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EFFECTS OF ADENYLATE CYCLASE ACTIVATORS ON PORCINE SKELETAL MUSCLE IN MALIGNANT HYPERPYREXIA
Br. J. Anaesth. (1987), 59, 1557-1562
EFFECTS OF ADENYLATE CYCLASE ACTIVATORS ON PORCINE SKELETAL MUSCLE IN MALIGNANT HYPERPYREXIA A. T. R. SIM, M. D. WHITE AND M. A. DENBOROUGH Malignant hyperpyrexia (MH) is an often fatal complication of general anaesthesia, which is SUMMARY attributed to a dysfunction of skeletal muscle The effect of adenylate cyclase activation on the (Denborough, 1980). While the precise site of the in vitro contractures of control and malignant abnormality associated with MH remains un- hyperpyrexia susceptible (MHS) porcine muscle known (Gronert, 1980), it is thought that an was investigated. While fluoride and molybdate increase in myoplasmic CaJ+ concentration can ions potentiated drug-induced contractures in account for most of the features of MH (Den- control muscle, other activators of adenylate borough, 1980). The concentration of Ca2+ in the cyclase (forskolin and noradrenaline) did not. myoplasm is regulated by the sarcoplasmic re- Furthermore, fluoride and molybdate had no ticulum (SR), and the enzyme adenylatc cyclase effect on MHS skeletal muscle contractility. has been found to influence SR Cai+-transport in Cyclic AMP content, basal adenylate cyclase cardiac muscle (Kirchberger and Tada, 1976) and activity and molybdate-stimulated adenylate slow skeletal muscle (Schwartz et al., 1976). cyclase activity of MHS skeletal muscle was not Furthermore, halothane, which precipitates an significantly different from that of control muscle. MH episode (Gronert, 1980) and induces MH- It is concluded that increased activity of adenylsusceptible (MHS) skeletal muscle to contract in ate cyclase does not represent the primary vitro (Ellis et al., 1971), activates the adenylate skeletal muscle defect which predisposes to cyclase system (Sprague, Yang and Ngai, 1974). porcine MH. Consequently, this link between adenylate cyclase and MH has been investigated. Although increased concentrations of cyclic AMP (cAMP), MATERIALS AND METHODS and increases in adenylate cyclase activity, have been reported in MHS human muscle (Willner, Pigs susceptible to MH were bred from crosses of Cerri and Wood, 1981; Ellis et al., 1984), no MHS Landrace and Large White breeds. Animals difference has been found between control and used as controls were either commercially purMHS porcine skeletal muscle, in relation to chased, unrelated animals or MH-negative sibadenylate cyclase activity (Ono, Topel and Althen, lings of MHS pigs. 1976, 1977). Since MHS skeletal muscle characSusceptibility to MH was assessed by the in teristically contracts in response to various re- vitro contracture testing of skeletal muscle, acagents in vitro (Moulds and Denborough, 1974a), cording to Okumura, Crocker and Denborough we have investigated the effect of adenylate cyclase (1979). activation on the contractility of control, and Anaesthesia and surgical procedures were MHS porcine, skeletal muscle in vitro. carried out using the method of Okumura, Crocker and Denborough (1979), except that the neurolept agent Stresnil (4-fluor-4-(4-(2A. T. R. SIM, PH.D.; M. D . WHITE, PH.D.; M. A. D E N BOBOUGH, M.D., D.PHIL., D.SC., F.R.c.p., F.R.A.C.P. ; Department pyridyl)-l-piperazinyl)-butyrophenone) was used of Medicine and Clinical Science, The John Curtin School of for premedication. Medical Research, The Australian National University, CanFor pharmacological studies, the method deberra, ACT, 2600, Australia. Accepted for Publication: July scribed by Moulds and Denborough (1974b) was 28, 1987. Correspondence to M.A.D. used. Muscle preparations which did not produce
BRITISH JOURNAL OF ANAESTHESIA
1558
TABLE I. The effect of adenylate cyclase activators on drug-induced contractures in porcine control skeletal muscle. Control muscle fibres were pretreated for 5 mm teith the appropriate adenylate cyclase activator and then exposed to the appropriate diagnostic reagent. Results are expressed as the mean contractures (g) ± SEM. For comparisons between prc-treatment and no pre-treatment: *P < 0.05 **P < 0.01; ***P < 0.001 (Student's paired t test) Pretreatment
Diagnostic reagent 3 % Halothane Caffeine 2 mmol litre"1 Suxamethonium 1 mmol litre"1 Potassium chloride 80 mmol litre"1
None
Sodium molybdate 20 mmol litre"1
0.01 ±0.005 (it = 21)
(it = 21)***
0.02±0.01
Sodium fluoride 20 mmol
litre-'
0.37 ±0.07
0.29±0.13 (» = 19)* 0.28 ±0.06 0.13±0.04 (it = 16)*** (» = 17)** 0.09 ±0.02 0.13±0.05
(n = 16) 0.01 ±0.005 (n - 19) 0.02 ±0.01
(it = 19)***
(it = 12)*
0.24±0.12
—
(it = 8)
(if = 8)
