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Malignant hyperthermia in pigs: Calcium ion uptake by mitochondria from skeletal muscle of susceptible animals given neuroleptic drugs and halothane
J. COW'.
PATH.
1982.
VOL.
191
92.
MALIGNANT HYPERTHERMIA UPTAKE BY MITOCHONDRIA MUSCLE OF SUSCEPTIBLE NEUROLEPTIC DRUGS
IN
PIGS: CALCIUM FROM SKELETAL ANIMALS GIVEN AND HALOTHANE
ION
BY
C. J. SOMERS* and J.V.
MCLOUGHLIN
Department of Physiology, Trinity College, Dublin 2, Ireland
INTRODUCTION
The anaesthetic halothane (2-bromo-2-chloro-l,l,l-trifluorethane), either alone or in combination with suxamethonium, is associated with the development of the malignant hyperthermia syndrome (MHS) in man and the pig. The syndrome usually develops very rapidly in the pig. The main clinical manifestations are rigidity of the skeletal muscles, hyperthermia, tachycardia and arhythmia, and respiratory and metabolic acidosis. (Sybesma and Eikelenboom, 1969; Berman, Harrison, Bull and Kench, 1970; Nelson, Jones, Venable and Kerr, 1972; Gronert and Theye, 1976; Hall, Lucke and Lister, 1976; Lucke, Hall and Lister, 1976; McLaughlin and Mothersill, 1976). Premeditation of MHS-susceptible animals with neuroleptic drugs such as azaperone, haloperidol and spiperone delays the onset of MHS (Ahern, Somers, Wilson and McLaughlin, 1977; McLaughlin, Somers, Ahern and Wilson, 1978). Prior anaesthesia with barbiturates, e.g. thiopentone (Gronert and Theye, 1976) and pentobarbitone (Ahern, Somers, Wilson and McLoughlin, 1980) or the steroid anaesthetic althesin (Ahern, McLaughlin, Wheatley and Wilson, 1979) has a similar action. There is a progressive loss of high-energy phosphates (“P) and an accumulation of lactate in skeletal muscle during this delay phase which precedes the onset of clinical signs. The depletion of =P in neuroleptic or barbiturate-modified MHS appears to occur more rapidly in red than in white fibres of skeletal muscle (McLaughlin et al.? 1978). This observation agrees with those of other workers which suggest that there may be some particular involvement of red or Type I myofibres in the syndrome. The myogenic aspect of MHS may be associated with increased concentrations of Ca2 + in the myoplasm. It is not known whether this may be caused by release of Ca2 + from intracellular storage sites such as the sarcoplasmic reticulum (SR) or mitochondria, a reduction in the capacity of these organelles to *Present address: The Agricultural 00” l-9975/82/020191 +08 $03.00/O
Institute, Dunsinea, Castleknock, Co. Dublin, Ireland. @ 1982 Academic Press Inc. (London) Limited
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take up Ca2 + or an influx of extracellular Ca2 + which leads to a calciuminduced calcium release from the SR. It seems likely, however, that alterations in Ca2 + uptake by the SR and mitochondria during MHS may be secondary to other intracellular changes such as a loss of ATP or a fall in pH (Britt, Endrenyi and Cadman, 1975). In the work described in this paper, normal and MHS-susceptible pigs were anaesthetized with halothane following premeditation with neuroleptic drugs. Other susceptible animals were anaesthetized without premeditation so that acute MHS developed. The uptake of Ca2 + by mitochondria from the predominantly red and white fibre areas of M. semitendinosus was investigated. The rate of Ca2 + uptake was determined both in the presence and absence of an adenine nucleotide (ATP). Ad enine nucleotides are known to enhance the uptake and/or retention of Ca 2 + by mitochondria (Carafoli, Rossi and Lehninger, 1965; Asimakis and Jordakl, 1977). MATERIALS
AND
METHODS
Pigs. Purebred Pietrain pigs were obtained from a herd kept for experimental purposes, purebred Landrace from the national breeding herd at the Department of Agricultural Testing Station, Dublin and crossbred Landrace-Large White from commercial sources. The liveweight of the pigs ranged from 30 to 50 kg. Halothane test for susceptibility to MHS. This test was carried out as described by Ahern et al. (1979). The Pietrain (5) and purebred Landrace pigs (2) gave a positive reaction, i.e., were susceptible to MHS. The Landrace-Large White crossbred pigs did not react. Anaesthesia. Halothane. Anaesthesia was induced with 5 per cent halothane (Hoechst, Germany) delivered in oxygen at a rate of 2 1 per min via a face mask. The concentration of halothane was regulated with a Fluotec vaporizer (Cyprane Ltd., Keighley, England) Following induction, the animals were intubated and anaesthesia maintained with 1 to 2 per cent