AbstractCalcium-induced calcium release and halothane-induced calcium release from pig sarcoplasmic reticulum (SR) were studied. The SR prepared from pig susceptible to malignant hyperthermia (MH) was shown to release calcium at a much lower level of calcium content than in normal pig SR. The concentration above which halothane can release calcium is 40 μM for both MH-SR and normal SR, although the latter required a high calcium content to demonstrate the calcium release. Dantrolene was shown to inhibit the halothane-induced calcium release. Results suggests that SR plays an importnat role in pathogenesis of MH

Gronert, Gerald A.

Taylor, Stuart

Tsuyoshi Ohnishi, S.

Elsevier - Publisher Connector 

Volume 161, number 1 FEBS 0771 September 1983 
Calcium-induced Ca2+ release from sarcoplasmic reticulum of 
pigs susceptible to malignant hyperthermia 
The effects of halothane and dantrolene 
S. Tsuyoshi Ohnishi, Stuart Taylor* and Gerald A. Gronert+ 
Department of Hematology and Medical Oncology, Hahnemann University School of Medicine, Philadelphia, 
PA 19102, *Department of Pharmacology and +Department of Anesthesiology, Mayo Medical School, Rochester, 
MN 55905, USA 
Received 1 June 1983 
Calcium-induced calcium release and halothane-induced calcium release from pig sarcoplasmic reticulum 
(SR) were studied. The SR prepared from pigs susceptible to malignant hyperthermia (MH) was shown 
to release calcium at a much lower level of calcium content han in normal pig SR. The concentration 
above which halothane can release calcium is 40 PM for both MH-SR and normal SR, although the latter 
required a high calcium content o demonstrate he calcium release. Dantrolene was shown to inhibit the 
halothane-induced calcium release. Results uggest that SR plays an important role in pathogenesis of MH. 
Calcium-induced calcium release Malignant hyperthermia Calcium release, from sarcoplasmic reticulum 
Halothane 
1. INTRODUCTION 
It is theorized that malignant hyperthermia 
(MH) is triggered by abnormally high levels of 
ionized calcium in muscle [I]. Since the sar- 
coplasmic reticulum (SR) plays a key role in 
regulating the level of Ca2+ in the cytoplasm of 
muscle, many studies have been done to determine 
the effects of halothane and other drugs on SR. 
However, previous reports of the effects of 
halothane on calcium fluxes across isolated SR 
vesicular membranes are inconsistent (for an ex- 
tensive list of references, see [2]). Because the 
results are so divergent, no clear-cut conclusion 
has been reached as to whether or not SR is really 
playing a role in triggering MH. Confusion in this 
area of study appears to result from several 
reasons; each investigator uses SR prepared by dif- 
ferent methods and measures calcium fluxes with 
different techniques under different conditions. In 
addition, the most physiological method to release 
Dantrolene 
calcium from isolated SR has not been established. 
Furthermore, there is no clear evidence to prove 
that halothane (which triggers MH) and dantrolene 
(which inhibits the symptom of MH) regulate the 
calcium fluxes of isolated SR in vitro [2]. In fact, 
it is reported that dantrolene does not inhibit either 
calcium-induced or membrane potential-induced 
calcium release from vesicles of isolated SR of rab- 
bits [3]. 
Since the contraction of skeletal muscle is caused 
by Ca2+ release from the SR, it seemed important 
to compare the effects of halothane and dantrolene 
on the Ca2+ release of SR preparations isolated 
from both normal and MH porcine muscles. 
Although calcium-induced Ca2+ release is pro- 
bably not the primary mechanism by which normal 
excitation-contraction coupling is initiated in 
vertebrate skeletal muscle, the presence of caffeine 
or other agents that enhance contractions might 
release additional calcium by this mechanism 
[7,13]. Therefore, we have used the calcium- 
Published by Elsevier Science Publishers B. V. 
00145793/83/$3.00  1983 Federation of European Biochemical Societies 103 
Volume 161, number 1 FEBS LETTERS September 1983 
induced calcium release phenomenon, which can cuvette. To the SR suspension (containing 
be observed in isolated SR at low Mg*+ concentra- 0.5 mg/ml of protein, 10 mM MgClz and 1OOpM 