a twitch response equivalent to a tension of 0.75 g or more, were rejected. Drugs and chemicals were added directly into the organ bath at thefinalconcentrations indicated in the text. Forskolin (Calbiochem) and dantrolene sodium (Norwich Eaton) were dissolved in dimethyl sulphoxide, final concentrations of which in the organ bath did not exceed 0.4% (v/v) and had no effect on muscle integrity. When used, thymol-free halothane was administered by passing carbogen through a Dragewick halothane vaporizer. Adenylate cyclase activity was determined by measuring the change in cAMP content of skeletal muscle homogenates in the presence of ATP and a phosphodiesterase inhibitor. Samples of muscle were obtained at biopsy, frozen immediately in liquid nitrogen and stored at — 70 °C until required. No sample was stored for more than 4 weeks, and control and MHS samples of equivalent storage age were assayed at the same time, in each study. Using a mortar and pestle, cooled to — 70 °C, approximately 0.5 g of muscle sample was ground to a fine powder and homogenized in 20 volumes of homogenization buffer using a Polytron at maximum setting for 2 x 10-s intervals. The homogenization buffer contained EDTA 4 mmol litre"1, potassium chloride 150 mmol litre"1, Tris-HCl 10 mmol litre"1, pH 7.4. Aliquots (50 ul) of muscle homogenate were added to 50 ul of assay buffer at 0 °C. The assay buffer contained ATP 1 mmol litre"1, creatine phosphate 20 mmol litre"1 and creatine phosphokinase 100 u ml"1, theophylline 20 mmol litre"1,
Forskolin 100 umol litre"1
Noradrenaline 100 umol litre"1
0.01 ±0.01 (n = 6) 0.0 (» = 5) —
0.0 (it = 4)
0.005 ±0.005 (if = 7)
0.01 ±0.01 ( « - 6) —
—
magnesium chloride 50 mmol litre 1, Tris-HCl 50 mmol litre"1, pH 7.4. The reaction was started by transferring the samples to a 37 °C water bath. After 10 min, the reaction was stopped by the addition of 5 % TCA 150 ul (w/v) and the protein precipitated by centrifugation at 2000 £ for 15 min. The supernatant was collected, extracted with water-saturated ether and assayed for cAMP content using a competitive-binding assay kit (Amersham Radiochemicals). Experiments were performed in duplicate. Dose-response curves for different chemicals were carried out on muscle from one animal. All adenylate cyclase activators used did not interfere with the cAMP assay. Adenylate cyclase activity was expressed as the amount of cAMP formed per mg of protein per min. Protein estimations were carried out using the method of Peterson (1977). RESULTS
Pharmacological action of adenylate cyclase activators on skeletal muscle
The results obtained when control porcine skeletal musclefibreswere pretreated with adenylate cyclase activators and then exposed to 3 % halothone, caffeine 2 mmol litre"1, suxamethonium 1 mmol (Anectine, Burroughs Wellcome) or potassium chloride 80 mmol litre"1 are shown in table I. These activators, alone, did not stimulate contractures in control or MHS skeletal muscle. Pre-treatment with 20 mmol litre"1 of fluoride or molybdate significantly
ADENYLATE CYCLASE AND MALIGNANT HYPERPYREXIA
1559
TABLE II. Effect of adenylate cyclase activators on drug-induced contractures in MHS porcine skeletal muscle fibres. MHS muscle fibres were pretreated for 5 min with the appropriate adenylate cyclase activator and then exposed to the appropriate diagnostic reagent. Results are expressed as the mean contractures (g) ± S.E.M. There was no significant difference between contractures in the absence or presence of any adenylate cyclase activators
Pretreatment
Diagnostic reagent 3% Halothane Caffeine 2 mmol litre"1 Suxamcthonium 1 mmol litre"1 Potassium chloride 80 mmol litre"1
None 0.85±0.1 (n = 14) 0.51 ±0.07 (n = ID 0.42 ±0.04 (n = 8) 2.04 ±0.43 (it =
8)
Sodium molybdate 20 mmol litre"1
Sodium fluoride 20 mmol litre"1
0.91 ±0.1 (» = 14) 0.38 ±0.08 (n = 11)
0.42 ±0.06 (n = 8) 1.86 ±0.46 (» = 8)
Forskolin lOOumol litre"1
Noradren aline 100 )imol litre"1
0.59±0.13 (» = 14) 0.31 ±0.08 (n = 7) 0.29±0.11 (» = 7)
0.95±0.21 (» = 5) 0.96 ±0.27 (« = 5)
1.35±0.19 (n = 5) 0.71 ±0.19 (n = 5) 0.23±0.19 (» = 5)
—
—
—
—
TABLE III. Effect of dantrolene on fluoride- and molybdate-potentiated contractures in control porcine skeletal muscle. Control muscle fibres were pre-treated with sodium fluoride 20 mol litre~l or molybdate 20 mmol litre'1 for 5 min followtd by dantrolene sodium 6 umol litre'1 for a further 5 min and then exposed to halothane orcaffeine.***P < 0.001 forcomparitonbetweencontracturesintheabscenceandpresenceofdantrolene (Student's paired t test)
Pretreatment Diagnostic reagent 3% Halothane Caffeine 2 mmol litre"1
None
Molybdate only
Molybdate + dantrolene
0.01 ±0.005 (n = 21) 0.02±0.01 (» = 16)
0.43 ±0.11 (n - 5) 0.29 ±0.03 (« - 5)
0.017±0.016
potentiated contractures in response to halothane, caffeine and suxamethonium (table I). Molybdate also increased the magnitude of the potassium chloride-induced contractures, but this was not statistically significant. Pretreatment with equivalent concentrations of sodium chloride in place of NaF + Na,MoO4 had no effect. Lower concentrations of molybdate also significantly potentiated drug-induced contractures in control muscle. Using molybdate 10 mmol litre"1, mean contractures of 0.26 ± 0.08 g (« = 7) were obtained in response to 3 % halothane, while molybdate 2.5 mmol litre"1 produced a mean contracture of 0.125 ±0.05 g (« = 3). While fluoride and molybdate potentiated drug-induced contractures in control skeletal muscle, forskolin and noradrenaline (100 umol litre"1) did not (table I). Furthermore, noradrenaline up to 2 mmol litre"1 failed to potentiate drug-induced contractures (results not shown).