halothane. Pentobarbitone. Anaesthesia was carried out as described by Ahern et al. (1980). Premeditation. Some animals were given the neuroleptic drugs azaperone (2 mg per kg bodyweight or spiperone (70 to 500 pg per kg bodyweight) by the intramuscular route approximately 30 min before the induction of anaesthesia. Both drugs were obtained from Janssen Pharmaceutics, Beerse, Belgium. Muscle. Biopsy specimens were taken from the predominantly red and white fibre areas of M. semitendinosus during anaesthesia and the loading of Ca2 + by mitochondria preparations was assayed. The concentration of creatine phosphate (CP) in the specimens was also determined. Assay of creatinephosphate. Muscular tissue was extracted and CP assayed as described by McLaughlin and Mothersill (1976). &feasurements of Ca2 + uptake by mitochondria. Specimens (2 g) of muscle were trimmed free of connective tissue, rinsed in sucrose solution (250 mM), finely minced and suspended in extraction medium containing sucrose (70 mM), mannitol (210 mM) trisamine buffer (18 mM, pH 7.4), b ovine serum albumin fraction V (Sigma) ( 10 per cent) and EDTA (10 mM) (Davidoff, 1974). Homogenization was carried out as described by Carafoli and Lehninger (1971). The homogenate was centrifuged at low (650 g) and high (8000 g) speeds for 10 min (Hedman, 1965). Mitochondrial pellets were washed in a medium (20 ml) containing sucrose (280 XIM) and trisamine buffer (1 mM, pH 7.4) and centrifuged (8000 g for 10 min). The pellets were suspended
CALCIUM
in washing chemically
UPTAKE
BY
PIG
MUSCLE
193
MITOCHONDRIA
medium and stored at 0 “C. The rate of Ca2 + uptake was measured radiofollowing the procedure of Reed and Bygrave’ (1974). Uptake was also
determined in the presence of a nucleotide (ATP 3 to 6 mM). The concentration of ATP was determined at 258 nM in a Pye-Unicam SP 1700 spectrophotometer and a molar absorption coefficient of 15.3 X 1Og M -I cm -I was used to calculate the value. Mitochondria were incubated for 3 min and incubation terminated by the addition of a solution (0.05 ml) containing ruthenium red (30 PM) and ethyleneglycolbis (Saminoethylether NJVI) tetracetic acid (EGTA, 100 mM). The suspensions were filtered with a Millipore Sampling Manifold and a 0.45 PM filter. The filters carrying particulate material were washed with assay medium, dried and placed in a scintillation cocktail (5 ml) containing Triton X. Samples of the preparations and standard solution of 45Ca2 + were read in a Packard 3375 Scintillation Counter (window setting 50 to 1000, gain 1 per cent). The counting efficiency of samples was 59 per cent and was determined by the channels ratio method. Calcium uptake was expressed as nmol Ca2+ per mg mitochondrial protein per min. Assay of mitochondrial protein. The total protein content of mitochondrial preparations was assayed by the biuret method of Gornall, Bardawill and David (1949) with bovine serum albumen as a standard. Assay of succinic dehydrogenase activity. (SDH). The activity of SDH in mitochondrial preparations was measured by the method of Tisdale (1967) with minor modifications. The specific activity of SDH was calculated using a molar absorption coefficient of 18.5 >’ IO6 M -l cm ml at 550 nM for cytochrome c. The enzyme activity was expressed as pmol cytochrome c reduced per min per mg mitochondrial protein.
RESULTS
The mean values &.E.M. for uptake of Ca 2 -+by preparations of mitochondria from M. semitendinosis of 4 MHZ&susceptible Pietrain and 7 non-susceptible Landrace Large White crossbred pigs premeditated with neuroleptic drugs and anaesthetized with halothane are given in Table 1. The Ca2 + uptake, TABLE “PTAKE
OF CR’+ BY MITOCHONDRIA NEUROLEPTIC-TREATED
-
..-~-~
FROM PIETRAIN
1
SKELETAL MUSCLE OF LANDRACE/LARGE PIGS ANAESTHETIZED WITH HALOTHANE
Myofibre we
Red XTP .4bsmt White Red ATP 3 to 6 mu White
Landrace large white
Pietrain
(n=7;l 166&20* 6.2&0.7t 110*1.3 5.611.3
(n=4) 174-r-25 7.8&25 69klO 5.3+0.9
721*100 26,-t40 376kl15 24+80
608f222 42530 342*30 2316.1
WHITE
___--
AND
Sign&znce~
._^
N.S. N.S.
N.S. N.S.
*nmol Ca* f per mg mitochondrial protein. tnmol Ca”+ per nmol cytochrome c reduced/min. $Landrace/Large White v. Pietrain.
expressed either on the basis of mg mitochondrial protein per min or SDH activity (pm01 per cytochrome c reduced per mg mitochondrial protein/min), was not significantly different between the MHS-susceptible and non-susceptible pigs. At the time when the biopsy specimens were taken (30 min anaes-
194
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SOMERS
AND
J.
