EFFECTS OF ADENYLATE CYCLASE ACTIVATORS ON PORCINE SKELETAL MUSCLE IN MALIGNANT HYPERPYREXIA

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1 Br. J. Anaesth. (1987), 59, 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 attributed to a dysfunction of skeletal muscle (Denborough, 1980). While the precise site of the abnormality associated with MH remains unknown (Gronert, 1980), it is thought that an increase in myoplasmic Ca J+ concentration can account for most of the features of MH (Denborough, 1980). The concentration of Ca 2+ in the myoplasm is regulated by the sarcoplasmic reticulum (SR), and the enzyme adenylatc cyclase has been found to influence SR Ca i+ -transport in cardiac muscle (Kirchberger and Tada, 1976) and slow skeletal muscle (Schwartz et al., 1976). Furthermore, halothane, which precipitates an MH episode (Gronert, 1980) and induces MHsusceptible (MHS) skeletal muscle to contract in vitro (Ellis et al., 1971), activates the adenylate cyclase system (Sprague, Yang and Ngai, 1974). Consequently, this link between adenylate cyclase and MH has been investigated. Although increased concentrations of cyclic AMP (camp), and increases in adenylate cyclase activity, have been reported in MHS human muscle (Willner, Cerri and Wood, 1981; Ellis et al., 1984), no difference has been found between control and MHS porcine skeletal muscle, in relation to adenylate cyclase activity (Ono, Topel and Althen, 1976, 1977). Since MHS skeletal muscle characteristically contracts in response to various reagents in vitro (Moulds and Denborough, 1974a), we have investigated the effect of adenylate cyclase activation on the contractility of control, and MHS porcine, skeletal muscle in vitro. A. T. R. SIM, PH.D.; M. D. WHITE, PH.D.; M. A. DEN- BOBOUGH, M.D., D.PHIL., D.SC., F.R.c.p., F.R.A.C.P. ; Department of Medicine and Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2600, Australia. Accepted for Publication: July 28, Correspondence to M.A.D. SUMMARY The effect of adenylate cyclase activation on the in vitro contractures of control and malignant hyperpyrexia susceptible (MHS) porcine muscle was investigated. While fluoride and molybdate ions potentiated drug-induced contractures in control muscle, other activators of adenylate cyclase (forskolin and noradrenaline) did not. Furthermore, fluoride and molybdate had no effect on MHS skeletal muscle contractility. Cyclic AMP content, basal adenylate cyclase activity and molybdate-stimulated adenylate cyclase activity of MHS skeletal muscle was not significantly different from that of control muscle. It is concluded that increased activity of adenylate cyclase does not represent the primary skeletal muscle defect which predisposes to porcine MH. MATERIALS AND METHODS Pigs susceptible to MH were bred from crosses of MHS Landrace and Large White breeds. Animals used as controls were either commercially purchased, unrelated animals or MH-negative siblings of MHS pigs. Susceptibility to MH was assessed by the in vitro contracture testing of skeletal muscle, according to Okumura, Crocker and Denborough (1979). Anaesthesia and surgical procedures were carried out using the method of Okumura, Crocker and Denborough (1979), except that the neurolept agent Stresnil (4-fluor-4-(4-(2- pyridyl)-l-piperazinyl)-butyrophenone) was used for premedication. For pharmacological studies, the method described by Moulds and Denborough (1974b) was used. Muscle preparations which did not produce

2 1558 BRITISH JOURNAL OF ANAESTHESIA 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 < (Student's paired t test) Pretreatment Diagnostic reagent molybdate fluoride litre-' Forskolin 100 umol Noradrenaline 100 umol 3% Halothane Caffeine 2 mmol Suxamethonium 1 mmol Potassium chloride 80 mmol 0.01 ±0.005 (it = 21) 0.02±0.01 (n = 16) 0.01 ±0.005 (n - 19) 0.02 ±0.01 (it = 8) 0.37 ±0.07 (it = 21)*** 0.28 ±0.06 (it = 16)*** 0.09 ±0.02 (it = 19)*** 0.24±0.12 (if = 8) 0.29±0.13 (» = 19)* 0.13±0.04 (» = 17)** 0.13±0.05 (it = 12)* 0.01 ±0.01 (n = 6) (it = 4) ±0.005 (if = 7) 0.01 ±0.01 («- 6) 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, potassium chloride 150 mmol, Tris-HCl 10 mmol, 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, creatine phosphate and creatine phosphokinase 100 u ml" 1, theophylline, magnesium chloride 50 mmol litre 1, Tris-HCl 50 mmol, 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, suxamethonium 1 mmol (Anectine, Burroughs Wellcome) or potassium chloride 80 mmol are shown in table I. These activators, alone, did not stimulate contractures in control or MHS skeletal muscle. Pre-treatment with of fluoride or molybdate significantly