tions [4-61 as a model to study the effects of Ca*+), 100 PM ATP was added. From the recorder 
halothane and dantrolene on Ca*’ release from trace of Ca*+ uptake and release [lo], the max- 
vesicles of porcine SR. We compared the imum amount of Ca*+ sequestered by the SR was 
Ca*+-induced Ca*+ release and halothane-induced measured and used as an index of Ca*+-uptake 
Ca*+ release from isolated SR of genetically sus- capacity of SR. The amount of endogenous Ca*+ 
ceptible MH-pigs and their littermates. The results content of the SR was determined from a separate 
suggest hat SR from normal and MH-pigs are dif- experiment whereby 10 pM A23 187 was added to 
ferent, and that SR may play an important role in the SR suspension and the amount of Ca*+ release 
the pathophysiology of MH. was measured. 
2. MATERIALS AND METHODS 3. RESULTS 
Three genetically susceptible MH-pigs and 3 lit- 
termate normal pigs (Pietrain: 20 to 50 kg) were 
used. Under thiopental anesthesia, we ventilated 
the animals and maintained arterial CO2 tension at 
40 mm Hg with a Harvard pump connected to an 
endotracheal tube. A mixture of 50% NOz-50% 
02 was used for ventilation while the semiten- 
dinosus muscle was removed surgically. The mus- 
cle was subsequently used for preparing isolated 
SR which began immediately after excision of the 
muscle. Heavy fraction of SR was prepared by a 
differential centrifugation method [8] with the 
following modifications: muscle weighing 100 g 
was homogenized in a Waring blender with 400 ml 
solution containing 20 mM MES buffer (2-(N- 
morpholino)ethanesulfonate; pH 6.8); and 
2.5 mM NaOH for 180 s (30 s x 6). The pH of the 
homogenate was adjusted to 6.8 (using BTB paper) 
by the addition of a small amount of 1 M NaOH. 
The fraction sedimented between 10000 x g for 
3 min and 17000 x g for 25 min was collected and 
wash-centrifuged once with a buffer solution con- 
taining 150 mM KC1 and 20 mM MES (pH 6.8). 
The pellets were resuspended in the same buffer 
and used for the experiment. The protein concen- 
tration was determined by the Biuret method. 
Fig. 1 demonstrates ATP-induced Ca*+ uptake 
and subsequent Ca*+ release induced by halothane 
(A) MH (B ) NORMAL 
ATP 
ATP 
1 HAL 
I 
I_-_- 
ATP 
60 
40 
20 
AJP 
Co 
I+-+ 
I MINUTE 
Calcium-induced Ca*+ release experiments were 
performed at 25°C as in [4,5] using arsenazo III as 
an indicator [9]. The ordinate of figures in this 
paper represents total [Ca*+] in the SR suspension 
as measured by the indicator. Thymol-free 
halothane was prepared by distilling commercial 
halothane solution. This was dissolved in ice-cold 
water to make a final concentration of 10 mM. 
The solution was made daily. This solution was 
added directly to the suspension of SR in the 
Fig.1. Halothane-induced Ca” release and 
Ca2+-induced Ca2+ release from SR prepared from (A) 
MH muscle and (B) normal muscle. Top and middle 
traces demonstrate the halothane effects and bottom 
traces Ca’+-induced Ca2+ release. Ca2+ concentrations 
were 40 pM (top), 90 PM (middle) and 60 PM (bottom). 
Additions of 0.5 mM ATE, 2OOpM halothane and 
20 pM Ca2+ were made at positions indicated by arrows. 
Experimental conditions: 150 mM KCl, 20 mM MES 
buffer (pH 6.8), 0.5 mM MgClz, 1.5 mg SR protein/ml, 
9pM arsenazo III, at 25°C. 
104 
Volume 161, number 1 FEBS LETTERS September 1983 
(200 ,&I) or 20 pM Ca”. The con~ntrations of 
Ca2+ added to the SR suspension prior to the addi- 
tion of ATP were 40 /IM (top traces), 90 FM (mid- 
dle traces) and 60 ,uM (bottom traces). When ATP 
is added to the SR suspension, Ca2’ in the suspen- 
sion is taken up by the SR (Ca2’-loading). When 
[Ca2’] reaches a near-zero level, 200 PM halothane 
(top and middle traces) or 20pM Ca” (bottom 
traces) are added to examine whether Ca2+ is 
released from inside the SR [4,5]. As shown, the 
SR prepared from normal pig (right-side traces) 
did not release Ca2+ by halothane at the 
Ca2+-loading of 40 FM (for 1.5 mglml SR pro- 
tein). It requires a Ca2+-loading of 9OpM to res- 
pond to the halothane addition. On the contrary, 
with SR prepared from susceptible pig (left-side 
traces), halothane released Ca2’ even at the 
CaLf-loading of 40,~M. The same phenomenon 
was observed with the Ca2”-induced Ca” release. 