V.
MCLOUGHLIN
thesia) the Pietrains had not yet shown clinical signs of MHS. Furthermore, the concentration of CP in the M. semitendinosus (red fibres 17.5 f 1.8, white 21.0 i 3.6 pmol per g) was similar to that found when the Pietrain pig is anaesthetized with an agent such as pentobarbitone which does not trigger the syndrome, an observation which suggested that the muscle was physiologically normal at the time of excision (Somers, Wilson, Ahern and McLaughlin, 1977). The mean values ~s.E.M. for a group of MHS-susceptible pigs exposed to halothane without premeditation with neuroleptic drugs are given in Table 2. The group TABLE Ca2+UpTAKE
BY MITOCHONDRIAFROM semitendinosus OF PIGS WHICH
Myojibre
2
THE PREDOMINANTLY RED AND WHITE DEVELOPED MHS DURING HALOTHANE
ape
ATP
Absent 44&&o* 1.9+0-q 45* 14* 2.4+0.7+
Red White *nmol Cat+ per tnmol Ca2 + per $.Data compared not develop MHS Mean values for
M.
Significance$
Ca2 + uptake ATP
FIBRE AREASOF ANAESTHESIA
3 to6mM 1 lo+26 5.6& 1-3 111*25 9.6k2.2
mg mitochondrial protein. nmol cytochrome c reduced/min. with corresponding data in Table under halothane anaesthesia. 7 animals.
1 for animals
P
which
did
consisted of 5 Pietrain and 2 pure-bred Landrace pigs. The biopsy specimens were taken after 30 min of anaesthesia, at which time muscular rigidity had developed. The development of MHS was associated with a significant reduction (Pt0.001) in the rate of Ca2 + uptake. The reduction was greater in red than in white myofibre preparations (P
Myojbre
type
Calcium Untreated
Red White
3 Gas+
BYPREPARATIONSOF
IN VITRO
uptake* Spiperone treated
58*8.7 26k4.4
87.5 f 10.6 35.8* 6.1
Signiflcancet PiO.05 N.S.
*nmol Ca2 f per mg mitochondria protein. tuntreated v. treated. Mean values&s.E.M. for 8 preparations.
neuroleptic drug significantly increased (P
CALCIUM
UPTAKE
BY
PIG
MUSCLE
195
MITOCHONDRIA
greater in preparations of mitochondria from red than from white myofibres, both in the absence (Pt0.01) and in the presence of adenine nucleotide (P
Caa+ RED
4
UPTAKEBYMITOCHONDRIALPREPARATIONS AND WHITE FIBRE AREAS OF PIG
Calcium uptake
hjwjibre ATP absent (1)
FROMTHE
&i. semitendinosus Al-P (3 to 6
mM)
Red
(2) White Significance (1) v. (21 *nmol tnmol
CaZ+ Ca2+
per mg mitochondrial per nmol cytochrome
protein. 6. reduced/min
DISCUSSION
The results indicate that mitochondria from the predominantly red myofibre area of M. semitendinosus of the pig accumulate Ca2 7. at a greater rate in vitro than do preparations of these organelles isolated from the predominantly white area. The difference between mitochondria from the two myofibre types was observed both in the presence and absence of an adenine nucleotide (ATP). When the rates of Ca2+ uptake were expressed on the basis of the succinic dehydrogenase activity of the muscle, there were no differences in activity between mitochondrial preparations from myofibre types. These observations support those of Kitchen, Van Winkle, Cruikshank, Spiegel and Zainis (1978) who reported that the rate of Ca2 + uptake was significantly higher in mitochondrial preparations from the soleus muscle of the rat (slow contracting) than in those from the tibialis (fast contracting). The results of these authors, when expressed on the basis of succinic dehydrogenase activity, likewise showed no difference in the rate of Ca2 + between the two muscles. Our results are in agreement with those of other workers. Differences in Ca2 + uptake by mitochondria from red and white myofibres of skeletal muscle were described by Patriarcai and Carafoli (1969). Following the administration of 45Ca2 + to rabbits, a higher concentration of the isotope was found in the mitochondrial fraction from red than from white myofibres. Lehninger (1970, 1974) suggested that the mitochondria from both red skeletal and cardiac myofibres may be involved in the regulation of Ca2 + concentrations in the sarcoplasm. In a comparative study of Ca2 + uptake by the sarcoplasmic reticulum and mitochondria from cardiac muscle, Kitagawa (1976) concluded that although the sarcoplasmic reticulum played a major role in controlling intracellular Ca2 + movements during physiological contractures, nevertheless during maximum contraction of cardiac muscle the sarcoplasmic reticulum alone could not bring about relaxation of its myofibres in the absence of Ca2
196
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MCLOUGHLIN
accumulation by mitochondria. Becker, Fiskum and Lehninger (1980) reported that at intracellular concentrations of Ca2 + both mitochondrial and endoplasmic reticulum preparations from liver actively participate in the regulation of the free Ca2 + concentration. These results also lend further support to the observations by McLaughlin et al. (1978) that neuroleptic drugs delay the onset of halothane-induced MHS. These authors reported that during the delay phase a progressive fall in the concentration of creatine phosphate and a rise in that of lactate occurred in M. semitendinosus. The loss of the high energy phosphate and the accumulation of lactate was more marked in the predominantly red than in the predominantly white fibre area of the muscle. The observation suggested that the red myofibre areas may be more susceptible to the actions of halothane than the white. The findings reported here show that when susceptible pigs developed MHS the mitochondria from red myofibres exhibited a greater reduction in Ca2 +-accumulating ability than did those from white. If the hypothesis that the trigger for MHS-associated rigidity and increased metabolic activity in skeletal muscle is a rise in the concentration of myoplasmic Ca2 + is accepted, then a reduction in the Ca2 +-binding capacity of mitochondria would perhaps affect the more mitochondrial-dependent red myofibres earlier than the sarcoplasmic reticulum-dependent white myofibres and stimulate both contractile and metabolic activity in the former.
