinical Science, John Curtin School of Medtcal Research, Australian National University, Canberra A CT 2601 (Australia) (Received
November
5th, 1981; revision February
23rd, 1982)
Summary The effects of halothane on adenylate kinase activity in porcine muscle have been examined. No abnormality in malignant hyperpyrexia susceptible muscle was found. At clinical concentrations of halothane only slight inhibition of adenylate kinase activity was observed. The inhibition increased with increasing concentrations of halothane and with decreasing concentrations of the substrates AMP or ADP. The inhibition was similar in both malignant hyperpyrexia susceptible and control muscle. It seems unlikely that adenylate kinase is involved directly in triggering malignant hyperpyrexia.
The anaesthetic complication malignant hyperpyrexia (MH) occurs in individuals and swine with an underlying disease of skeletal muscle. Malignant hyperpyrexia in swine is a good model for malignant hyperpyrexia in man [ 11. The reaction is usually triggered by succinylcholine or halothane, and is characterised by muscular rigidity, metabolic acidosis and a steep rise in body temperature 121. During the acute episode, skeletal muscle becomes depleted in ATP [3], and muscle adenylate kinase (AK) deficiency has been reported in a family in which two members died from MH [4]. As adenylate kinase catalyses the reversible reaction 2 ADP = AMP + ATP and plays an important role in maintaining cellular levels of adenine nucleotides [5], and as a specific binding site for halothane has been demonstrated in the AK molecule at the site normally occupied by AMP or ADP [6], the suggestion has been made that a deficiency of AK might play a central role in the development of MH ([4] Schmidt, K. personal communication). With this in mind, the effect of halothane on AK activity has been examined in muscle from swine which are susceptible to MH and in controls. * To whom correspondence
0009-8981/82/0000-0000/$02,75
should
be addressed.
Q 1982 Elsevier Biomedical
Press
226
Methods Susceptibility to MH This was determined in swine by pharmacological described previously [l].
methods which have been
Measurement of AK activity Gracilis muscle was removed from swine under thiopentone-nitrous oxide anaesthesia and immediately frozen in liquid nitrogen. The frozen muscle samples were washed in 154 mmol/l NaCl twice, and extracted in 5 mmol/l Tris-HClf0.5 mmol/l EDTA buffer (pH 8.0) at 0-4’C by homogenisation briefly (2 X 15 s) with a Polytron PT 10 homogenizer at maximal output. The muscle homogenates were centrifuged at 40000 X g for 30 min and the clear supernatants stored at - 15°C. The AK activity was measured by either ADP consumption or ADP formation using the coupled reaction systems described by Fildes and Harris [7]. The concentrations of the reactants in a standard reaction mixture (forward direction; 2 ADP -, ATP + AMP) were: 10 mmolfl glucose, 20 mmol/l MgCl,, 0.6 mmol/l NADP, 4 mmol/l ADP and 2.5 units of both glucose-&phosphate dehydrogenase and hexokinase in 100 mmol/l Tris-HCl buffer, pH 8.0. The standard reaction mixture in the backward direction contained 20 mmol/l MgCl,, 0.33 mmol/l phosphoenolpyruvate, 0.3 mmol/l NADH,, I mmol/l ATP, 0.5 mmol/l AMP and 10 units of both lactate dehydrogenase and pyruvate kinase in 100 mmol/l triethanolamine buffer, pH 7.5. Since halothane is a volatile anaesthetic special care was taken to ensure reproducible halothane concentrations in the reaction mixture. Special round-top cuvettes were used which were tightly sealed with rubber caps. All the reactants including halothane were added before the cuvette was sealed. The volume of the reactants was such that no air space was left in the cuvette. The contents were mixed and incubated at 25OC or 37OC in a waterbath for one min before the addition