(„ = 5 )***
0.012±0.012 (n = 5)***
Fluoride only
Fluoride + dantrolene
0.38±0.11 0.05 ±0.04 („ = 5)*** (» = 5) 0.0 0.12±0.03 (n = 5) , (n - 5 ) * "
When these experiments were carried out with MHS porcine muscle, the results were different from those obtained with control muscle (table II). Fluoride and molybdate 20 mmol litre"1 produced no significant changes in drug-induced contractures. Forskolin and noradrenaline also failed to have any effect on contractures induced by the same drugs in MHS tissue (table II). The effect of dantrolene on fluoride and molybdate-potentiated contractures was also examined (table III). Dantrolene was added to the organ bath after fluoride and molybdate and before the diagnostic reagent. Treatment of muscle fibres with dantrolene 6 umol litre"1 prevented fluoride- and molybdatc-potentiation of contractures in response to 3 % halothane and caffeine 2 mmol litre"1 (table III). Dantrolene was equally effective in reversing fluoride- and molybdate-potentiated drug-induced contractures (not shown).
BRITISH JOURNAL OF ANAESTHESIA
1560
TABLE IV. Adenylau cyclase activity of control and MHS porcine skeletal muscle. Where appropriate, sodium molybdale 20 mmol litre'1 with or without dantrolene 6 fttnol Htre'1 mas included in the assay buffer. Results are expressed as the mean adenylau cyclase activity ±SEM. There was no significant difference between control and MHS samples Adenylate cyclase activity (pmol cAMP/mg protein/min)
Sample
None
Molybdate 20 mmol litre"1
Control
6.79 ±0.97 (» - 8) 8.06 ±1.34 (n - 8)
30.17±6.55 (n - 8 ) 28.12±4.31 (n = 7)
MHS
Adenylate cyclase activity of control and MHS skeletal muscle Cyclic-AMP content of control samples varied between 8.46 and 30 pmol/mg protein and between 12.8 and 41.3 pmol/mg protein for MHS samples. The mean cAMP content of MHS skeletal muscle homogenates (22.39 ±3.7 pmol/ mg protein) was not significantly different from that of control muscle (18.19±2.49 pmol/mg protein). Furthermore, basal adenylate cyclase activity in MHS skeletal muscle homogenates was not significantly different from the basal adenylate cyclase activity of control samples (table IV). The effect of adenylate cyclase activators on skeletal muscle adenylate cyclase activity was also studied. Dose-response curves showed that the inclusion of fluoride, molybdate, forskolin and noradrenaline in the assay buffer produced a concentration-dependent increase in adenylate cyclase activity in control samples (fig. 1). These agents were thus shown .to activate adenylate cyclase in porcine skeletal muscle as they do in other tissues. However, there was no significant difference between control and MHS samples in relation to molybdate-stimulated adenylate cyclase activity (table IV). Furthermore, dantrolene had no significant effect on molybdate-stimulated adenylate cyclase activity in control or MHS samples (table IV).
DISCUSSION
Adenylate cyclase activity has been reported to be increased in MHS humans (Willner, Cerri and Wood, 1981; Ellis et al., 1984), but not in MHS pigs (Ono, Topel and Althen, 1976, 1977). While the results of these different investigations may reflect a real difference and species variability, it
Molybdate 20 mmol litre ' + dantrolene 6 |imol litre"1
24.08 ±5.25 (n - 5)
25.23 ±3.1 (n - 5)
may be noted that the use of an animal model allows more stringent attention to the choice of controls, muscle type and experimental conditions. In the investigation of human MH, controls are obtained from various sources and may involve different muscle types (Willner, Cerri and Wood, 1981) and anaesthetic conditions (Gronert and Van Dyke, 1984). However, variations in the concentrations of circulating catecholamines (Halter, Pflug and Porter, 1977) and adenylate cyclase activity in different muscle types (Festoff, Oliver and Reddy, 1977) may lead to spurious differences between subjects. The animals used in the present study were subject to the same experimental conditions and results showed that there was no difference between control and MHS porcine muscle in relation to cAMP content and basal adenylate cyclase activity. While fluoride and molybdate increased the sensitivity of control muscle to the diagnostic reagents, other activators of adenylate cyclase (forskolin and noradrenaline) did not. Fluoride has been shown to activate adenylate cyclase only in broken cell preparations (Perkins and Moore, 1971) while forskolin is a general activator of adenylate cyclase in all systems, including whole cell preparations (Seamon and Daly, 1981). Since the muscle preparations used in this study were essentially intact, it is unlikely that fluoride increased the sensitivity of control muscle through a direct action on adenylate cyclase. Furthermore, it is not known whether molybdate stimulates adenylate cyclase in whole cell preparations, but it is generally believed to have a mechanism of action similar to that of fluoride (Richards and Swislocki, 1979). Although fluoride and molybdate were both found to stimulate adenylate cyclase activity in skeletal muscle homogenates,
ADENYLATE CYCLASE AND MALIGNANT HYPERPYREXIA
8
1561
related to activation of adenylate cyclase activity. It is also concluded that the primary defect associated with MH does not reside in the adenylate cyclase system. The finding that fluoride and molybdate did not increase the contractile response of MHS muscle suggests that MHS muscle has a decreased sensitivity to fluoride and molybdate. Both fluoride and molybdate are potent inhibitors of protein phosphatases (Hollander, 1971) and, as such, might interfere with the phosphorylation state of certain proteins involved in muscle contractility. Experiments to explore this hypothesis have been started.