SUMMARY
The uptake of calcium ions by mitochondria from skeletal muscle was similar for malignant hyperthermia syndrome (MHS)-susceptible pigs given neuroleptic drugs and normal pigs. The onset of MHS in the former was associated with a significant reduction in calcium ion uptake. Spiperone increased the binding of calcium by a preparation of mitochondria in vitro. Mitochondria from the predominantly red fibre area of M. semitendinosus had a greater calcium binding capacity than those from the predominantly white fibre area.
ACKNOWLEDGMENTS
The authors wish to thank the Agricultural Institute for the award of a grant. Mrs Mary Butler provided skilled technical assistance.
REFERENCES
Ahern, C. P., Somers, C. J., Wilson, P., and McLaughlin, J. V. (1977). The prevention of acute malignant hyperthermia in halothane-sensitive Pietrain pigs by low dosesof neuroleptic drugs. Proceedings of the Third International Conference on Production Diseases in Farm Animals, Pudoc, Wageningen, pp. 169-171. Ahern, C. P., McLaughlin, J. V., Wheatley, A., and Wilson, P. (1979). Althesin as an anaesthetic in experimental animals susceptible to halothane-initiated’ malignant hyperthermia. British Journal of Pharmacology, 67, 425.
CALCIUM
UPTAKE
BY
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MUSCLE
MITOCHONDRIA
197
Ahern, C. P., Somers, C. J,, Wilson, P., and McLaughlin, J. V. (1980). Halothaneinduced malignant hyperthermia: creatine phosphate concentration in skeletal muscle as an early indicator of the onset of the syndrome. Journal of Comparative Pathology, 90, 177-186. Asimakis, G. K., and Jordakl, L. A. (1977). Effects of atractylanide and palmityl coenzyme A on calcium transport in cardiac mitochondria. Archives of Biochemistry and Biophysics,79, ZOO-210. Becker, G. L., Fiskum, G. and Lehninger, A. L. (1980). Regulation of free Ca2 + by liver mitochondria and endoplasmic reticulum TheJournal of BiologicalChemistry,
255,9009-9012. Berman, H. C., Harrison, G. G., Bull, A. B., and Kench, J. E. (1970). Changes underlying halothane-induced malignant hyperpyrexia in Landrace pigs. Nature,
225, 635-655. Britt,
B. A., Endrenyi, I., and Cadman, D. I. (1975). Calcium uptake in muscle sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: in vitro and in vivo studies with and without halothane. British Journal of Anaesthesia, 47,
650-693. (:arafoli, E., Rossi, C. S., and Lehninger, A. L. (1965). Uptake of adenine nucleotides by respiratory mitochondria during active accumulation of Ca2 + and phosphate. Journal of Biological Chemistry,249, 2254-226 1. C:arafoli, E., and Lehninger, A. L. (1971). A survey of the interaction of calcium inns with mitochondria from different tissues and species. BiochemicalJournal. 122, 681-690. Davidoff, F. (1974). Effects of guanidine derivatives on mitochondria function. Journal vf Biological Chemistry,249, 6406-6415 ( 1974). Gornall, A. G. C., Bardawill, C. J., and David, M. M. (1949). Determination of strum proteins by means of the biuret reaction. Journal of Biological Chemistpt?