of the substrate (ADP or AMP) through the cap. Control experiments in the absence of AK using ATP (forward reaction) or ADP (backward reaction) as the substrate were performed in order to test the effect of halothane on the coupling enzyme system above. No inhibition of the activity of the coupling enzymes by halothane {up to 7.5 mmol/l) was observed when the incubation period was one minute. After the initial rates were recorded in a Varian Techtron 635 spectrophotometer at 340 nm, one ml of the contents was quickly transferred under 1 ml of Ccl,, mixed and centrifuged to separate the two layers. The halothane concentration in the sample was measured by gas chromatography in a Packard 7300 gas chromatograph from a 2-~1 sample of the Ccl, layer by comparing with a halothane standard curve prepared in the same conditions. Chloroform was used as an internal standard. Results During the in vitro tests for MH susceptibility the muscle was maintained at 37°C in a modified Krebs-Ringer solution saturated with 95% O/5% CO, carbogen. The
227
I a control muscle 25X
c
37’C
Control muscle
~~OI/I
1 b
MH muscle 25°C
d
MH muscle 37°C
rmolll
AMP
AMP
Fig. 1. The effect of halothane on adenylate kinase activity in porcine muscle as a function of AMP concentration. The activity was measured in control and MH-su~eptible muscle at 25°C or 3’7°C in the presence of 1.O mmol/l (a, b) or 0.6 mmol/l (c, d) halothane (0) or in the absence of halothane (X). In (a) the effect of 2.75 mmol/l halothane is also included (A). The values in all figures are the means of
duplicate experiments.
introduction of 3% halothane (v/v) in carbogen causes a contracture in MH-susceptible muscle [I]. The concentration of halothane in the medium during the contracture was 1.04 mmol/I at 25°C and 0.61 mmol/l at 37°C. The effect of 0.6 mmol/l and 1.O mmol/l halothane on muscle AK activity using the reaction ATP + AMP -, 2 ADP is shown in Fig. 1. The concentration of AMP was varied as halothane appears to bind to the same site as AMP 161.Haiothane did
f1 2
mmoi/l
Fig. 2. The adenylate
4
6
8
Ho&hone
kinase
activity
of porcine
activity was measured at a fixed concentration muscle; (X), control muscle.
muscle
as a function
of AMP (100 pmol/l)
of halothane
at 37%
The MH-susceptible
concentration.
(0),
228
mmot/t tialothone
Fig. 3. The adenylate kinase activity of porcine muscle as a function of temperature and halothane concentration. The activity was measured at 100 pmol/l AMP at 25’C (X) or 37’C (0) using MH-susceptible muscle. The concentrations of halothane in the pharmacological experiments at 25’C and 37°C are indicated by the arrows.
not inhibit AK activity at either 25°C or 37°C in either MH-susceptible or control muscle. The same set of experiments was repeated using the reaction 2 ADP --*ATP + AMP and varying the concentration of ADP (50-500 pmol/l). Again, no inhibition of AK activity by halothane was observed at either temperature in either MH-susceptible or control muscle. Higher concentrations of halothane inhibited AK activity. The effect of 2.75 mmol/l halothane is shown in Fig. 1. There was partial inhibition below a concentration of 200 pmol/l AMP. The extent of the inhibition increased when the concentration of AMP was reduced. Similar results were obtained in the forward reaction by varying the concentration of ADP in the presence of 2.75 mmol/l halothane. The effect of halothane on AK activity at a fixed, low concentration of AMP as a function of halothane concentration is shown in Fig. 2. The concentration of AMP (100 pmol/l) is the K, value for AMP in the presence of 1 mmol/l ATP. The same K, for AMP (100 pmol/l) was recorded for both MH susceptible and control muscle AK. The inhibition of the reaction by halothane appeared to be directly proportional to the concentration of halothane. Both MH susceptible and control muscle showed similar responses to halothane. The effect of halothane concentration on MH-susceptible muscle AK activity at 37’C and at 25’C is shown in Fig. 3. The concentration-dependent inhibition by halothane was observed at both temperatures. Discussion Susceptibility to MH is not exclusively a human condition but is found also in a number of animals [2]. Swine have been used extensively in studies of the MH