6
3 2
2
4
6
8
10
Concentration (mmol litre'1)
REFERENCES Denborough, M. A. (1980). The pathopharmacology of malignant hyperpyrexia. Pharmacol. Ther., 9, 357. Ellis, F. R., Halsall. P. J., Allan, P., and Hay, E. (1984). A biochemical abnormality found in muscle from unstressed malignant hyperpyrexia susceptible humans. Biochem. Soc. Trans., 12, 357. Harriman, D. G. F., Keaney, N. P., Kyei-Mensah, K., and Tyrell, J. H. (1971). Halothane induced muscle contracture as a cause of hyperpyrexia. Br. J. Anaesth., 43, 721. Festoff, B. W., Oliver, K. L., and Reddy, N. B. (1977). In vitro studies of skeletal muscle membranes: Adenylate cyclase of fast and slow twitch muscle and the effects of denervation. J. Memb. Biol., 32, 331. Gronert, G. A. (1980). Malignant hyperthermia. Anes-
10
•
•
S *
I S
thesiology, 53, 395.
100
200
300
400
S00
Concentration (pmol litre"1)
FIG. 1. Effect of adenylate cyclase activators on adenylate cyclase activity in control skeletal muscle, A: Sodium fluoride (A) and sodium molybdate (-AT) were included in the assay buffer at the concentrations indicated (mmol litre"1). B: Forskolin ( • ) and noradrenaline ( # ) were included in the assay buffer at the concentrations indicated (umol litre"1). Each point on the curves represents one individual experiment. Adenylate cyclase activity results are expressed as the ratio of «timiilati-H: basal activity.
there was no significant difference between control and MHS muscle in relation to molybdatestimulated adenylate cyclase activity. Furthermore, dantrolene, which antagonized and prevented fluoride- and molybdate-potentiation of drug-induced contractures, had no effect on molybdate-stimulated adenylate cyclase activity. For these reasons, it is unlikely that the pharmacological action of fluoride and molybdate is
Van Dyke, R. A. (1984). (Letter to the editor.) Anesth. Analg., 63, 89. Halter, J. B., Pflug, A. E., and Porter, D. (1977). Mechanism of plasma catecholamine increases during surgical stress in man. J. Clin. Endocrinol. Metab., 45, 936. Hollander, V. P. (1971). The Enzymes, p. 450. New York: Academic Press. Kirchberger, M. A., and Tada, M. (1976). Effects of 3', 5'monophosphate dependent protein kinase on SR isolated from cardiac and slow and fast contracting skeletal muscles. Biochim. Biophys. Acta, 628, 438. Moulds, R. F. W., and Denborough, M. A. (1974a). Biochemical basis of malignant hyperpyrexia. Br. Med. J., 2, 241. (1974b). A study of the action of caffeine, halothane, potassium chloride and procaine on normal human skeletal muscle. Clin. Exp. Pharmacol. Physiol, 1, 197. Okumura, F., Crocker, B. D., and Denborough. M. A. (1979). Identification of susceptibility to malignant hyperpyrexia in swine. Br. J. Anaesth., 51, 171. Ono, K., Topcl, D. G., and Althen, T. G. (1976). Cyclic AMP in longissimus muscle from control and stress susceptible pigs. J. Food Sri., 41, 108. (1977). Adenylate cyclase and cyclic 3'-5'nucleotide phosphodiesterase activities in muscles from stress susceptible and control pigs. J. Food Sci., 42, 111. Perkins, J. P., and Moore, M. M. (1971). Adenylate cyclase of rat cerebral cortex: Activation by sodium fluoride and detergents. J. Biol. Chem., 246, 62.
1562 Peterson, G. L. (1977). A simplification of the protein assay method of Lowry which is more generally applicable. Analyt. Biodum., 83, 346. Richards, J. M., and Swislocki, N. I. (1979). Activation of adenylate cyclase by molybdate. J. Biol. Chem., 254, 6857. Schwartz, A., Entman, M. L., Karichi, K., Lane., L. K., Winkle, W. E. V., and Bornet, E. P. (1976). The rate of calcium uptake into sarcoplasmic reticulum of cardiac and skeletal muscle. Biochem. Biophys. Acta, 426, 57. Seamon, K. B., and Daly, J. W. (1981). Forskolin: A unique
BRITISH JOURNAL OF ANAESTHESIA diterpene activator of cyclic-AMP-generating systems. Cyc. Nuc. Res., 7, 201. Sprague, D. H., Yang, J. C , and Ngai, S. H. (1974). Effects of isoflurane and halothane on contractility and the cyclic 3'5'-adenosine monophosphate system in the rat aorta. Anathaiology., 40, 162. WiUner, J. H., Cerri, C. G., and Wood, D. S. (1981). High skeletal muscle adenylate cyclase in malignant hypothermia. J. Clin. Invest., 68, 1119.