3 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 molybdate fluoride Forskolin looumol Noradr en aline 100 )imol 3% Halothane Caffeine 2 mmol Suxamcthonium 1 mmol Potassium chloride 80 mmol 0.85±0.1 (n = 14) 0.51 ±0.07 (n = ID 0.42 ±0.04 (n = 8) 2.04 ±0.43 (it = 8) 0.91 ±0.1 (» = 14) 0.38 ±0.08 (n = 11) 0.42 ±0.06 (n = 8) 1.86 ±0.46 (» = 8) 0.59±0.13 (» = 14) 0.31 ±0.08 (n = 7) 0.29±0.11 (» = 7) 0.95± ±0.27 («= 5) 1.35±0.19 (n = 5) 0.71 ±0.19 (n = 5) 0.23±0.19 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 < forcomparitonbetweencontracturesintheabscenceandpresenceofdantrolene (Student's paired t test) Pretreatment Diagnostic reagent Molybdate only Molybdate + dantrolene Fluoride only Fluoride + dantrolene 3% Halothane Caffeine 2 mmol 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 ( = 5)*** 0.012±0.012 (n = 5)*** 0.38± ±0.03 (n = 5), 0.05 ±0.04 ( = 5)*** 0.0 (n - 5)*" 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,MoO 4 had no effect. Lower concentrations of molybdate also significantly potentiated drug-induced contractures in control muscle. Using molybdate 10 mmol, mean contractures of 0.26 ± 0.08 g («= 7) were obtained in response to 3% halothane, while molybdate 2.5 mmol produced a mean contracture of ±0.05 g («= 3). While fluoride and molybdate potentiated drug-induced contractures in control skeletal muscle, forskolin and noradrenaline (100 umol ) did not (table I). Furthermore, noradrenaline up to 2 mmol failed to potentiate drug-induced contractures (results not shown). 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 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 prevented fluoride- and molybdatc-potentiation of contractures in response to 3 % halothane and caffeine 2 mmol (table III). Dantrolene was equally effective in reversing fluoride- and molybdate-potentiated drug-induced contractures (not shown).

4 1560 BRITISH JOURNAL OF ANAESTHESIA TABLE IV. Adenylau cyclase activity of control and MHS porcine skeletal muscle. Where appropriate, sodium molybdale 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 Molybdate Molybdate litre ' + dantrolene 6 imol Control MHS 6.79 ±0.97 (» - 8) 8.06 ±1.34 (n - 8) 30.17±6.55 (n -8) 28.12±4.31 (n = 7) ±5.25 (n - 5) ±3.1 (n - 5) 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 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,

5 ADENYLATE CYCLASE AND MALIGNANT HYPERPYREXIA S * I S Concentration (mmol litre' 1 ) S00 Concentration (pmol ) FIG. 1. Effect of adenylate cyclase activators on adenylate cyclase activity in control skeletal muscle, A: fluoride (A) and sodium molybdate (-AT) were included in the assay buffer at the concentrations indicated (mmol ). B: Forskolin ( ) and noradrenaline (#) were included in the assay buffer at the concentrations indicated (umol ). 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 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. 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. Anesthesiology, 53, 395. 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 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.

6 1562 BRITISH JOURNAL OF ANAESTHESIA Peterson, G. L. (1977). A simplification of the protein assay diterpene activator of cyclic-amp-generating systems. Cyc. method of Lowry which is more generally applicable. Nuc. Res., 7, 201. Analyt. Biodum., 83, 346. Sprague, D. H., Yang, J. C, and Ngai, S. H. (1974). Effects of Richards, J. M., and Swislocki, N. I. (1979). Activation of isoflurane and halothane on contractility and the cyclic 3'- adenylate cyclase by molybdate. J. Biol. Chem., 254, '-adenosine monophosphate system in the rat aorta. Schwartz, A., Entman, M. L., Karichi, K., Lane., L. K., Anathaiology., 40, 162. Winkle, W. E. V., and Bornet, E. P. (1976). The rate of WiUner, J. H., Cerri, C. G., and Wood, D. S. (1981). High calcium uptake into sarcoplasmic reticulum of cardiac and skeletal muscle adenylate cyclase in malignant hypothermia. skeletal muscle. Biochem. Biophys. Acta, 426, 57. J. Clin. Invest., 68, Seamon, K. B., and Daly, J. W. (1981). Forskolin: A unique