229
myopathy since the pharmacological properties of MH swine muscle appear to be identical with MH human muscle [I]. Recent experiments on the contractile response of MH porcine skeletal muscle to halothane have shown that the response is temperature-dependent [8]. The contracture occurs at 37°C but is absent at 25°C whereas in control swine no contracture occurs at either temperature. A possible relationship between MH and AK deficiency was reported by Schmitt et al in 1974 [4]. More recently a specific binding site for halothane in the AK molecule was shown by Sachsenheimer et al [6]. These observations suggested that AK might be directly involved in the triggering of MH by halothane. Our earlier studies have established that no statistically significant abnormality exists in MH muscle in respect to AK activity or AK isoenzyme pattern [9]. Similar results were recently reported by Cerri et al [lOJ. The present investigation was carried out to determine whether or not the effect of halothane on AK activity was abnormal in MH-susceptible muscle. If AK is directly involved in MH one might expect to see the effect of halothane expressed in a temperature-dependent manner. Direct evidence for an interaction between halothane and AK has been published only by Sachsenheimer et al [6]. In their experiments 2.5 mmol/l halothane caused 50% in~bition of the AK activity in the presence of 100 pmol/l AMP and 1 mmol/l ATP while no inhibition was observed with 500 ~mol/l AMP. Our results are similar in general terms as no inhibition was present above 200 pmol/l AMP. Fifty percent inhibition of the AK activity was observed at about 30 pmol/l AMP concentrations (Fig. 1, a), No significant difference was observed between MH susceptible and control muscle in response to low concentrations of halothane (Fig. 1). There has been uncertainty about the actual concentrations of halothane in tissues during clinical anaesthesia but the levels employed in the present study (0.6- 1.O mmol/l, Fig. 1) are similar to many of the recent estimates on clinical concentrations of halothane [l l- 131 and were the concentrations used routinely in the in vitro pharmacological tests for MH susceptibility in this laboratory. Higher concentrations of halothane inhibited AK activity in a manner dependent on the concentration of the substrate and halothane (Figs. 1 and 2, respectively). The data are consistent with the view that halothane and AMP are competing for the same site on the AK molecule. Similar results were obtained with ADP which appears to share the same site on the AK molecule with AMP [5]. The inhibition of AK activity by halothane might become significant in conditions where the level of ADP or AMP is very low and the concentration of halothane is high. On the other hand, AK is plentiful in tissues such as skeletal muscle [5]. Due to the high lipid solubility of halothane [ 141 the concentration of halothane in muscle might be higher than that in the surrounding medium. The concentration of ADP and AMP in muscle does not appear to change during an episode of MH [ 151. The most significant observation, however, was that the effect of halothane on AK in MH-susceptible and control muscle did not differ (Fig. 2). fn addition, the response was similar at 25’C and 37°C (Fig. 3). These results do not support the suggestion that MH is triggered by an interaction between halothane and the AK molecule.
