Br.J. Anaesth.

(1980), 52, 319

EFFECTS OF TEMPERATURE, ADENOSINE TRIPHOSPHATE AND

MAGNESIUM CONCENTRATIONS ON THE CONTRACTION OF
ACTOMYOSIN ISOLATED FROM HALOTHANE-SENSITIVE
AND -INSENSITIVE GERMAN LANDRACE PIGS
R. A. GREEN, G. MITCHELL AND J. J. A. HEFFRON

SUMMARY

The effects of temperature, adenosine triphosphate and magnesium on the calcium sensitivity of

Downloaded from http://bja.oxfordjournals.org/ at University of California, Santa Barbara on June 30, 2015
actomyosin were investigated on actomyosin obtained from six halothane-sensitive and five
halothane-resistant German Landrace pigs. No difference in the contractile properties was found
in actomyosin from the two types of pig. However, in both types calcium sensitivity of the acto-
myosin was lost at temperatures slightly greater than those occurring physiologically. Both ATP
and Mg l + protect against the loss of calcium sensitivity. These results suggest that the basic lesion
in halothane-induced malignant hyperthermia does not lie in the contractile proteins. It is likely,
however, that the decreases in intracellular ATP and Mg*+ concentrations which occur in pigs
during malignant hyperthermia, contribute to the development of the syndrome.

Malignant hypothermia (MH) is a rare syndrome
occurring in humans exposed to certain anaesthetic
agents, especially halothane. A similar or identical
syndrome occurs in some breeds of pig (Britt and
Kalow, 1970). MH is characterized by a rapid
increase in body temperature (> 2 °C h" 1 ), lactica-
cidosis, muscle rigidity and death (Relton, Britt and
Steward, 1973).
The site of the primary lesion is considered to be
the skeletal muscle as this is the most likely source of
heat, lactic acid and increased concentrations of
serum creatine phosphokinase (c.p.k.) (de Villafranca
and Waksmonski, 1970). A central cause has been
eliminated on the basis of clinical findings (Britt
and Kalow, 1970).
Until recently the emphasis of research has been
on the sarcoplasmic reticulum (SR), which was
thought to be especially sensitive to anaesthetic agents
in susceptible individuals, the dominant theory
being that the SR released calcium into the sarco-
plasm in response to an effect of the triggering agent
(Britt and Kalow, 1970; Hall, Lucke and Lister,
1975).
Increases in the calcium concentration (approx-
imately 5 x 10"6 mol litre"1) would account for the
R. A. GRHEN, B.SC.; G. MITCHELL, B.SC., B.V.SC, PH.D.;
J. J. A. HEFFRON,* PH.D.; Departments of Physiology and
Physiological Chemistry,* University of the Witwatersrand,
Medical School, Hospital Street, Johannesburg.
Correspondence to G. Mitchell, Department of Physio-
logy, School of Veterinary Science, Park Row, Bristol BS1
5LS (until December 31, 1980).
0007-0912/80/030319-05 $01.00
muscle rigidity, as a result of increased actomyosin
interaction, activation of phosphorylase kinase, and
an increase in the rate of glycolysis which could in
turn account for the generation of heat and the
production of lactate. However, the fact that mem-
brane stabilizing agents, which affect calcium move-
ments out of and into the SR cannot reverse the
established syndrome, suggests that the SR is not
the site of the primary lesion (Hall, Trim and
Woolf, 1972; Mitchell and Heffron, 1975).
Recently, Fuchs (1975) suggested that there may
be a loss of calcium sensitivity in the contractile
proteins of sensitive muscle during MH rather than
an increase in myoplasmic calcium concentrations.
Fuchs (1975) and Fuchs, Hartshorne and Barns
(1975) showed also that when the temperature was
increased to slightly greater than that occurring
physiologically, there was a loss of calcium dependence
for the occurrence of superprecipitation in frog,
rabbit and human muscle natural actomyosin (NAM).
It has been shown also that increased concentrations
of adenosine triphosphate (ATP) can "protect"
against this loss of calcium sensitivity (Levy and
Fleisher, 1965). Furthermore, it has been shown that
halothane inhibits porcine mitochondrial respiration
when NAD-linked substrates are used (Eikelenboom
and van der Bergh, 1971). This could result in a
reduction in ATP synthesis. Therefore, it seems
possible that, if there is an underlying deficiency in
the production of ATP, muscle contraction will occur
independent of calcium concentrations, as the
temperature increases.
© Macmillan Journals Ltd 1980
320 BRITISH JOURNAL OF ANAESTHESIA

