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Dysfunction of sarcoplasmic reticulum in rabbit and human steroid myopathy
EXPERIMENTAL
NEUROLOGY
Dysfunction and SHIN’ICHI
SHOJI,
Department
AKIO
51,
304-309
(1976)
of Sarcoplasmic Human Steroid TAKAGI,
HIDEO
Reticulum Myopathy SUGITA,
AND
of Neurology, Institute of Brain Research, University of Tokyo, Tokyo, Japan Received
December
in Rabbit
YASUO
Faculty
TOYOKURA of
1
Medicine,
17,197s
Experimental steroid myopathy was induced in rabbits by injections of triamcinolone acetonide. Sarcoplasmic reticulum was prepared from quadriceps muscle. Calcium-dependent ATPase activity decreased in the steroidtreated group. Calcium uptake and binding of sarcoplasmic reticulum were also reduced in the steroid-treated group. By the same methods, sarcoplasmic reticulum in human steroid myopathy was analysed. Again, decreased calcium-dependent ATPase activity, calcium uptake, and binding were revealed in steroid myopathy. These results are consistent with the view that the effects of glucocorticoid on skeletal muscle are mediated, in part, through its action on the calcium transport system.
INTRODUCTION Some authors reported that the sarcoplasmicreticulum (SR) was dilated in human steroid myopathy muscle (2). The function of this organelle in this myopathy has been studied only by Peter et al. (6). They examined triamcinolone-treated rats and found no difference from the controls. We studied its function in rabbits and in a patient with steroid myopathy. MATERIALS
AND METHODS
Male albino rabbits, weighing about 2 kg, were used for this experiment. All were given chow diet and water ad lib. Triamcinolone acetonide (%1 We express our sincere thanks to Dr. M. Tomonaga, of Section of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, for his morpho!ogical examinations, and to Dr. R. J. T. Pennington, Honorary Reader in Neurochemistry, University of Newcastle upon Tyne, for his critical review of the manuscript. Dr. Shoji’s present address is : Neurochemistry Department, Regional Neurological Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, United Kingdom. Reprint requests should be sent to Dr. Shoji’s present address. 304 Copyright All rights
1976 by Academic Press, Inc. o3 reproduction in any form reserved.
STEROID
MYOPATHY
30.5
fluoro-16a-hydroxy-prednisolone acetonide) was given daily in doses of 10 mg/kg body wt by subcutaneous injection (40 mg/ml solution) for 7 or 14 days. An equal volume of 0.9% NaCl solution was injected into control animals. Twenty-four hours after the last injection, the rabbits were anesthetized by ether and killed by exsanguination from the carotid arteries. The quadriceps muscles were quickly dissected and the lateral vastus muscles were separated on ice. Sarcoplasmic reticulum fractions were prepared according to a modified Harigaya’s method (3). All procedures were carried out at 0°C on ice. After fat and connective tissue had been cautionsly removed, 7 to 12 g muscle (lateral vastus) was minced with stainless steel scissors and suspended in 9 volumes ice-cold 10 mM NaHC03 solution. The muscle was then homogenized for 15 set three times, with 30-set intervals, using a Polytron homogenizer. The homogenate was then centrifuged for 5 min at 4OOOg, after which the speed was increased to 9000g for 20 min. The supernatant was again centrifuged for 20 min at 9000g. The resulting supernatant was passed through a Whatman No. 41 filter paper to remove fat and air bubbles. The filtrate was centrifuged for 30 min at 40,OOOy in a Spinco ultracentrifuge. The sediment was suspended in about 6 ml of a solution containing 0.6 M KC1 and 20 m&f Tris (pH 6.8) using a small Teflon pestle. The solution was centrifuged again for 30 min at 40,OOOg. The sediment was suspended in about 2 ml of a solution containing 50 mM KC1 and 20 111~ Tris (pH 6.8) using a small Teflon pestle again. The suspension was used as the sar,coplasmic reticulum fraction. Biopsied muscle was obtained from the lateral vastus of a patient with cutaneous disease and steroid myopathy. Control muscle was obtained from the same muscle during the surgery of a patient without muscle disease, of the same age and sex. From l- to 2-g samples of these muscles, sarcoplasmic reticulum was prepared by the same method as mentioned above. Protein was assayed by the Biuret method. ATPase activity was determined by the increase in Pi after incubation at 25°C for 5 min. Inorganic phosphate was measured by the Fiske-SubbaRow technique ( 1). The reaction mixture, 1.5 ml in total, contained 20 mM Tris-malate buffer (pH 6.8), 4 mM MgSOJ, 0.1 M KCl, 0.1 M EGTA, 2 mM ATP, and 24 to 108 lug SR protein. The reaction was started by adding the ATP and was stopped by adding 1 ml of 20% trichloroacetic acid solution. Mg-ATPase activity was measured in this medium. Mg, CaATPase activity was assayed with addition of CaCI, (0.1 mM). The Ca-ATPase activity was calculated as the difference between Mg, CaATPase activity and Mg-ATPase activity, Calcium binding was measured by the Millipore filter method (5). The
306
SHOJI
ET AL.