EFFECTS OF ADENYLATE CYCLASE ACTIVATORS ON PORCINE SKELETAL MUSCLE IN MALIGNANT HYPERPYREXIA A. T. R. SIM, M. D. WHITE AND M. A. DENBOROUGH Malignant hyperpyrexia (MH) is an often fatal complication of general anaesthesia, which is SUMMARY attributed to a dysfunction of skeletal muscle The effect of adenylate cyclase activation on the (Denborough, 1980). While the precise site of the in vitro contractures of control and malignant abnormality associated with MH remains un- hyperpyrexia susceptible (MHS) porcine muscle known (Gronert, 1980), it is thought that an was investigated. While fluoride and molybdate increase in myoplasmic CaJ+ concentration can ions potentiated drug-induced contractures in account for most of the features of MH (Den- control muscle, other activators of adenylate borough, 1980). The concentration of Ca2+ in the cyclase (forskolin and noradrenaline) did not. myoplasm is regulated by the sarcoplasmic re- Furthermore, fluoride and molybdate had no ticulum (SR), and the enzyme adenylatc cyclase effect on MHS skeletal muscle contractility. has been found to influence SR Cai+-transport in Cyclic AMP content, basal adenylate cyclase cardiac muscle (Kirchberger and Tada, 1976) and activity and molybdate-stimulated adenylate slow skeletal muscle (Schwartz et al., 1976). cyclase activity of MHS skeletal muscle was not Furthermore, halothane, which precipitates an significantly different from that of control muscle. MH episode (Gronert, 1980) and induces MH- It is concluded that increased activity of adenylsusceptible (MHS) skeletal muscle to contract in ate cyclase does not represent the primary vitro (Ellis et al., 1971), activates the adenylate skeletal muscle defect which predisposes to cyclase system (Sprague, Yang and Ngai, 1974). porcine MH. Consequently, this link between adenylate cyclase and MH has been investigated. Although increased concentrations of cyclic AMP (cAMP), MATERIALS AND METHODS and increases in adenylate cyclase activity, have been reported in MHS human muscle (Willner, Pigs susceptible to MH were bred from crosses of Cerri and Wood, 1981; Ellis et al., 1984), no MHS Landrace and Large White breeds. Animals difference has been found between control and used as controls were either commercially purMHS porcine skeletal muscle, in relation to chased, unrelated animals or MH-negative sibadenylate cyclase activity (Ono, Topel and Althen, lings of MHS pigs. 1976, 1977). Since MHS skeletal muscle characSusceptibility to MH was assessed by the in teristically contracts in response to various re- vitro contracture testing of skeletal muscle, acagents in vitro (Moulds and Denborough, 1974a), cording to Okumura, Crocker and Denborough we have investigated the effect of adenylate cyclase (1979). activation on the contractility of control, and Anaesthesia and surgical procedures were MHS porcine, skeletal muscle in vitro. carried out using the method of Okumura, Crocker and Denborough (1979), except that the neurolept agent Stresnil (4-fluor-4-(4-(2A. T. R. SIM, PH.D.; M. D . WHITE, PH.D.; M. A. D E N BOBOUGH, M.D., D.PHIL., D.SC., F.R.c.p., F.R.A.C.P. ; Department pyridyl)-l-piperazinyl)-butyrophenone) was used of Medicine and Clinical Science, The John Curtin School of for premedication. Medical Research, The Australian National University, CanFor pharmacological studies, the method deberra, ACT, 2600, Australia. Accepted for Publication: July scribed by Moulds and Denborough (1974b) was 28, 1987. Correspondence to M.A.D. used. Muscle preparations which did not produce
BRITISH JOURNAL OF ANAESTHESIA
1558
TABLE I. The effect of adenylate cyclase activators on drug-induced contractures in porcine control skeletal muscle. Control muscle fibres were pretreated for 5 mm teith the appropriate adenylate cyclase activator and then exposed to the appropriate diagnostic reagent. Results are expressed as the mean contractures (g) ± SEM. For comparisons between prc-treatment and no pre-treatment: *P < 0.05 **P < 0.01; ***P < 0.001 (Student's paired t test) Pretreatment
Diagnostic reagent 3 % Halothane Caffeine 2 mmol litre"1 Suxamethonium 1 mmol litre"1 Potassium chloride 80 mmol litre"1
None
Sodium molybdate 20 mmol litre"1
0.01 ±0.005 (it = 21)
(it = 21)***
0.02±0.01
Sodium fluoride 20 mmol
litre-'
0.37 ±0.07
0.29±0.13 (» = 19)* 0.28 ±0.06 0.13±0.04 (it = 16)*** (» = 17)** 0.09 ±0.02 0.13±0.05
(n = 16) 0.01 ±0.005 (n - 19) 0.02 ±0.01
(it = 19)***
(it = 12)*
0.24±0.12
—
(it = 8)
(if = 8)