In addition, Hall, Lucke and Lister (1975) have
shown that infusions of Mg 2+ can decrease the
muscle rigidity characteristic of the syndrome. This
suggests that Mg 2 + , which leaks out of myoplasm
during the syndrome (Bemoan et al., 1970), may
play a regulatory role in excitation-contraction
coupling, and its leakage could, therefore, precipitate
contraction independent of the calcium concentra-
tion. This effect may also be temperature dependent.
age loss of calcium sensitivity was calculated using
the response to ATP-Mg addition in the absence of
calcium (EGTA added), and the maximum response
when calcium was added.
Analytical grade reagents and double-distilled
water were used throughout the experiment. ATP
(disodium salt) was obtained from Boehringer.
RESULTS

Therefore, we have examined tie effect of tempera- Figure 1 shows a typical tracing of the superprecipita-
ture on natural actomyosin (NAM) obtained from tion phenomenon when full calcium sensitivity was

Downloaded from http://bja.oxfordjournals.org/ at University of California, Santa Barbara on June 30, 2015
halothane-sensitive and -resistant pigs, and the retained. The response decreased after an initial
effects of various concentrations of ATP and Mg 2 + rapid increase following the addition of calcium.
on the calcium sensitivity of excitation-contraction Figure 2 shows the percentage loss of calcium
coupling.
0.4 n
MATERIALS AND METHODS
Natural actomyosin was prepared from 11 pigs (six 0.3-
halothane susceptible, five resistant), by extracting o
w
homogenized biopsied muscle with 4 volumes of a
solution containing: potassium chloride 0.6 mol < 0.2-
litre- 1 , Tris-HCl 50mmol litre- 1 (pH 7.5) and ATP- MgiOmrnol litre"1
dithreital 1 mmol litre- 1 (DTT) for 24 h at ( M °C. CaP'OSmmolItre"
O.l-
This was followed by dilution with a further 4
volumes and centrifugation at 20 000 £ for 30 min to
remove non-solubilized material. The supernatant O 2 3 4 5 6 7 8 9 IO

was diluted with 9 volumes of double-distilled water, Time

and the actomyosin collected at 5000 £ and resus-
FIG. 1. A typical tracing of precipitation of actomyosin by
pended in 3 volumes of a solution containing: calcium; temperature = 35 °C. The precipitation results in
Tris-HCl 25 mmol litre- 1 (pH 7.3), D T T 1 mmol increased absorbance.
litre- 1 and potassium chloride 0.6 mol litre- 1 . The
purification procedure was carried out twice. The
protein concentration in the final suspension was 5/ 6
lOO-i Nor mo I
measured by the biuret method with a bovine 99- Holothane Sensitive
serum albumin standard and adjusted to 5 mg ml" 1 .
80
All preparations were glycerinated (50%) and
stored at 0 °C until used. 70-
60-
Changes in turbidity associated with precipitation /J=8
50-
of actomyosin (Spicer and Gergely, 1951) were T "

measured on a Beckman Acta C III spectrophoto- 40-

meter, at a wavelength of 660 nm and temperatures 30-
were monitored with a platinum resistance probe in 20-
one of the cuvettes. The incubation solution con- 10 •
tained imidazole HC1 25 mmol litre- 1 (pH7.1), 0
potassium chloride 50 mmol litre- 1 , D T T 1 mmol -10
litre- 1 , protein 0.2 mg ml" 1 , 4% glycerol, ethylene 30.5 35.5 40.0 43.5 46.0
glycol bis (p-aminoethyl ether) N,N,N',N'-tetra- Temperature (°c)
acetic acid (EGTA) 0.8 mmol litre- 1 , and calcium
chloride 0.8 mmol litre- 1 , magnesium sulphate and FIG. 2. The effect of temperature on calcium sensitivity.
ATP as indicated. Each preparation of actomyosin contained ATP 1 mmol
litre" 1 and Mg 1+ 1 mmol litre"1. The percentage loss of
Cuvette contents were continuously stirred mag- calcium sensitivity is shown as mean and standard error of
netically to avoid settling of the precipitate. Percent- the mean for all preparations at each temperature.
MALIGNANT HYPERTHERMIA: ATP, Mg2+ AND ACTOMYOSIN 321

B
100 - n=5 n-S

i
90 •
80 -
70-
60 -
50 •
40 -

i
30-
20-
10 -

Downloaded from http://bja.oxfordjournals.org/ at University of California, Santa Barbara on June 30, 2015
05 10 20 40 0.5 I.O 20 4 0 Q5 10 4.0
(Mg ATP) mmol litre'1 (ATP) mmol litre"1 (Mg) mmol litre"1
(Mg)=IO mmol litre (ATP)-IOmmol litre'1
FIG. 3. The effects of varying concentrations of ATP + Mg on calcium sensitivity. Percentage loss of
calcium was measured at a cuvette temperature of 43.5 °C.