177, 75 l-766. Gronert, G. A., and Theye, R. A. (1976). Halothane-induced porcine malignant hyperthermia. Anaesthesiology, 44, 36-43. Hall, G. M., Lucke, J. N., and Lister, N. (1976). Porcine malignant hyperthermia, IV. Neuromuscular blockade. British Journal of Anaestksia, 48, I 136-I 141. Hedman, R. (1965). Properties of isolated skeletal muscle mitochondria from rat. ExperimentalCell Research,38, 1-12. Kitagawa, T. ( 1976). Physiological significance of Ca*+ uptake by mitochondria in the heart in comparison with that by the sarcoplasmic reticulum. Journal oj‘ Biochemistry(Tokyo), 80, 1129-l 147. Kitchen, S. E., Van Winkle, W. B., Cruickshank, G. S., Spiegel, G., and Zaimis, E. (1978). Mitochondria and sarcoplasmic reticulum fragments isolated from slow and fast contracting muscles of the cat. In The Biochemistryof Myasthenia Gravis and Muscular Dystrophy. G. G. Lunt and R. M. Marchbanks, Eds, Academic Press, London. Lehninger, A. L. (1970). Mitochondria and calcium ion transport. Biochemical Journal, 119, 129-l 38. Lehninger, A. L. (1974). Ca 2 + transport by mitochondria and its possible role in the cardiac contraction-relaxation cycle. Supplementto Circulation Research,34 and 35, 11 l--83 and 11 l-90. Lucke, J. N., Hall, G. M., and Lister, D. (1976). Porcine malignant hyperthermia 1: metabolic and physiological changes. British Journal of Anaesthesia,48, 297-304. McLaughlin, J. V., Somers, C. J., Ahern, A. P., and Wilson, P. (1978). The influence of neuroleptic drugs on the development of muscular rigidity in halothane sensitive pigs. In The Biochemistryof Myasthenia Grauis and Muscular Dystrophy. G. G. Lunt and R. M. Marchbanks, Eds, Academic Press, London, pp, 361-365. McLaughlin, J. V., and Mothersill, C. (1976). Halothane-induced rigidity and associated glycolytic changes in red and white fibres of skeletal muscle of the pig.
Journal of ComparativePathology,86, 465-476.
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Nelson, T. E., Jones, E. W., Venable, J. H., and Kerr, D. D. (1972). Malignant hyperthermia of Poland China swine. Anaesthesiology, 36, 52-56. Patriarcia, P., and Carafoli, E. (1969). A comparative study of the intracellular Ca2 + movements in white and red muscle. Experientia, 2516, 598-599. Reed, K. C., and Bygrave, F. L. (1974). Methodology for in vitro studies of Ca2 f transport. Analytical Biochemistry, 67, 44-54. Somers, C. J., Wilson, P., Ahern, C. P., and McLaughlin, J. V. (1977). Energy phosphate turnover and glycolysis in skeletal muscle of the pietrain pig: the effects of premeditation with azaparone and pentobarbitone anaesthesia. Journal of Comparative Pathology, 87, 177-183. Sybesma, W., and Eikelenboom, G. (1969). Malignant hyperthermia syndrome in pigs. Netherlands Journal of Veterinary Science, 2, 155-160. Tisdale, H. B. (1967). Preparation and properties of succinic-cytochrome c reductase (Complex ZZ-ZZZ). In Methods of Enzymology. R. W. Estabrook and M. Pulman, Eds, Vol. 10, Academic Press, New York, pp. 2 13-2 16. [Received for publication,
February 1 Oth, 198 I]
PATH.
1982.
VOL.
191
92.
MALIGNANT HYPERTHERMIA UPTAKE BY MITOCHONDRIA MUSCLE OF SUSCEPTIBLE NEUROLEPTIC DRUGS
IN
PIGS: CALCIUM FROM SKELETAL ANIMALS GIVEN AND HALOTHANE
ION
BY
C. J. SOMERS* and J.V.
MCLOUGHLIN
Department of Physiology, Trinity College, Dublin 2, Ireland
INTRODUCTION
The anaesthetic halothane (2-bromo-2-chloro-l,l,l-trifluorethane), either alone or in combination with suxamethonium, is associated with the development of the malignant hyperthermia syndrome (MHS) in man and the pig. The syndrome usually develops very rapidly in the pig. The main clinical manifestations are rigidity of the skeletal muscles, hyperthermia, tachycardia and arhythmia, and respiratory and metabolic acidosis. (Sybesma and Eikelenboom, 1969; Berman, Harrison, Bull and Kench, 1970; Nelson, Jones, Venable and Kerr, 1972; Gronert and Theye, 1976; Hall, Lucke and Lister, 1976; Lucke, Hall and Lister, 1976; McLaughlin and Mothersill, 1976). Premeditation of MHS-susceptible animals with neuroleptic drugs such as azaperone, haloperidol and spiperone delays the onset of MHS (Ahern, Somers, Wilson and McLaughlin, 1977; McLaughlin, Somers, Ahern and Wilson, 1978). Prior anaesthesia with barbiturates, e.g. thiopentone (Gronert and Theye, 1976) and pentobarbitone (Ahern, Somers, Wilson and McLoughlin, 1980) or the steroid anaesthetic althesin (Ahern, McLaughlin, Wheatley and Wilson, 1979) has a similar action. There is a progressive loss of high-energy phosphates (“P) and an accumulation of lactate in skeletal muscle during this delay phase which precedes the onset of clinical signs. The depletion of =P in neuroleptic or barbiturate-modified MHS appears to occur more rapidly in red than in white fibres of skeletal muscle (McLaughlin et al.? 1978). This observation agrees with those of other workers which suggest that there may be some particular involvement of red or Type I myofibres in the syndrome. The myogenic aspect of MHS may be associated with increased concentrations of Ca2 + in the myoplasm. It is not known whether this may be caused by release of Ca2 + from intracellular storage sites such as the sarcoplasmic reticulum (SR) or mitochondria, a reduction in the capacity of these organelles to *Present address: The Agricultural 00” l-9975/82/020191 +08 $03.00/O
Institute, Dunsinea, Castleknock, Co. Dublin, Ireland. @ 1982 Academic Press Inc. (London) Limited
192
C.