a twitch response equivalent to a tension of 0.75 g or more, were rejected. Drugs and chemicals were added directly into the organ bath at thefinalconcentrations indicated in the text. Forskolin (Calbiochem) and dantrolene sodium (Norwich Eaton) were dissolved in dimethyl sulphoxide, final concentrations of which in the organ bath did not exceed 0.4% (v/v) and had no effect on muscle integrity. When used, thymol-free halothane was administered by passing carbogen through a Dragewick halothane vaporizer. Adenylate cyclase activity was determined by measuring the change in cAMP content of skeletal muscle homogenates in the presence of ATP and a phosphodiesterase inhibitor. Samples of muscle were obtained at biopsy, frozen immediately in liquid nitrogen and stored at — 70 °C until required. No sample was stored for more than 4 weeks, and control and MHS samples of equivalent storage age were assayed at the same time, in each study. Using a mortar and pestle, cooled to — 70 °C, approximately 0.5 g of muscle sample was ground to a fine powder and homogenized in 20 volumes of homogenization buffer using a Polytron at maximum setting for 2 x 10-s intervals. The homogenization buffer contained EDTA 4 mmol litre"1, potassium chloride 150 mmol litre"1, Tris-HCl 10 mmol litre"1, pH 7.4. Aliquots (50 ul) of muscle homogenate were added to 50 ul of assay buffer at 0 °C. The assay buffer contained ATP 1 mmol litre"1, creatine phosphate 20 mmol litre"1 and creatine phosphokinase 100 u ml"1, theophylline 20 mmol litre"1,
Forskolin 100 umol litre"1
Noradrenaline 100 umol litre"1
0.01 ±0.01 (n = 6) 0.0 (» = 5) —
0.0 (it = 4)
0.005 ±0.005 (if = 7)
0.01 ±0.01 ( « - 6) —
—
magnesium chloride 50 mmol litre 1, Tris-HCl 50 mmol litre"1, pH 7.4. The reaction was started by transferring the samples to a 37 °C water bath. After 10 min, the reaction was stopped by the addition of 5 % TCA 150 ul (w/v) and the protein precipitated by centrifugation at 2000 £ for 15 min. The supernatant was collected, extracted with water-saturated ether and assayed for cAMP content using a competitive-binding assay kit (Amersham Radiochemicals). Experiments were performed in duplicate. Dose-response curves for different chemicals were carried out on muscle from one animal. All adenylate cyclase activators used did not interfere with the cAMP assay. Adenylate cyclase activity was expressed as the amount of cAMP formed per mg of protein per min. Protein estimations were carried out using the method of Peterson (1977). RESULTS
Pharmacological action of adenylate cyclase activators on skeletal muscle
The results obtained when control porcine skeletal musclefibreswere pretreated with adenylate cyclase activators and then exposed to 3 % halothone, caffeine 2 mmol litre"1, suxamethonium 1 mmol (Anectine, Burroughs Wellcome) or potassium chloride 80 mmol litre"1 are shown in table I. These activators, alone, did not stimulate contractures in control or MHS skeletal muscle. Pre-treatment with 20 mmol litre"1 of fluoride or molybdate significantly
ADENYLATE CYCLASE AND MALIGNANT HYPERPYREXIA
1559
TABLE II. Effect of adenylate cyclase activators on drug-induced contractures in MHS porcine skeletal muscle fibres. MHS muscle fibres were pretreated for 5 min with the appropriate adenylate cyclase activator and then exposed to the appropriate diagnostic reagent. Results are expressed as the mean contractures (g) ± S.E.M. There was no significant difference between contractures in the absence or presence of any adenylate cyclase activators
Pretreatment
Diagnostic reagent 3% Halothane Caffeine 2 mmol litre"1 Suxamcthonium 1 mmol litre"1 Potassium chloride 80 mmol litre"1
None 0.85±0.1 (n = 14) 0.51 ±0.07 (n = ID 0.42 ±0.04 (n = 8) 2.04 ±0.43 (it =
8)
Sodium molybdate 20 mmol litre"1
Sodium fluoride 20 mmol litre"1
0.91 ±0.1 (» = 14) 0.38 ±0.08 (n = 11)
0.42 ±0.06 (n = 8) 1.86 ±0.46 (» = 8)
Forskolin lOOumol litre"1
Noradren aline 100 )imol litre"1
0.59±0.13 (» = 14) 0.31 ±0.08 (n = 7) 0.29±0.11 (» = 7)
0.95±0.21 (» = 5) 0.96 ±0.27 (« = 5)
1.35±0.19 (n = 5) 0.71 ±0.19 (n = 5) 0.23±0.19 (» = 5)
—
—
—
—
TABLE III. Effect of dantrolene on fluoride- and molybdate-potentiated contractures in control porcine skeletal muscle. Control muscle fibres were pre-treated with sodium fluoride 20 mol litre~l or molybdate 20 mmol litre'1 for 5 min followtd by dantrolene sodium 6 umol litre'1 for a further 5 min and then exposed to halothane orcaffeine.***P < 0.001 forcomparitonbetweencontracturesintheabscenceandpresenceofdantrolene (Student's paired t test)
Pretreatment Diagnostic reagent 3% Halothane Caffeine 2 mmol litre"1
None
Molybdate only
Molybdate + dantrolene
0.01 ±0.005 (n = 21) 0.02±0.01 (» = 16)
0.43 ±0.11 (n - 5) 0.29 ±0.03 (« - 5)
0.017±0.016
potentiated contractures in response to halothane, caffeine and suxamethonium (table I). Molybdate also increased the magnitude of the potassium chloride-induced contractures, but this was not statistically significant. Pretreatment with equivalent concentrations of sodium chloride in place of NaF + Na,MoO4 had no effect. Lower concentrations of molybdate also significantly potentiated drug-induced contractures in control muscle. Using molybdate 10 mmol litre"1, mean contractures of 0.26 ± 0.08 g (« = 7) were obtained in response to 3 % halothane, while molybdate 2.5 mmol litre"1 produced a mean contracture of 0.125 ±0.05 g (« = 3). While fluoride and molybdate potentiated drug-induced contractures in control skeletal muscle, forskolin and noradrenaline (100 umol litre"1) did not (table I). Furthermore, noradrenaline up to 2 mmol litre"1 failed to potentiate drug-induced contractures (results not shown).