reaction mixture, 2.0 ml in total, contained 20 mM Tris (pH 6X), 2 mM MgC12, 0.144 M KCl, 4 mM ATP, 0.1 mM CaC12 with radioactive calcium (2 &i/2 ml) and 150 to 230 pg/ml SR protein. When 5 mM potassium oxalate was included in the reaction mixture (Ca-uptake), the amount of sarcoplasmic reticulum protein was reduced to 5 to 23 pg/ml. The reaction was started by adding the ATP. After incubating for 15 min at 25°C the reaction was stopped by passing the medium through a Millipore filter (GS 0.22 mm). Aliquots of both filtrate and incubation medium were dried on planchets and the radioactivity of each sample was counted by a gas flow counter. The calcium binding and uptake were calculated from the difference between the radioactivity of the filtrates of sarcoplasmic reticulum-free incubation medium and of the sarcoplasmic reticulumcontaining samples. RESULTS Lateral vastus muscles of rabbits and patient were examined by histoand electron microscopical methods. Histological logical, histochemical, examinations showed slight to moderate nonspecific myopathic changes, histochemical stains demonstrated a Type II fiber predominance and a
FIG.
tron
1. Fragmented sarcoplasmic micrograph. X 70,000.
reticulum
with
other
unidentified
organelles.
Elec
STEROID
TABLE Sarcoplasmic A Yield
B ATPase
group group
1
Reticulum
of SR
Controls 7-Day l&Day
307
MYOPATHY
of Rabbits
-
(mg SR protein/g
wet vjt muscle) 0.76 0.59h 0.47”
(4),’ (3) (3)
activity
(pmole
Pi/mg
SR protein/min)
Mg-ATPase Controls 7-D ay group 14.Day group C Calcium
uptake
(bmole
I Controls 7-D ay group l&Day group D Calcium
4.66 2.60
binding
group group
Ca/mg SR protein/l5 Experiment
min)
III
I\
5.25 1.86
7.95
5.56
4.15
3.86
ipmole
0.143 0.122
0.615 0.151* 0.3336
II
I Controls 7-Day l&Day
Ca-ATPase
0.484 0.282 0.361
(4) (3) (3)
Ca/mg SK protein/l5 Experiment III
I\
0.211 0.084
0.194
0.220
0.154
0.158
analysed.
2.96 2.69 min)
II
u Numbers in parentheses show number of rabbits of each group. b P < 0.05 (Student’s t) compared with controls.
v
In A, B, data
\ 0.153 0.105 show
means
preferential Type II fiber atrophy, and electron microscopy revealed a dilation of sarcoplasmic reticulum in these muscles. Extracted sarcoplasmic reticulum was also examined by electron microscopy. These fractions showed membranous structures (fragmented sarcoplasmic reticulum) with other unidentified organelles or structures (Fig. 1). Exfievimeutal Steroid Afyo~a.tlzy of Rabbit. The yield of sarcoplasmic reticulum was significantly decreased in steroid-treated rabbits (Table 1, A). Mg,Ca-ATPase and Ca-ATPase were significantly reduced in the steroid-treated groups (Table 1, B). In order to examine the possibility
308
SHO JI
TABLE Sarcoplasmic
Reticulum
ET AL.