230
References 1 Okumura F, Cracker BD, Denborough MA. Identification of susceptibility to malignant hyperpyrexia in swine. Br J Anaesth 1979; 51: 171-176. 2 Denborough MA. The pathopharmacology of malignant hyperpyrexia. Pharmacol Ther 1980; 9: 357-365. 3 Harrison GG, Saunders SJ, Biebuyck JF, Hickman R, Dent DM, Weaver V, Terblanche J. Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Br J Anaesth 1969; 41: 844-855. 4 Schmitt J, Schmidt K, Ritter H. Hereditary malignant hyperpyrexia associated with muscle adenylate kinase deficiency. Hum Genet 1974; 24: 253-257. 5 Noda L. Adenylate kinase. In: Boyer PD, ed., The enzymes, Vol. 8, Part A. New York: Academic Press, 1973: 279-305. 6 Sachsenheimer W, Pai EF, Schulz GE, Schirmer RH. Halothane binds in the adenine-specific niche of crystalline adenylate kinase. FEBS Lett 1977; 79: 310-312. 7 Fildes RA, Harris H. Genetically determined variation of adenylate kinase in man. Nature 1966; 209: 261-263. 8 Sullivan JS, Denborough MA. Temperature dependence of muscle function in malignant hyperpyrexia susceptible swine. Br J Anaesth 1981; 53: 173-177. 9 Marjanen LA, Denborough MA. Adenylate kinase in malignant hyperpyrexia. Br J Anaesth 1982; in press. IO Cerri CG, Willner JH, Britt BA, Wood DS. Adenylate kinase deficiency and malignant hyperthermia. Human Genet 1981; 57: 325-326. 11 Rosenberg PH, Eible H, Stier A. Biphasic effects of halothane on phospholipids and synaptic plasma membranes: a spin label study. Mol Pharmacol 1975; 11: 8799882. 12 Boggs JM, Yoong T, Hsia JC. Site and mechanism of anesthetic action. 1. Effect of anaesthetic and pressure fluidity of spin-labelled lipid vesicles. Mol Pharmacol 1976; 12: 136- 143. 13 Gingerick R, Wright PH, Paradise RR. Effects of halothane on glucose-stimulated insulin secretion and glucose oxidation in isolated rat pancreatic islets. Anesthesiology 1980; 53: 219-222. 14 Mastrangelo DJ, Trudell JR, Edmunds HN, Cohen EN. Effect of clinical concentrations of halothane on phospholipid-cholesterol membrane fluidity. Mol Pharmacol 1978; 14: 463-467 15 Clark MG, Williams CH, Pfeifer WF, Bloxham DP, Holland PC. Taylor CA, Lardy HA. Accelerated substrate cycling of fructose-6-phosphate in muscle of malignant hyperthermic pigs. Nature 1973; 245: 999101.
Clmica Chimica Aeta, 122 (1982) 225-230 Elsevier Biomedical Press
CCA 2167
Effect of halothane on adenylate kinase in porcine malignant hyperpyrexia Leo Department
A. Marjanen * and Michael A. ~enborough
of Medicine and Clinical Science, John Curtin School of Medtcal Research, Australian National University, Canberra A CT 2601 (Australia) (Received
November
5th, 1981; revision February
23rd, 1982)
Summary The effects of halothane on adenylate kinase activity in porcine muscle have been examined. No abnormality in malignant hyperpyrexia susceptible muscle was found. At clinical concentrations of halothane only slight inhibition of adenylate kinase activity was observed. The inhibition increased with increasing concentrations of halothane and with decreasing concentrations of the substrates AMP or ADP. The inhibition was similar in both malignant hyperpyrexia susceptible and control muscle. It seems unlikely that adenylate kinase is involved directly in triggering malignant hyperpyrexia.
The anaesthetic complication malignant hyperpyrexia (MH) occurs in individuals and swine with an underlying disease of skeletal muscle. Malignant hyperpyrexia in swine is a good model for malignant hyperpyrexia in man [ 11. The reaction is usually triggered by succinylcholine or halothane, and is characterised by muscular rigidity, metabolic acidosis and a steep rise in body temperature 121. During the acute episode, skeletal muscle becomes depleted in ATP [3], and muscle adenylate kinase (AK) deficiency has been reported in a family in which two members died from MH [4]. As adenylate kinase catalyses the reversible reaction 2 ADP = AMP + ATP and plays an important role in maintaining cellular levels of adenine nucleotides [5], and as a specific binding site for halothane has been demonstrated in the AK molecule at the site normally occupied by AMP or ADP [6], the suggestion has been made that a deficiency of AK might play a central role in the development of MH ([4] Schmidt, K. personal communication). With this in mind, the effect of halothane on AK activity has been examined in muscle from swine which are susceptible to MH and in controls. * To whom correspondence
0009-8981/82/0000-0000/$02,75
should
be addressed.