0.3-1 Temperature
Mg 2 * 1 mmd litre"1
Mg 2 * 4 mmol litre"1
Mg 1 mmot litre'1 +•
ATP 4 mmol litre'1
0.2- •50
o
ID
ID •451?
o
ID
O.I •
ATP and Mg2* added •40 5 CO

I •35 %
©

-30

10 15 20
Time (min)

FIG. 4. The effect of Mg l + on calcium sensitivity. The absorbance of the system was measured at
1-min intervals. Increased concentrations of magnesium delay the onset of precipitation.

sensitivity as temperature was increased from 30 to
46 °C in both halothane-sensitive and non-sensitive
pigs. The large SEM at 40 °C is probably attributable
to the proximity to the critical temperature at which
a large change in calcium sensitivity occurs. Thus at
this temperature, because of slight biological varia-
tion, a large range of responses resulted. There were
no significant differences between the responses of
sensitive and non-sensitive pigs at any temperature
(t test).
Figure 3A shows the effect of different concentra-
tions of magnesium-ATP on percentage loss of
calcium sensitivity at a temperature of 43.5 °C. It is
clear that increasing the concentration of Mg-ATP
maintains calcium sensitivity.
Figure 3B shows the effects of different concentra-
tions of ATP with a constant magnesium concentra-
tion (1 mmol litre"1) at the same temperature. These
results show that increasing ATP concentrations
protected the actomyosin against temperature-
induced calcium insensitivity.
Varying magnesium concentrations alone (fig. 3c)
with a constant ATP concentration gave no protection
against loss of calcium sensitivity. Thus at 43.5 °C
ATP conferred protection against loss of calcium
sensitivity, whereas magnesium did not.
322 BRITISH JOURNAL OF ANAESTHESIA

However, figure 4 shows a typical result found
when the temperature of the cuvettes was allowed to
increase from approximately 30 °C to approximately
50 °C in the absence of calcium but with ATP
1 mmol litre" 1 added. Suspensions containing mag-
nesium 4 mmol litre" 1 underwent superprecipitation
at greater temperatures than those with magnesium
1 mmol litre" 1 . These critical temperatures (estimated tures.
as the temperature at which 50% superprecipitation
occurs) for the five pigs tested are shown in table I.
malfunctioning. Further, the results show that the
effects of aberrant ATP and Mg 2 + concentrations are
potentiated as the temperature increases. It is dear
that at greater temperatures adequate ATP (4 mmol
litre" 1 fig. 4) prevents superprecipitation. However,
if ATP concentrations are inadequate, then loss of
Mg 2 + will cause superprecipitation at lower tempera-
It has been shown that during the syndrome
muscle ATP concentrations are small (Isaacs and
Heffron, 1975) or decline and that Mg 2 + leaks out of