2
in Human Control
Yield of SRa Calcium uptakeb Calcium bindingb ATPase activityc Mg-ATPase Ca-ATPase a Results r, Results c Results
in milligrams in micromoles in micromoles
Steroid
Myopathy Steroid
myopathy
1.46 3.89 0.081
0.59 2.51 0.053
0.193 0.774
0.246 0.250
of SR protein per grams wet weight. of Ca per milligrams SR protein per 15 min. Pi per milligram SR protein per min.
of mitochondrial contamination, we measured ATPase with addition of CaCla (0.1 mM) and azide (1 mM). This activity was 94% of the Mg,CaATPase in the controls and 97-98s in the steroid-treated groups. Both calcium uptake and calcium binding were decreased in steroidtreated rabbits (Tables 1 C, D). Comparison of the 7-day group and the 14-day group was done in Experiment V. In this experiment, the 14-day group showed a greater decrease in calcium uptake and binding than the 7-day group. Hzmmz Steroid Myopathy. Sarcoplasmic reticulum isolated from a patient with steroid myopathy revealed the same tendency as shown in rabbit experiments. The yield of sarcoplasmic reticulum was 40% of the control. Ca-ATPase, calcium uptake, and calcium binding were 32, 65, and 65% of each control value, respectively (Table 2). DISCUSSION Peter, Verhaag, and Worsfold (6) reported that the function of sarcoplasmi’creticulum in triamcinolone-treated rats was not altered. Of course, the difference in species of animals might make the comparison of their results and ours difficult. In their rat steroid myopathy the ratio of the weight of the gastrocnemius muscle, which they used for biochemical analysis, to body weight was not changed throughout the experiment. In our rabbits, however, the average weight of lateral vastus, which we used for isolation of sarcoplasmic reticulum, was 0.8870 of body weight in controls, 0.61% in the 7-day group, and 0.49% in the 14-day group. In this sense,the myopathy seemedto be more advanced in our experimental condition. One of the histochemical characteristics of steroid myopathy is a preferential Type II fiber atrophy. This means that Type II fibers are involved more strongly than Type I fibers. The lateral vastus of quadriceps
STEROID
MYOPATHY
309
muscle is a white muscle, which has a Type II fiber predominance. From this point, the lateral vastus appeared to be more suitable for analysis than a mixed muscle, e.g., the gastrocnemius muscle. We demonstrated that the yield of sarcoplasmic reticulum was less and the function of the isolated sarcoplasmic reticulum was moderately disturbed in experimental and human steroid myopathy. These results might be related to the morphological changes of the sarcoplasmic reticulum in the disease. The disturbance of sarcoplasmic reticulum function is supposedly one of the causative factors in muscular weakness, although the exact extent of its contribution is difficult to evaluate. Martonosi (4) reported a direct effect of steroid in vitro on sarcoplasmic reticulum prepared from rabbit skeletal muscle. His results were that steroids which have the cis conformation of ring A and B, and have also at least two hydrophilic groups in the molecule, inhibit calcium uptake and activate ATPase activity. Triamcinolone, which we used in our experiment, fits these molecular conditions. We checked the in z&o effect of triamcinolone on sarcoplasmic reticulum by the same method as that of Martonosi. Calcium uptake was decreased to 62% of control when the same weight of triamcinolone was added to sarcoplasmic reticulum protein, but calcium-dependent ATPase activity was not changed. It seemed improbable that such a high concentration of triamcinolone was obtained in viva. Therefore, the possibility that there is the “direct” effect of steroid on calcium uptake might be ruled out. The question as to why the sarcoplasmicreticulum is disturbed in steroid myopathy remains to be answered, but the catabolic action of glucocorticoid might somehow play a role in the destruction of the protein membrane of the reticulum (7). REFERENCES and Y. SUBBAROW. 1925. The calorimetric determinationof phosBiol. Chew. 66: 375-400. GIJLDING, D. N., S. M. MURRAY, G. W. PEARCE, and M. THO~\IPSON. 1961. Corticosteroid myopathy. A+w. Phys. Med. 6: 171-177. HARIGAYA, S., Y. OGAWA, and H. SUGITA. 1968. Calcium binding activity of microsomal fraction of rabbit red muscle. J. Biochcm. 63 : 324-331. MARTONOSI, A. 1968. Steroid effects on the calcium ion transport of rabbit skeletal muscle microsomes. Arch. Biochem. Biophys. 125 : 295-302. MARTONOSI, A., and R. FERETOS. 1964. Sarcoplasmic reticulum. I. The uptake of Ca++ by sarcoplasmic reticulum fragments. J. Biol. Chem. 239: 648-658. PETER, J. B., D. A. VERHAAG, and M. WORSFOLD. 1970. Studies of steroid myopathy examination of the possible effect of triamcinolone on mitochondria and sarcotubular vesicles of rat skeletal muscle. Biochem. Pharwacol. 19: 1627-1636. SHOJI, S., A. TAKAGI, H. SUGITA, and Y. TOYOKURA. 1974. Muscle giycogen metabolism in steroid-induced myopathy of rabbits. Exp. Neural. 45 : l-7.