Q 1982 Elsevier Biomedical
Press
226
Methods Susceptibility to MH This was determined in swine by pharmacological described previously [l].
methods which have been
Measurement of AK activity Gracilis muscle was removed from swine under thiopentone-nitrous oxide anaesthesia and immediately frozen in liquid nitrogen. The frozen muscle samples were washed in 154 mmol/l NaCl twice, and extracted in 5 mmol/l Tris-HClf0.5 mmol/l EDTA buffer (pH 8.0) at 0-4’C by homogenisation briefly (2 X 15 s) with a Polytron PT 10 homogenizer at maximal output. The muscle homogenates were centrifuged at 40000 X g for 30 min and the clear supernatants stored at - 15°C. The AK activity was measured by either ADP consumption or ADP formation using the coupled reaction systems described by Fildes and Harris [7]. The concentrations of the reactants in a standard reaction mixture (forward direction; 2 ADP -, ATP + AMP) were: 10 mmolfl glucose, 20 mmol/l MgCl,, 0.6 mmol/l NADP, 4 mmol/l ADP and 2.5 units of both glucose-&phosphate dehydrogenase and hexokinase in 100 mmol/l Tris-HCl buffer, pH 8.0. The standard reaction mixture in the backward direction contained 20 mmol/l MgCl,, 0.33 mmol/l phosphoenolpyruvate, 0.3 mmol/l NADH,, I mmol/l ATP, 0.5 mmol/l AMP and 10 units of both lactate dehydrogenase and pyruvate kinase in 100 mmol/l triethanolamine buffer, pH 7.5. Since halothane is a volatile anaesthetic special care was taken to ensure reproducible halothane concentrations in the reaction mixture. Special round-top cuvettes were used which were tightly sealed with rubber caps. All the reactants including halothane were added before the cuvette was sealed. The volume of the reactants was such that no air space was left in the cuvette. The contents were mixed and incubated at 25OC or 37OC in a waterbath for one min before the addition of the substrate (ADP or AMP) through the cap. Control experiments in the absence of AK using ATP (forward reaction) or ADP (backward reaction) as the substrate were performed in order to test the effect of halothane on the coupling enzyme system above. No inhibition of the activity of the coupling enzymes by halothane {up to 7.5 mmol/l) was observed when the incubation period was one minute. After the initial rates were recorded in a Varian Techtron 635 spectrophotometer at 340 nm, one ml of the contents was quickly transferred under 1 ml of Ccl,, mixed and centrifuged to separate the two layers. The halothane concentration in the sample was measured by gas chromatography in a Packard 7300 gas chromatograph from a 2-~1 sample of the Ccl, layer by comparing with a halothane standard curve prepared in the same conditions. Chloroform was used as an internal standard. Results During the in vitro tests for MH susceptibility the muscle was maintained at 37°C in a modified Krebs-Ringer solution saturated with 95% O/5% CO, carbogen. The
227
I a control muscle 25X
c
37’C
Control muscle
~~OI/I
1 b
MH muscle 25°C
d
MH muscle 37°C
rmolll
AMP
AMP
Fig. 1. The effect of halothane on adenylate kinase activity in porcine muscle as a function of AMP concentration. The activity was measured in control and MH-su~eptible muscle at 25°C or 3’7°C in the presence of 1.O mmol/l (a, b) or 0.6 mmol/l (c, d) halothane (0) or in the absence of halothane (X). In (a) the effect of 2.75 mmol/l halothane is also included (A). The values in all figures are the means of
duplicate experiments.
introduction of 3% halothane (v/v) in carbogen causes a contracture in MH-susceptible muscle [I]. The concentration of halothane in the medium during the contracture was 1.04 mmol/I at 25°C and 0.61 mmol/l at 37°C. The effect of 0.6 mmol/l and 1.O mmol/l halothane on muscle AK activity using the reaction ATP + AMP -, 2 ADP is shown in Fig. 1. The concentration of AMP was varied as halothane appears to bind to the same site as AMP 161.Haiothane did
f1 2
mmoi/l
Fig. 2. The adenylate
4
6
8
Ho&hone
kinase
activity
of porcine
activity was measured at a fixed concentration muscle; (X), control muscle.
muscle
as a function
of AMP (100 pmol/l)
of halothane
at 37%
The MH-susceptible
concentration.