At the Ca2+-loading level of 60pM, MH-SR 
released 24 FM Ca2+ from inside on addition of 
20 PM Ca2’ to the SR suspension (bottom left). At 
the same condition, normal SR did not release any 
Ca2+ from inside; added Ca2’ (20 PM) was simply 
sequestered by the SR. 
Fig.2 shows the relationship between the level of 
r.3 
E 
\ 
I 
z 
30 
.5 MH ,__--o”- 
. 7-- 
+ 
N 
0 
v 
1 I I 
cl 40 80 I20 
f I Ca2+ L I n moles fmg 1 
c 
j y; *_____~~~ 
u 
I I I 
0 50 100 150 200 
HALOTHANE (JJM) Fig.2. Relationship between the level of Ca2+-loading 
and the amount of halothane-induced Ca2+ release. 
Experimental conditions are the same as those in fig.1 
(top or bottom traces), except for the Ca2+ 
concentration which was varied. The halothane 
concentration used to induce the Ca2+ release was 
2OOfiM. halothane concentration which was varied. 
Fig.3. The dose-response relationship of halothane- 
induced Ca2+ release. The levels of Ca2+-loading 
fnmol/mg) are indicated in the figure. The experimental 
conditions are the same as those in fig. 1, except for the 
Ca2+-loading and the amount of Ca” release upon 
the addition of 2OO/rM halothane. There is a 
threshold value, below which halothane was 
unable to release Ca2+ from the SR. Above the 
threshold value, the amount of Ca2+ release in- 
creased with the increased level of Ca2+-loading, 
but the amount quickly reaches a plateau. By ex- 
trapolating the initial increasing portion of the 
curve, the threshold value could be estimated. The 
major difference between MH-SR and normal SR 
is that the threshold value for halothane-induced 
Ca2’ release of MH-SR is much less than that of 
normal SR. For the addition of 200 gM halothane, 
the threshold values of Ca2+-loading for Ca2’ 
release are 13.3 + 3.0 nmol/mg SR protein for 
MH-SR and 47.0 f 6.5 for normal SR, respective- 
ly @ < 0.001). The Ca2+ uptake capacities are 
155.0 2 15.5 nmol/mg SR protein for MH-SR 
and 160.5 f 12.3 for normal SR (p > 0.2); the en- 
dogenous Ca2+ contents are 52.0 f 4.0 nmol/mg 
SR protein for MH-SR and 52.7 f. 6.4 for normal 
SR, respectively 0, > 0.2). 
Fig.3 indicates dose-response relationship of 
halothane-induced Ca2+ release. At the Ca2+ 
-loading level of 27 nmollmg, MH-SR started to 
release calcium at the halothane concentration of 
40 ,uM, whereas no release is observed with normal 
SR. When the level of Ca2+-loading of normal SR 
is increased (e.g., 60 nmol/mg) halothane-induced 
105 
Volume 161, number 1 FEBS LETTERS September 1983 
DANTROLENE 
I HiLOTHANE 100 JIM 
I MINUTE 
Fig.4. Inhibition of halothane-induced Ca*+ release by 
dantrolene. Concentration of halothane: 100 PM. 
Concentrations of dantrolene: O-40 pM. Other 
experimental conditions: 150 mM KCl, 20 mM MES 
(pH 6.8), 2 mM M&12, 1.5 mg SR protein/ml, 9/1M 
arsenazo III, 40pM CaCl2 and 1 mM ATP. 
Ca2+ release was initiated again at 40 PM 
halothane. 
Fig.4 demonstrates the effect of dantrolene on 
the halothane-induced Ca2+ release. Both the 
velocity and the amount of Ca2+ release were in- 
hibited by dantrolene. 
4. DISCUSSION 
At low Mg2+ concentrations (e.g., 0.5 mM), 
striated muscle demonstrates the phenomenon call- 
ed calcium-induced Ca2+ release [l 1,121. This 
phenomenon has been extensively studied in skin- 
ned muscle fibers of skeletal muscle [ 131 and car- 
diac muscle [ 141. This concentration of Mg2+ is not 
a physiological one. 
However, since a reproducible calcium-induced 
Ca2+ release and halothane-induced Ca2+ release 
from isolated SR is observed with this condition, 
we have been using this as an in vitro model for 
Ca2+ release [4,5]. 
It has been proposed that the mechanisms of 
halothane-induced and calcium-induced Ca2+ 
release involve the same channel [5]. These release 
phenomena appear to be transient. The fact that 
released Ca2+ is sequestered again by the SR sug- 
gests that the channel closes shortly after it opens. 
However, we cannot rule out the possibility that 
the halothane-induced Ca2+ release occurs in a 
subpopulation of SR and the released Ca2+ is then 
reaccumulated by another (halothane-insensitive) 
subpopulation. 
Here, the halothane-induced and Ca2+-induced 
Ca2+ release phenomena of SR preparations 
isolated from normal pigs and MH pigs are 