1. FISKE,
C. H.,
phorus.J.
2. 3.
4. 5.
6.
7.
NEUROLOGY
Dysfunction and SHIN’ICHI
SHOJI,
Department
AKIO
51,
304-309
(1976)
of Sarcoplasmic Human Steroid TAKAGI,
HIDEO
Reticulum Myopathy SUGITA,
AND
of Neurology, Institute of Brain Research, University of Tokyo, Tokyo, Japan Received
December
in Rabbit
YASUO
Faculty
TOYOKURA of
1
Medicine,
17,197s
Experimental steroid myopathy was induced in rabbits by injections of triamcinolone acetonide. Sarcoplasmic reticulum was prepared from quadriceps muscle. Calcium-dependent ATPase activity decreased in the steroidtreated group. Calcium uptake and binding of sarcoplasmic reticulum were also reduced in the steroid-treated group. By the same methods, sarcoplasmic reticulum in human steroid myopathy was analysed. Again, decreased calcium-dependent ATPase activity, calcium uptake, and binding were revealed in steroid myopathy. These results are consistent with the view that the effects of glucocorticoid on skeletal muscle are mediated, in part, through its action on the calcium transport system.
INTRODUCTION Some authors reported that the sarcoplasmicreticulum (SR) was dilated in human steroid myopathy muscle (2). The function of this organelle in this myopathy has been studied only by Peter et al. (6). They examined triamcinolone-treated rats and found no difference from the controls. We studied its function in rabbits and in a patient with steroid myopathy. MATERIALS
AND METHODS
Male albino rabbits, weighing about 2 kg, were used for this experiment. All were given chow diet and water ad lib. Triamcinolone acetonide (%1 We express our sincere thanks to Dr. M. Tomonaga, of Section of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, for his morpho!ogical examinations, and to Dr. R. J. T. Pennington, Honorary Reader in Neurochemistry, University of Newcastle upon Tyne, for his critical review of the manuscript. Dr. Shoji’s present address is : Neurochemistry Department, Regional Neurological Centre, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, United Kingdom. Reprint requests should be sent to Dr. Shoji’s present address. 304 Copyright All rights
1976 by Academic Press, Inc. o3 reproduction in any form reserved.
STEROID
MYOPATHY
30.5
fluoro-16a-hydroxy-prednisolone acetonide) was given daily in doses of 10 mg/kg body wt by subcutaneous injection (40 mg/ml solution) for 7 or 14 days. An equal volume of 0.9% NaCl solution was injected into control animals. Twenty-four hours after the last injection, the rabbits were anesthetized by ether and killed by exsanguination from the carotid arteries. The quadriceps muscles were quickly dissected and the lateral vastus muscles were separated on ice. Sarcoplasmic reticulum fractions were prepared according to a modified Harigaya’s method (3). All procedures were carried out at 0°C on ice. After fat and connective tissue had been cautionsly removed, 7 to 12 g muscle (lateral vastus) was minced with stainless steel scissors and suspended in 9 volumes ice-cold 10 mM NaHC03 solution. The muscle was then homogenized for 15 set three times, with 30-set intervals, using a Polytron homogenizer. The homogenate was then centrifuged for 5 min at 4OOOg, after which the speed was increased to 9000g for 20 min. The supernatant was again centrifuged for 20 min at 9000g. The resulting supernatant was passed through a Whatman No. 41 filter paper to remove fat and air bubbles. The filtrate was centrifuged for 30 min at 40,OOOy in a Spinco ultracentrifuge. The sediment was suspended in about 6 ml of a solution containing 0.6 M KC1 and 20 m&f Tris (pH 6.8) using a small Teflon pestle. The solution was centrifuged again for 30 min at 40,OOOg. The sediment was suspended in about 2 ml of a solution containing 50 mM KC1 and 20 111~ Tris (pH 6.8) using a small Teflon pestle again. The suspension was used as the sar,coplasmic reticulum fraction. Biopsied muscle was obtained from the lateral vastus of a patient with cutaneous disease and steroid myopathy. Control muscle was obtained from the same muscle during the surgery of a patient without muscle disease, of the same age and sex. From l- to 2-g samples of these muscles, sarcoplasmic reticulum was prepared by the same method as mentioned above. Protein was assayed by the Biuret method. ATPase activity was determined by the increase in Pi after incubation at 25°C for 5 min. Inorganic phosphate was measured by the Fiske-SubbaRow technique ( 1). The reaction mixture, 1.5 ml in total, contained 20 mM Tris-malate buffer (pH 6.8), 4 mM MgSOJ, 0.1 M KCl, 0.1 M EGTA, 2 mM ATP, and 24 to 108 lug SR protein. The reaction was started by adding the ATP and was stopped by adding 1 ml of 20% trichloroacetic acid solution. Mg-ATPase activity was measured in this medium. Mg, CaATPase activity was assayed with addition of CaCI, (0.1 mM). The Ca-ATPase activity was calculated as the difference between Mg, CaATPase activity and Mg-ATPase activity, Calcium binding was measured by the Millipore filter method (5). The
306
SHOJI
ET AL.