(0),
228
mmot/t tialothone
Fig. 3. The adenylate kinase activity of porcine muscle as a function of temperature and halothane concentration. The activity was measured at 100 pmol/l AMP at 25’C (X) or 37’C (0) using MH-susceptible muscle. The concentrations of halothane in the pharmacological experiments at 25’C and 37°C are indicated by the arrows.
not inhibit AK activity at either 25°C or 37°C in either MH-susceptible or control muscle. The same set of experiments was repeated using the reaction 2 ADP --*ATP + AMP and varying the concentration of ADP (50-500 pmol/l). Again, no inhibition of AK activity by halothane was observed at either temperature in either MH-susceptible or control muscle. Higher concentrations of halothane inhibited AK activity. The effect of 2.75 mmol/l halothane is shown in Fig. 1. There was partial inhibition below a concentration of 200 pmol/l AMP. The extent of the inhibition increased when the concentration of AMP was reduced. Similar results were obtained in the forward reaction by varying the concentration of ADP in the presence of 2.75 mmol/l halothane. The effect of halothane on AK activity at a fixed, low concentration of AMP as a function of halothane concentration is shown in Fig. 2. The concentration of AMP (100 pmol/l) is the K, value for AMP in the presence of 1 mmol/l ATP. The same K, for AMP (100 pmol/l) was recorded for both MH susceptible and control muscle AK. The inhibition of the reaction by halothane appeared to be directly proportional to the concentration of halothane. Both MH susceptible and control muscle showed similar responses to halothane. The effect of halothane concentration on MH-susceptible muscle AK activity at 37’C and at 25’C is shown in Fig. 3. The concentration-dependent inhibition by halothane was observed at both temperatures. Discussion Susceptibility to MH is not exclusively a human condition but is found also in a number of animals [2]. Swine have been used extensively in studies of the MH
229
myopathy since the pharmacological properties of MH swine muscle appear to be identical with MH human muscle [I]. Recent experiments on the contractile response of MH porcine skeletal muscle to halothane have shown that the response is temperature-dependent [8]. The contracture occurs at 37°C but is absent at 25°C whereas in control swine no contracture occurs at either temperature. A possible relationship between MH and AK deficiency was reported by Schmitt et al in 1974 [4]. More recently a specific binding site for halothane in the AK molecule was shown by Sachsenheimer et al [6]. These observations suggested that AK might be directly involved in the triggering of MH by halothane. Our earlier studies have established that no statistically significant abnormality exists in MH muscle in respect to AK activity or AK isoenzyme pattern [9]. Similar results were recently reported by Cerri et al [lOJ. The present investigation was carried out to determine whether or not the effect of halothane on AK activity was abnormal in MH-susceptible muscle. If AK is directly involved in MH one might expect to see the effect of halothane expressed in a temperature-dependent manner. Direct evidence for an interaction between halothane and AK has been published only by Sachsenheimer et al [6]. In their experiments 2.5 mmol/l halothane caused 50% in~bition of the AK activity in the presence of 100 pmol/l AMP and 1 mmol/l ATP while no inhibition was observed with 500 ~mol/l AMP. Our results are similar in general terms as no inhibition was present above 200 pmol/l AMP. Fifty percent inhibition of the AK activity was observed at about 30 pmol/l AMP concentrations (Fig. 1, a), No significant difference was observed between MH susceptible and control muscle in response to low concentrations of halothane (Fig. 1). There has been uncertainty about the actual concentrations of halothane in tissues during clinical anaesthesia but the levels employed in the present study (0.6- 1.O mmol/l, Fig. 1) are similar to many of the recent estimates on clinical concentrations of halothane [l l- 131 and were the concentrations used routinely in the in vitro pharmacological tests for MH susceptibility in this laboratory. Higher concentrations of halothane inhibited AK activity in a manner dependent on the concentration of the substrate and halothane (Figs. 1 and 2, respectively). The data are consistent with the view that halothane and AMP are competing for the same site on the AK molecule. Similar results were obtained with ADP which appears to share the same site on the AK molecule with AMP [5]. The inhibition of AK activity by halothane might become significant in conditions where the level of ADP or AMP is very low and the concentration of halothane is high. On the other hand, AK is plentiful in tissues such as skeletal muscle [5]. Due to the high lipid solubility of halothane [ 141 the concentration of halothane in muscle might be higher than that in the surrounding medium. The concentration of ADP and AMP in muscle does not appear to change during an episode of MH [ 151. The most significant observation, however, was that the effect of halothane on AK in MH-susceptible and control muscle did not differ (Fig. 2). fn addition, the response was similar at 25’C and 37°C (Fig. 3). These results do not support the suggestion that MH is triggered by an interaction between halothane and the AK molecule.