Downloaded from http://bja.oxfordjournals.org/ at University of California, Santa Barbara on June 30, 2015
TABLE I. Critical temperatures for solutions containing the myoplasm into the plasma (Berman et al., 1970).
magnesium 1.0 mmol litre-1 and 4.0 mmol litre'1. *Prepara- Hyperthermia is a characteristic of the syndrome.
tion obtained from halothane-scnsinve pigs Therefore, it is conceivable that the rigor in MH is
Temperatures (°C) initiated or potentiated by a loss in calcium sensitivity
as a result of reduced muscle ATP and Mg 2 + and
Mg«+ Mg«+ hyperthermia which occurs during the syndrome.
Preparation 1 mmol litre" 1 4 mmol litre" 1 In these circumstances attempts to reverse the
1* 43.5 45.0 established syndrome by decreasing myoplasmic
2 44.8 46.3 calcium concentrations must be futile. On the other
3 45.0 46.3 hand, as reported by Hall, Lucke and Lister (1975),
4 45.0 46.3 infusions of Mg 2 + will tend to reverse rigidity.
5* 42.5 44.0 Apart from the apparent protecting effect of Mg 2+
alone on loss of calcium sensitivity (table I), Mg 2+
The differences in temperatures were significant at infusions will also depress glycolysis, stimulate
the 5% level (Wilcoxon test) for all pigs tested, but oxidative phosphorylation and increase uptake by
there were no significant differences between halo- SR (Relton, Britt and Steward, 1973).
thane-susceptible and -resistant pigs, although All these observations suggest that, although the
critical temperatures for sensitive pigs were clearly contractile proteins are not the site of the basic
less than those for insensitive pigs. Figure 4 lesion in MH, they become involved secondarily,
shows also that, in the presence of adequate ATP lose their calcium sensitivity and potentiate an already
(4 mmol litre" 1 ), superprecipitation did not occur as uncontrollable reaction.
the temperature increased, even if Mg 2 + concentra-
tions were small (1 mmol litre" 1 ). REFERENCES
Berman, M. C , Harrison, G. G., Bull, A. B., and Kench,
J. E. (1970). Changes underlying halothane-induced
DISCUSSION malignant hyperpyrexia in Landrace pigs. Nature
(Land.), 225, 653.
These results show that NAM isolated from pig Britt, B. A., and Kalow, W. (1970). Malignant hyperthermia
muscle behaved in the same way as that of frog, aetiology unknown. Can. Anaesth. Soc.J., 17, 316.
rabbit and human muscle (Fuchs, Hartshorne and Eikelenboom, G., and van der Berg, S. G. (1971). Aberrant
mitochondrial energy metabolism in stress-susceptible
Barns, 1975) in that it lost its sensitivity to calcium pigs, in Second International Symposium on Condition and
at temperatures greater than those occurring physio- Meat Quality in Pigs (eds J. C. M. Hassel-de-Heer, G. R.
logically. Schmidt, W. Sybesma and others), p. 66. Wageningen,
In addition, the data indicate that the basic lesion Holland: Pudock Press.
Fuchs, F. (1975). Thermal inactivation of the calcium
in this syndrome does not lie in the contractile regulatory mechanism of human skeletal muscle acto-
proteins as there are no significant differences myosin: a possible contributing factor in the rigidity of
between halothane-susceptible and -resistant pigs malignant hyperthermia. Anesthesiology, 42, 584.
(fig. 2). However, the results suggest that, although Hartshorne, D. J., and Barnes, E. M. (1975). ATPase
the contraction mechanism is similar in all pigs and is activity and superprecipitation of skeletal muscle acto-
myosin of frog and rabbit: effect of temperature on
dependent on adequate ATP and Mg 2 + concentra- calcium sensitivity. Comp. Biochem. PkysioL, 51B, 165.
tions for proper functioning, disturbances in the Hall, G. M., Lucke, J. M., and Lister, D. (1975). Treatment
concentrations of ATP and Mg 2 + will lead to of malignant hyperthermia. Anaesthesia, 30, 308.
MALIGNANT HYPERTHERMIA: ATP, Mg2+ AND ACTOMYOSIN
Hall, L. W., Trim, C. M., and Woolf, N. (1972). Further
studies of porcine malignant hyperthermia. Br. Med. J.,
2,145.
Isaacs, H., and Heffron, J. J. A. (1975). Morphological and
biochemical defects in muscle of human carriers of the
MH syndrome. Br. J. Anaesth., 47, 475.
Levy, H. M., and Fleisher, M. (1965). Studies of the
superprecipitation of actomyosin suspension as measured