reaction mixture, 2.0 ml in total, contained 20 mM Tris (pH 6X), 2 mM MgC12, 0.144 M KCl, 4 mM ATP, 0.1 mM CaC12 with radioactive calcium (2 &i/2 ml) and 150 to 230 pg/ml SR protein. When 5 mM potassium oxalate was included in the reaction mixture (Ca-uptake), the amount of sarcoplasmic reticulum protein was reduced to 5 to 23 pg/ml. The reaction was started by adding the ATP. After incubating for 15 min at 25°C the reaction was stopped by passing the medium through a Millipore filter (GS 0.22 mm). Aliquots of both filtrate and incubation medium were dried on planchets and the radioactivity of each sample was counted by a gas flow counter. The calcium binding and uptake were calculated from the difference between the radioactivity of the filtrates of sarcoplasmic reticulum-free incubation medium and of the sarcoplasmic reticulumcontaining samples. RESULTS Lateral vastus muscles of rabbits and patient were examined by histoand electron microscopical methods. Histological logical, histochemical, examinations showed slight to moderate nonspecific myopathic changes, histochemical stains demonstrated a Type II fiber predominance and a
FIG.
tron
1. Fragmented sarcoplasmic micrograph. X 70,000.
reticulum
with
other
unidentified
organelles.
Elec
STEROID
TABLE Sarcoplasmic A Yield
B ATPase
group group
1
Reticulum
of SR
Controls 7-Day l&Day
307
MYOPATHY
of Rabbits
-
(mg SR protein/g
wet vjt muscle) 0.76 0.59h 0.47”
(4),’ (3) (3)
activity
(pmole
Pi/mg
SR protein/min)
Mg-ATPase Controls 7-D ay group 14.Day group C Calcium
uptake
(bmole
I Controls 7-D ay group l&Day group D Calcium
4.66 2.60
binding
group group
Ca/mg SR protein/l5 Experiment
min)
III
I\
5.25 1.86
7.95
5.56
4.15
3.86
ipmole
0.143 0.122
0.615 0.151* 0.3336
II
I Controls 7-Day l&Day
Ca-ATPase
0.484 0.282 0.361
(4) (3) (3)
Ca/mg SK protein/l5 Experiment III
I\
0.211 0.084
0.194
0.220
0.154
0.158
analysed.
2.96 2.69 min)
II
u Numbers in parentheses show number of rabbits of each group. b P < 0.05 (Student’s t) compared with controls.
v
In A, B, data
\ 0.153 0.105 show
means
preferential Type II fiber atrophy, and electron microscopy revealed a dilation of sarcoplasmic reticulum in these muscles. Extracted sarcoplasmic reticulum was also examined by electron microscopy. These fractions showed membranous structures (fragmented sarcoplasmic reticulum) with other unidentified organelles or structures (Fig. 1). Exfievimeutal Steroid Afyo~a.tlzy of Rabbit. The yield of sarcoplasmic reticulum was significantly decreased in steroid-treated rabbits (Table 1, A). Mg,Ca-ATPase and Ca-ATPase were significantly reduced in the steroid-treated groups (Table 1, B). In order to examine the possibility
308
SHO JI
TABLE Sarcoplasmic
Reticulum
ET AL.