(„ = 5 )***
0.012±0.012 (n = 5)***
Fluoride only
Fluoride + dantrolene
0.38±0.11 0.05 ±0.04 („ = 5)*** (» = 5) 0.0 0.12±0.03 (n = 5) , (n - 5 ) * "
When these experiments were carried out with MHS porcine muscle, the results were different from those obtained with control muscle (table II). Fluoride and molybdate 20 mmol litre"1 produced no significant changes in drug-induced contractures. Forskolin and noradrenaline also failed to have any effect on contractures induced by the same drugs in MHS tissue (table II). The effect of dantrolene on fluoride and molybdate-potentiated contractures was also examined (table III). Dantrolene was added to the organ bath after fluoride and molybdate and before the diagnostic reagent. Treatment of muscle fibres with dantrolene 6 umol litre"1 prevented fluoride- and molybdatc-potentiation of contractures in response to 3 % halothane and caffeine 2 mmol litre"1 (table III). Dantrolene was equally effective in reversing fluoride- and molybdate-potentiated drug-induced contractures (not shown).
BRITISH JOURNAL OF ANAESTHESIA
1560
TABLE IV. Adenylau cyclase activity of control and MHS porcine skeletal muscle. Where appropriate, sodium molybdale 20 mmol litre'1 with or without dantrolene 6 fttnol Htre'1 mas included in the assay buffer. Results are expressed as the mean adenylau cyclase activity ±SEM. There was no significant difference between control and MHS samples Adenylate cyclase activity (pmol cAMP/mg protein/min)
Sample
None
Molybdate 20 mmol litre"1
Control
6.79 ±0.97 (» - 8) 8.06 ±1.34 (n - 8)
30.17±6.55 (n - 8 ) 28.12±4.31 (n = 7)
MHS
Adenylate cyclase activity of control and MHS skeletal muscle Cyclic-AMP content of control samples varied between 8.46 and 30 pmol/mg protein and between 12.8 and 41.3 pmol/mg protein for MHS samples. The mean cAMP content of MHS skeletal muscle homogenates (22.39 ±3.7 pmol/ mg protein) was not significantly different from that of control muscle (18.19±2.49 pmol/mg protein). Furthermore, basal adenylate cyclase activity in MHS skeletal muscle homogenates was not significantly different from the basal adenylate cyclase activity of control samples (table IV). The effect of adenylate cyclase activators on skeletal muscle adenylate cyclase activity was also studied. Dose-response curves showed that the inclusion of fluoride, molybdate, forskolin and noradrenaline in the assay buffer produced a concentration-dependent increase in adenylate cyclase activity in control samples (fig. 1). These agents were thus shown .to activate adenylate cyclase in porcine skeletal muscle as they do in other tissues. However, there was no significant difference between control and MHS samples in relation to molybdate-stimulated adenylate cyclase activity (table IV). Furthermore, dantrolene had no significant effect on molybdate-stimulated adenylate cyclase activity in control or MHS samples (table IV).
DISCUSSION
Adenylate cyclase activity has been reported to be increased in MHS humans (Willner, Cerri and Wood, 1981; Ellis et al., 1984), but not in MHS pigs (Ono, Topel and Althen, 1976, 1977). While the results of these different investigations may reflect a real difference and species variability, it
Molybdate 20 mmol litre ' + dantrolene 6 |imol litre"1
24.08 ±5.25 (n - 5)
25.23 ±3.1 (n - 5)
may be noted that the use of an animal model allows more stringent attention to the choice of controls, muscle type and experimental conditions. In the investigation of human MH, controls are obtained from various sources and may involve different muscle types (Willner, Cerri and Wood, 1981) and anaesthetic conditions (Gronert and Van Dyke, 1984). However, variations in the concentrations of circulating catecholamines (Halter, Pflug and Porter, 1977) and adenylate cyclase activity in different muscle types (Festoff, Oliver and Reddy, 1977) may lead to spurious differences between subjects. The animals used in the present study were subject to the same experimental conditions and results showed that there was no difference between control and MHS porcine muscle in relation to cAMP content and basal adenylate cyclase activity. While fluoride and molybdate increased the sensitivity of control muscle to the diagnostic reagents, other activators of adenylate cyclase (forskolin and noradrenaline) did not. Fluoride has been shown to activate adenylate cyclase only in broken cell preparations (Perkins and Moore, 1971) while forskolin is a general activator of adenylate cyclase in all systems, including whole cell preparations (Seamon and Daly, 1981). Since the muscle preparations used in this study were essentially intact, it is unlikely that fluoride increased the sensitivity of control muscle through a direct action on adenylate cyclase. Furthermore, it is not known whether molybdate stimulates adenylate cyclase in whole cell preparations, but it is generally believed to have a mechanism of action similar to that of fluoride (Richards and Swislocki, 1979). Although fluoride and molybdate were both found to stimulate adenylate cyclase activity in skeletal muscle homogenates,
ADENYLATE CYCLASE AND MALIGNANT HYPERPYREXIA
8
1561
related to activation of adenylate cyclase activity. It is also concluded that the primary defect associated with MH does not reside in the adenylate cyclase system. The finding that fluoride and molybdate did not increase the contractile response of MHS muscle suggests that MHS muscle has a decreased sensitivity to fluoride and molybdate. Both fluoride and molybdate are potent inhibitors of protein phosphatases (Hollander, 1971) and, as such, might interfere with the phosphorylation state of certain proteins involved in muscle contractility. Experiments to explore this hypothesis have been started.