by change in turbidity. I: Effects of adenosine triphosphate
concentration and temperature. BioMm. Biophys. Ada,
100, 479.
Mitchell, G., and Heffron, J. J. A. (1975). Procaine in
porcine malignant hyperthermia. Br.J. Anaesth., 47, 667.
Relton, J. E. S., Britt, B. A., and Steward, D. J. (1973).
Malignant hyperpyrexia. Br. J. Anaesth., 45, 269.
Spicer, S. S., and Gergely, J. (1951). Studies on the
combination of myosin and actin. J. Biol. Chem., 188,179.
de Villafranca, G. W., and Waksmonski, C. A. (1970).
Superprecipitation of horseshoe crab and rabbit myosin
B. Int. J. Biochem., 1, 29.
EFFETS DE LA TEMPERATURE ET DES
CONCENTRATIONS DU TRIPHOSPATE
D'ADENOSINE ET DE MAGNESIUM SUR LA
CONTRACTION DE L'ACTOMYOSINE PRELEVEE
SUR DES COCHONS SENSIBLES A
L'HALOTHANE ET SUR DES COCHONS
LANDRACE ALLEMANDS QUI Y SONT
INSENSIBLES
RESUMB
On a fait des recherches sur l'actomyosine obtenue de six
cochons sensibles a 1'halothane et de cinq cochons Landrace
allemands resistants a 1'halothane, pour etudier les effets
de la temperature, du triphosphate d'adenosine et du
magnesium sur la sensibility au calcium de l'actomyosine.
On n'a trouve dans l'actomyosine emanant des deux types
de cochons aucune difference dans les proprietes con-
tractiles. Quoi qu'il en soit, la sensibiliti au calcium de
l'actomyosine a etc, dans les deux types, perdue a des
temperatures legerement superieures a celles qui se pro-
duisent physiologiquement. L'ATP tout comme le Mg 1+
protigent contre la perte de sensibilite au calcium. Les
resultats obtenus laissent penser que la lesion de base qui se
produit dans l'hyperthermie maligns provoquee par
1'halothane ne se trouve pas dans les proteines contractiles.
II est toutefois probable que les diminutions dans les
concentrations intracellulaires d'ATP et de Mg 1+ qui se
produisent chez les cochons pendant l'hypeithennie
maligne contribuent au developpement du syndrome.
323
WIRKUNGEN VON TEMPERATUR,
ADENOSINTRIPHOPHAT UND MAGNESIUM
AUF DIE KONTRAKTION VON ACTOMYOSIN
AUS HALOTHANEMPFINDLICHEN UND
HALOTHANUNEMPFINDLICHEN DEUTSCHEN
LANDRACE-SCHWEINEN
ZDSAMMENFASSUNG
Die Wirkungen von Temperatur, Adenosintriphophat und
Magnesium auf die Kalziumempnndlichkeit von Actomyosin
wurden bei Actomyosin unteTSUcht, das aus sechs halothan-
empfincUichen nnH fflnf halnthnniinprnpfinrllir^pn deutschen
Landrace-Schweinen enmommen wurde. Im Actomyosin
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aus beiden Gruppen von Schweinen fend man keine
Unterschiede der Kontraktionseigenschaften. Bei beiden
Gruppen aber ging die Kalziumempfindh'chkeit bei
Temperaturen verloren, die leicht liber den physiologisch
gegeben lagen. Sowohl ATP und Mg 1+ schutzen vor dem
Verlust von Kalziumempnndlichkeit. Diese Resultate
zeigen, dass die grundlegende Lasion bei durch Halothan
bewirkter, maligner Hyerthermie nicht in den kontraktilen
Proteinen liegt. Es ist aber warhscheinlich, dass das
Absinken intrazellulSrer ATP-und Mg'+-Konzentrarionen,
zu dem es bei Schweinen wahrend maligner Hyperthermie
kommt, zur Entwicklung des Syndroms beitrfigt.
EFECTOS DE LA TEMPERATURA,
CONCENTRACIONES DEL TRIFOSFATO DE
ADENOSINA Y DE MAGNESIO SOBRE
CONTRACCION DE ACTOMIOSINA AISLADA A
PARTIR DE PUERCOS ALEMANES LANDRACE
SENSIBLES E INSENSIBLES AL HALOTANO
SUMARIO
Se averigu6 los efectos de la temperature, del trifosfeto de
adenosina y del magnesio sobre la sensibilidad al calcio de
la actomiosina mediante actomiosina obtenida a partir de
seis puercos alemanes Landrace sensibles al halotano y
cinco resistantes al mismo. No se observaron diferencias
algunas en las propiedades contractiles de la actomiosina
de los dos tipos de puercos. Sin embargo, en ambos tipos, se
perdi6 la sensibilidad al calcio de la actomiosina a tempera-
tures ligeramente mas altas que las que ocurren fisiologica-
mente. Ambos el ATP y el Mg t+ protegen contra la perdida
de sensibilidad al calcio. Estos resultados hacen pensar que
la Iesi6n basica de la hipertermia maligna indudda por
halotano no se debe a las proteinas contractiles. Sin embargo
es probable que las reducciones de las concentraciones
intracelulares en ATP y Mg 1+ que ocurren en los puercos
en el curso de la hipertermina mnligna contribuyan al
desarrollo del sfndrome.