J.
SOMERS
AND
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V.
MCLOUGHLIN
take up Ca2 + or an influx of extracellular Ca2 + which leads to a calciuminduced calcium release from the SR. It seems likely, however, that alterations in Ca2 + uptake by the SR and mitochondria during MHS may be secondary to other intracellular changes such as a loss of ATP or a fall in pH (Britt, Endrenyi and Cadman, 1975). In the work described in this paper, normal and MHS-susceptible pigs were anaesthetized with halothane following premeditation with neuroleptic drugs. Other susceptible animals were anaesthetized without premeditation so that acute MHS developed. The uptake of Ca2 + by mitochondria from the predominantly red and white fibre areas of M. semitendinosus was investigated. The rate of Ca2 + uptake was determined both in the presence and absence of an adenine nucleotide (ATP). Ad enine nucleotides are known to enhance the uptake and/or retention of Ca 2 + by mitochondria (Carafoli, Rossi and Lehninger, 1965; Asimakis and Jordakl, 1977). MATERIALS
AND
METHODS
Pigs. Purebred Pietrain pigs were obtained from a herd kept for experimental purposes, purebred Landrace from the national breeding herd at the Department of Agricultural Testing Station, Dublin and crossbred Landrace-Large White from commercial sources. The liveweight of the pigs ranged from 30 to 50 kg. Halothane test for susceptibility to MHS. This test was carried out as described by Ahern et al. (1979). The Pietrain (5) and purebred Landrace pigs (2) gave a positive reaction, i.e., were susceptible to MHS. The Landrace-Large White crossbred pigs did not react. Anaesthesia. Halothane. Anaesthesia was induced with 5 per cent halothane (Hoechst, Germany) delivered in oxygen at a rate of 2 1 per min via a face mask. The concentration of halothane was regulated with a Fluotec vaporizer (Cyprane Ltd., Keighley, England) Following induction, the animals were intubated and anaesthesia maintained with 1 to 2 per cent halothane. Pentobarbitone. Anaesthesia was carried out as described by Ahern et al. (1980). Premeditation. Some animals were given the neuroleptic drugs azaperone (2 mg per kg bodyweight or spiperone (70 to 500 pg per kg bodyweight) by the intramuscular route approximately 30 min before the induction of anaesthesia. Both drugs were obtained from Janssen Pharmaceutics, Beerse, Belgium. Muscle. Biopsy specimens were taken from the predominantly red and white fibre areas of M. semitendinosus during anaesthesia and the loading of Ca2 + by mitochondria preparations was assayed. The concentration of creatine phosphate (CP) in the specimens was also determined. Assay of creatinephosphate. Muscular tissue was extracted and CP assayed as described by McLaughlin and Mothersill (1976). &feasurements of Ca2 + uptake by mitochondria. Specimens (2 g) of muscle were trimmed free of connective tissue, rinsed in sucrose solution (250 mM), finely minced and suspended in extraction medium containing sucrose (70 mM), mannitol (210 mM) trisamine buffer (18 mM, pH 7.4), b ovine serum albumin fraction V (Sigma) ( 10 per cent) and EDTA (10 mM) (Davidoff, 1974). Homogenization was carried out as described by Carafoli and Lehninger (1971). The homogenate was centrifuged at low (650 g) and high (8000 g) speeds for 10 min (Hedman, 1965). Mitochondrial pellets were washed in a medium (20 ml) containing sucrose (280 XIM) and trisamine buffer (1 mM, pH 7.4) and centrifuged (8000 g for 10 min). The pellets were suspended
CALCIUM
in washing chemically
UPTAKE
BY
PIG
MUSCLE
193
MITOCHONDRIA
medium and stored at 0 “C. The rate of Ca2 + uptake was measured radiofollowing the procedure of Reed and Bygrave’ (1974). Uptake was also