studied. All pigs used here were examined via biop- 
sy and halothane exposure to determine that mus- 
cle was susceptible to halothane. The control pigs 
were littermates with a normal response to 
anesthesia. We observed that both the Ca2+ uptake 
capacity and the endogenous Ca2+ content of 
MH-SR and normal SR preparations were the 
same. The difference between MH and normal SR 
seemed to be the level of Ca2+-loading at which 
halothane started inducing Ca2+ release. 
Halothane released Ca2+ from MH-SR at a low 
level of loading (13.3 nmol/mg compared to 
47 nmol/mg for normal SR). If the level of 
Ca2+-loading was less than 47 nmol/mg SR pro- 
tein, Ca2+ release was not observed with normal 
SR by halothane triggering. According to [13], the 
level of Ca2+ in SR in vivo is less than 0.25 of its 
full capacity. It is, therefore, tempting to speculate 
that the level of Ca2+ in SR of normal pigs is such 
that halothane does not trigger Ca2+ release, 
whereas at the same level of Ca’+, halothane in- 
duces the Ca2+ release in MH-SR. 
At an elevated level of Ca2+-loading, normal SR 
also released Ca2+ by halothane; the maximum 
amount of the release was the same as that from 
MH-SR. It is interesting to note that the threshold 
concentration of halothane required to trigger the 
release was also the same: 40 PM for both MH and 
normal SR. (The threshold concentration of 
halothane for Ca2+ release from rabbit skeletal SR 
is also 40 pM [5]. At room temperature, 40 PM 
halothane in water is in equilibrium with about 
0.08% halothane vapor (v/v) in the air [15], which 
is well under the anesthetic concentration 
(0.3-l%).) This suggests that the affinity of 
halothane receptors on the putative Ca2+ channel 
[5] of both MH and normal SR is the same. A 
possible difference between MH and normal SR 
may be that the permeability of the Ca2+ channel 
106 
Volume 161, number 1 FEBS LETTERS September 1983 
of MH-SR is greater than that of normal SR. This 
would allow the Ca’+-efflux in MH-SR to start at 
a lower level of Ca2+-loading than in normal SR. 
A phenomenon of regulation of Ca” by the inter- 
nal Ca” concentration has been studied in rabbit 
SR [ 16,171. An alternative explanation for this dif- 
ference is that MH-SR is more labile than control 
SR so that MH-SR is damaged during the 
preparation procedure. Further study is needed to 
clarify this point. 
Dantrolene has been used as a drug to effectively 
manage the MH symptom in both man and pig [ 11. 
The drug was shown to slow down the spontaneous 
release of Ca2+ from the isolated SR [ 181. We have 
obtained more direct evidence to show that dan- 
trolene inhibits the halothane-induced Ca2+ release 
from the isolated SR (fig.4). Further support for 
the idea that dantrolene suppresses Ca2+ release 
was shown in isolated intact skeletal muscle fibers 
injected with Aequorin and inactivated via mem- 
brane depolarization [ 191. 
Since SR is not the only organelle in muscle that 
influences the level of ionized calcium in the 
myoplasm, we cannot rule out the possibility that 
abnormalities of other organelles, or membrane 
systems in intact muscle may also be involved in 
the pathogenesis of MH [20]. However, the data 
presented here suggest hat SR itself plays an im- 
portant role in MH. 
ACKNOWLEDGEMENTS 
Authors would like to extend their sincere ap- 
preciation for the assistance provided by Mr 
Jeffrey L. Flick, Mr Jack Pyenson, Mr Joseph J. 
Sabadish, Dr Kazumi Horiuchi and Dr Shyh-rong 
G. Fang of Hahnemann University School of 
Medicine, and Mr James H. Milde of Mayo 
Medical School. Thanks are also due to Dr 
Thomas E. Nelson, University of Texas Medical 
Branch, Galveston, for his participation in the 
preliminary stage of this work. This work was sup- 
ported in part by NIH grants GM30703, HL23788, 
AAO4320, GM21729 and NS14268. 
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Calcium-induced Ca2+ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia The effects of halothane and dantrolene

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Calcium-induced Ca2+ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia The effects of halothane and dantrolene

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