6
3 2
2
4
6
8
10
Concentration (mmol litre'1)
REFERENCES Denborough, M. A. (1980). The pathopharmacology of malignant hyperpyrexia. Pharmacol. Ther., 9, 357. Ellis, F. R., Halsall. P. J., Allan, P., and Hay, E. (1984). A biochemical abnormality found in muscle from unstressed malignant hyperpyrexia susceptible humans. Biochem. Soc. Trans., 12, 357. Harriman, D. G. F., Keaney, N. P., Kyei-Mensah, K., and Tyrell, J. H. (1971). Halothane induced muscle contracture as a cause of hyperpyrexia. Br. J. Anaesth., 43, 721. Festoff, B. W., Oliver, K. L., and Reddy, N. B. (1977). In vitro studies of skeletal muscle membranes: Adenylate cyclase of fast and slow twitch muscle and the effects of denervation. J. Memb. Biol., 32, 331. Gronert, G. A. (1980). Malignant hyperthermia. Anes-
10
•
•
S *
I S
thesiology, 53, 395.
100
200
300
400
S00
Concentration (pmol litre"1)
FIG. 1. Effect of adenylate cyclase activators on adenylate cyclase activity in control skeletal muscle, A: Sodium fluoride (A) and sodium molybdate (-AT) were included in the assay buffer at the concentrations indicated (mmol litre"1). B: Forskolin ( • ) and noradrenaline ( # ) were included in the assay buffer at the concentrations indicated (umol litre"1). Each point on the curves represents one individual experiment. Adenylate cyclase activity results are expressed as the ratio of «timiilati-H: basal activity.
there was no significant difference between control and MHS muscle in relation to molybdatestimulated adenylate cyclase activity. Furthermore, dantrolene, which antagonized and prevented fluoride- and molybdate-potentiation of drug-induced contractures, had no effect on molybdate-stimulated adenylate cyclase activity. For these reasons, it is unlikely that the pharmacological action of fluoride and molybdate is
Van Dyke, R. A. (1984). (Letter to the editor.) Anesth. Analg., 63, 89. Halter, J. B., Pflug, A. E., and Porter, D. (1977). Mechanism of plasma catecholamine increases during surgical stress in man. J. Clin. Endocrinol. Metab., 45, 936. Hollander, V. P. (1971). The Enzymes, p. 450. New York: Academic Press. Kirchberger, M. A., and Tada, M. (1976). Effects of 3', 5'monophosphate dependent protein kinase on SR isolated from cardiac and slow and fast contracting skeletal muscles. Biochim. Biophys. Acta, 628, 438. Moulds, R. F. W., and Denborough, M. A. (1974a). Biochemical basis of malignant hyperpyrexia. Br. Med. J., 2, 241. (1974b). A study of the action of caffeine, halothane, potassium chloride and procaine on normal human skeletal muscle. Clin. Exp. Pharmacol. Physiol, 1, 197. Okumura, F., Crocker, B. D., and Denborough. M. A. (1979). Identification of susceptibility to malignant hyperpyrexia in swine. Br. J. Anaesth., 51, 171. Ono, K., Topcl, D. G., and Althen, T. G. (1976). Cyclic AMP in longissimus muscle from control and stress susceptible pigs. J. Food Sri., 41, 108. (1977). Adenylate cyclase and cyclic 3'-5'nucleotide phosphodiesterase activities in muscles from stress susceptible and control pigs. J. Food Sci., 42, 111. Perkins, J. P., and Moore, M. M. (1971). Adenylate cyclase of rat cerebral cortex: Activation by sodium fluoride and detergents. J. Biol. Chem., 246, 62.
1562 Peterson, G. L. (1977). A simplification of the protein assay method of Lowry which is more generally applicable. Analyt. Biodum., 83, 346. Richards, J. M., and Swislocki, N. I. (1979). Activation of adenylate cyclase by molybdate. J. Biol. Chem., 254, 6857. Schwartz, A., Entman, M. L., Karichi, K., Lane., L. K., Winkle, W. E. V., and Bornet, E. P. (1976). The rate of calcium uptake into sarcoplasmic reticulum of cardiac and skeletal muscle. Biochem. Biophys. Acta, 426, 57. Seamon, K. B., and Daly, J. W. (1981). Forskolin: A unique
BRITISH JOURNAL OF ANAESTHESIA diterpene activator of cyclic-AMP-generating systems. Cyc. Nuc. Res., 7, 201. Sprague, D. H., Yang, J. C , and Ngai, S. H. (1974). Effects of isoflurane and halothane on contractility and the cyclic 3'5'-adenosine monophosphate system in the rat aorta. Anathaiology., 40, 162. WiUner, J. H., Cerri, C. G., and Wood, D. S. (1981). High skeletal muscle adenylate cyclase in malignant hypothermia. J. Clin. Invest., 68, 1119.