determined in the presence of a nucleotide (ATP 3 to 6 mM). The concentration of ATP was determined at 258 nM in a Pye-Unicam SP 1700 spectrophotometer and a molar absorption coefficient of 15.3 X 1Og M -I cm -I was used to calculate the value. Mitochondria were incubated for 3 min and incubation terminated by the addition of a solution (0.05 ml) containing ruthenium red (30 PM) and ethyleneglycolbis (Saminoethylether NJVI) tetracetic acid (EGTA, 100 mM). The suspensions were filtered with a Millipore Sampling Manifold and a 0.45 PM filter. The filters carrying particulate material were washed with assay medium, dried and placed in a scintillation cocktail (5 ml) containing Triton X. Samples of the preparations and standard solution of 45Ca2 + were read in a Packard 3375 Scintillation Counter (window setting 50 to 1000, gain 1 per cent). The counting efficiency of samples was 59 per cent and was determined by the channels ratio method. Calcium uptake was expressed as nmol Ca2+ per mg mitochondrial protein per min. Assay of mitochondrial protein. The total protein content of mitochondrial preparations was assayed by the biuret method of Gornall, Bardawill and David (1949) with bovine serum albumen as a standard. Assay of succinic dehydrogenase activity. (SDH). The activity of SDH in mitochondrial preparations was measured by the method of Tisdale (1967) with minor modifications. The specific activity of SDH was calculated using a molar absorption coefficient of 18.5 >’ IO6 M -l cm ml at 550 nM for cytochrome c. The enzyme activity was expressed as pmol cytochrome c reduced per min per mg mitochondrial protein.
RESULTS
The mean values &.E.M. for uptake of Ca 2 -+by preparations of mitochondria from M. semitendinosis of 4 MHZ&susceptible Pietrain and 7 non-susceptible Landrace Large White crossbred pigs premeditated with neuroleptic drugs and anaesthetized with halothane are given in Table 1. The Ca2 + uptake, TABLE “PTAKE
OF CR’+ BY MITOCHONDRIA NEUROLEPTIC-TREATED
-
..-~-~
FROM PIETRAIN
1
SKELETAL MUSCLE OF LANDRACE/LARGE PIGS ANAESTHETIZED WITH HALOTHANE
Myofibre we
Red XTP .4bsmt White Red ATP 3 to 6 mu White
Landrace large white
Pietrain
(n=7;l 166&20* 6.2&0.7t 110*1.3 5.611.3
(n=4) 174-r-25 7.8&25 69klO 5.3+0.9
721*100 26,-t40 376kl15 24+80
608f222 42530 342*30 2316.1
WHITE
___--
AND
Sign&znce~
._^
N.S. N.S.
N.S. N.S.
*nmol Ca* f per mg mitochondrial protein. tnmol Ca”+ per nmol cytochrome c reduced/min. $Landrace/Large White v. Pietrain.
expressed either on the basis of mg mitochondrial protein per min or SDH activity (pm01 per cytochrome c reduced per mg mitochondrial protein/min), was not significantly different between the MHS-susceptible and non-susceptible pigs. At the time when the biopsy specimens were taken (30 min anaes-
194
C.
J,
SOMERS
AND
J.
V.
MCLOUGHLIN
thesia) the Pietrains had not yet shown clinical signs of MHS. Furthermore, the concentration of CP in the M. semitendinosus (red fibres 17.5 f 1.8, white 21.0 i 3.6 pmol per g) was similar to that found when the Pietrain pig is anaesthetized with an agent such as pentobarbitone which does not trigger the syndrome, an observation which suggested that the muscle was physiologically normal at the time of excision (Somers, Wilson, Ahern and McLaughlin, 1977). The mean values ~s.E.M. for a group of MHS-susceptible pigs exposed to halothane without premeditation with neuroleptic drugs are given in Table 2. The group TABLE Ca2+UpTAKE
BY MITOCHONDRIAFROM semitendinosus OF PIGS WHICH
Myojibre
2
THE PREDOMINANTLY RED AND WHITE DEVELOPED MHS DURING HALOTHANE
ape
ATP
Absent 44&&o* 1.9+0-q 45* 14* 2.4+0.7+
Red White *nmol Cat+ per tnmol Ca2 + per $.Data compared not develop MHS Mean values for
M.
Significance$
Ca2 + uptake ATP
FIBRE AREASOF ANAESTHESIA
3 to6mM 1 lo+26 5.6& 1-3 111*25 9.6k2.2
mg mitochondrial protein. nmol cytochrome c reduced/min. with corresponding data in Table under halothane anaesthesia. 7 animals.
1 for animals
P
which
did
consisted of 5 Pietrain and 2 pure-bred Landrace pigs. The biopsy specimens were taken after 30 min of anaesthesia, at which time muscular rigidity had developed. The development of MHS was associated with a significant reduction (Pt0.001) in the rate of Ca2 + uptake. The reduction was greater in red than in white myofibre preparations (P
Myojbre
type
Calcium Untreated
Red White
3 Gas+
BYPREPARATIONSOF
IN VITRO
uptake* Spiperone treated
58*8.7 26k4.4
87.5 f 10.6 35.8* 6.1
Signiflcancet PiO.05 N.S.