230
References 1 Okumura F, Cracker BD, Denborough MA. Identification of susceptibility to malignant hyperpyrexia in swine. Br J Anaesth 1979; 51: 171-176. 2 Denborough MA. The pathopharmacology of malignant hyperpyrexia. Pharmacol Ther 1980; 9: 357-365. 3 Harrison GG, Saunders SJ, Biebuyck JF, Hickman R, Dent DM, Weaver V, Terblanche J. Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Br J Anaesth 1969; 41: 844-855. 4 Schmitt J, Schmidt K, Ritter H. Hereditary malignant hyperpyrexia associated with muscle adenylate kinase deficiency. Hum Genet 1974; 24: 253-257. 5 Noda L. Adenylate kinase. In: Boyer PD, ed., The enzymes, Vol. 8, Part A. New York: Academic Press, 1973: 279-305. 6 Sachsenheimer W, Pai EF, Schulz GE, Schirmer RH. Halothane binds in the adenine-specific niche of crystalline adenylate kinase. FEBS Lett 1977; 79: 310-312. 7 Fildes RA, Harris H. Genetically determined variation of adenylate kinase in man. Nature 1966; 209: 261-263. 8 Sullivan JS, Denborough MA. Temperature dependence of muscle function in malignant hyperpyrexia susceptible swine. Br J Anaesth 1981; 53: 173-177. 9 Marjanen LA, Denborough MA. Adenylate kinase in malignant hyperpyrexia. Br J Anaesth 1982; in press. IO Cerri CG, Willner JH, Britt BA, Wood DS. Adenylate kinase deficiency and malignant hyperthermia. Human Genet 1981; 57: 325-326. 11 Rosenberg PH, Eible H, Stier A. Biphasic effects of halothane on phospholipids and synaptic plasma membranes: a spin label study. Mol Pharmacol 1975; 11: 8799882. 12 Boggs JM, Yoong T, Hsia JC. Site and mechanism of anesthetic action. 1. Effect of anaesthetic and pressure fluidity of spin-labelled lipid vesicles. Mol Pharmacol 1976; 12: 136- 143. 13 Gingerick R, Wright PH, Paradise RR. Effects of halothane on glucose-stimulated insulin secretion and glucose oxidation in isolated rat pancreatic islets. Anesthesiology 1980; 53: 219-222. 14 Mastrangelo DJ, Trudell JR, Edmunds HN, Cohen EN. Effect of clinical concentrations of halothane on phospholipid-cholesterol membrane fluidity. Mol Pharmacol 1978; 14: 463-467 15 Clark MG, Williams CH, Pfeifer WF, Bloxham DP, Holland PC. Taylor CA, Lardy HA. Accelerated substrate cycling of fructose-6-phosphate in muscle of malignant hyperthermic pigs. Nature 1973; 245: 999101.