*nmol Ca2 f per mg mitochondria protein. tuntreated v. treated. Mean values&s.E.M. for 8 preparations.
neuroleptic drug significantly increased (P
CALCIUM
UPTAKE
BY
PIG
MUSCLE
195
MITOCHONDRIA
greater in preparations of mitochondria from red than from white myofibres, both in the absence (Pt0.01) and in the presence of adenine nucleotide (P
Caa+ RED
4
UPTAKEBYMITOCHONDRIALPREPARATIONS AND WHITE FIBRE AREAS OF PIG
Calcium uptake
hjwjibre ATP absent (1)
FROMTHE
&i. semitendinosus Al-P (3 to 6
mM)
Red
(2) White Significance (1) v. (21 *nmol tnmol
CaZ+ Ca2+
per mg mitochondrial per nmol cytochrome
protein. 6. reduced/min
DISCUSSION
The results indicate that mitochondria from the predominantly red myofibre area of M. semitendinosus of the pig accumulate Ca2 7. at a greater rate in vitro than do preparations of these organelles isolated from the predominantly white area. The difference between mitochondria from the two myofibre types was observed both in the presence and absence of an adenine nucleotide (ATP). When the rates of Ca2+ uptake were expressed on the basis of the succinic dehydrogenase activity of the muscle, there were no differences in activity between mitochondrial preparations from myofibre types. These observations support those of Kitchen, Van Winkle, Cruikshank, Spiegel and Zainis (1978) who reported that the rate of Ca2 + uptake was significantly higher in mitochondrial preparations from the soleus muscle of the rat (slow contracting) than in those from the tibialis (fast contracting). The results of these authors, when expressed on the basis of succinic dehydrogenase activity, likewise showed no difference in the rate of Ca2 + between the two muscles. Our results are in agreement with those of other workers. Differences in Ca2 + uptake by mitochondria from red and white myofibres of skeletal muscle were described by Patriarcai and Carafoli (1969). Following the administration of 45Ca2 + to rabbits, a higher concentration of the isotope was found in the mitochondrial fraction from red than from white myofibres. Lehninger (1970, 1974) suggested that the mitochondria from both red skeletal and cardiac myofibres may be involved in the regulation of Ca2 + concentrations in the sarcoplasm. In a comparative study of Ca2 + uptake by the sarcoplasmic reticulum and mitochondria from cardiac muscle, Kitagawa (1976) concluded that although the sarcoplasmic reticulum played a major role in controlling intracellular Ca2 + movements during physiological contractures, nevertheless during maximum contraction of cardiac muscle the sarcoplasmic reticulum alone could not bring about relaxation of its myofibres in the absence of Ca2
196
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accumulation by mitochondria. Becker, Fiskum and Lehninger (1980) reported that at intracellular concentrations of Ca2 + both mitochondrial and endoplasmic reticulum preparations from liver actively participate in the regulation of the free Ca2 + concentration. These results also lend further support to the observations by McLaughlin et al. (1978) that neuroleptic drugs delay the onset of halothane-induced MHS. These authors reported that during the delay phase a progressive fall in the concentration of creatine phosphate and a rise in that of lactate occurred in M. semitendinosus. The loss of the high energy phosphate and the accumulation of lactate was more marked in the predominantly red than in the predominantly white fibre area of the muscle. The observation suggested that the red myofibre areas may be more susceptible to the actions of halothane than the white. The findings reported here show that when susceptible pigs developed MHS the mitochondria from red myofibres exhibited a greater reduction in Ca2 +-accumulating ability than did those from white. If the hypothesis that the trigger for MHS-associated rigidity and increased metabolic activity in skeletal muscle is a rise in the concentration of myoplasmic Ca2 + is accepted, then a reduction in the Ca2 +-binding capacity of mitochondria would perhaps affect the more mitochondrial-dependent red myofibres earlier than the sarcoplasmic reticulum-dependent white myofibres and stimulate both contractile and metabolic activity in the former.
SUMMARY
The uptake of calcium ions by mitochondria from skeletal muscle was similar for malignant hyperthermia syndrome (MHS)-susceptible pigs given neuroleptic drugs and normal pigs. The onset of MHS in the former was associated with a significant reduction in calcium ion uptake. Spiperone increased the binding of calcium by a preparation of mitochondria in vitro. Mitochondria from the predominantly red fibre area of M. semitendinosus had a greater calcium binding capacity than those from the predominantly white fibre area.
ACKNOWLEDGMENTS
The authors wish to thank the Agricultural Institute for the award of a grant. Mrs Mary Butler provided skilled technical assistance.
REFERENCES
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