2
in Human Control
Yield of SRa Calcium uptakeb Calcium bindingb ATPase activityc Mg-ATPase Ca-ATPase a Results r, Results c Results
in milligrams in micromoles in micromoles
Steroid
Myopathy Steroid
myopathy
1.46 3.89 0.081
0.59 2.51 0.053
0.193 0.774
0.246 0.250
of SR protein per grams wet weight. of Ca per milligrams SR protein per 15 min. Pi per milligram SR protein per min.
of mitochondrial contamination, we measured ATPase with addition of CaCla (0.1 mM) and azide (1 mM). This activity was 94% of the Mg,CaATPase in the controls and 97-98s in the steroid-treated groups. Both calcium uptake and calcium binding were decreased in steroidtreated rabbits (Tables 1 C, D). Comparison of the 7-day group and the 14-day group was done in Experiment V. In this experiment, the 14-day group showed a greater decrease in calcium uptake and binding than the 7-day group. Hzmmz Steroid Myopathy. Sarcoplasmic reticulum isolated from a patient with steroid myopathy revealed the same tendency as shown in rabbit experiments. The yield of sarcoplasmic reticulum was 40% of the control. Ca-ATPase, calcium uptake, and calcium binding were 32, 65, and 65% of each control value, respectively (Table 2). DISCUSSION Peter, Verhaag, and Worsfold (6) reported that the function of sarcoplasmi’creticulum in triamcinolone-treated rats was not altered. Of course, the difference in species of animals might make the comparison of their results and ours difficult. In their rat steroid myopathy the ratio of the weight of the gastrocnemius muscle, which they used for biochemical analysis, to body weight was not changed throughout the experiment. In our rabbits, however, the average weight of lateral vastus, which we used for isolation of sarcoplasmic reticulum, was 0.8870 of body weight in controls, 0.61% in the 7-day group, and 0.49% in the 14-day group. In this sense,the myopathy seemedto be more advanced in our experimental condition. One of the histochemical characteristics of steroid myopathy is a preferential Type II fiber atrophy. This means that Type II fibers are involved more strongly than Type I fibers. The lateral vastus of quadriceps
STEROID
MYOPATHY
309
muscle is a white muscle, which has a Type II fiber predominance. From this point, the lateral vastus appeared to be more suitable for analysis than a mixed muscle, e.g., the gastrocnemius muscle. We demonstrated that the yield of sarcoplasmic reticulum was less and the function of the isolated sarcoplasmic reticulum was moderately disturbed in experimental and human steroid myopathy. These results might be related to the morphological changes of the sarcoplasmic reticulum in the disease. The disturbance of sarcoplasmic reticulum function is supposedly one of the causative factors in muscular weakness, although the exact extent of its contribution is difficult to evaluate. Martonosi (4) reported a direct effect of steroid in vitro on sarcoplasmic reticulum prepared from rabbit skeletal muscle. His results were that steroids which have the cis conformation of ring A and B, and have also at least two hydrophilic groups in the molecule, inhibit calcium uptake and activate ATPase activity. Triamcinolone, which we used in our experiment, fits these molecular conditions. We checked the in z&o effect of triamcinolone on sarcoplasmic reticulum by the same method as that of Martonosi. Calcium uptake was decreased to 62% of control when the same weight of triamcinolone was added to sarcoplasmic reticulum protein, but calcium-dependent ATPase activity was not changed. It seemed improbable that such a high concentration of triamcinolone was obtained in viva. Therefore, the possibility that there is the “direct” effect of steroid on calcium uptake might be ruled out. The question as to why the sarcoplasmicreticulum is disturbed in steroid myopathy remains to be answered, but the catabolic action of glucocorticoid might somehow play a role in the destruction of the protein membrane of the reticulum (7). REFERENCES and Y. SUBBAROW. 1925. The calorimetric determinationof phosBiol. Chew. 66: 375-400. GIJLDING, D. N., S. M. MURRAY, G. W. PEARCE, and M. THO~\IPSON. 1961. Corticosteroid myopathy. A+w. Phys. Med. 6: 171-177. HARIGAYA, S., Y. OGAWA, and H. SUGITA. 1968. Calcium binding activity of microsomal fraction of rabbit red muscle. J. Biochcm. 63 : 324-331. MARTONOSI, A. 1968. Steroid effects on the calcium ion transport of rabbit skeletal muscle microsomes. Arch. Biochem. Biophys. 125 : 295-302. MARTONOSI, A., and R. FERETOS. 1964. Sarcoplasmic reticulum. I. The uptake of Ca++ by sarcoplasmic reticulum fragments. J. Biol. Chem. 239: 648-658. PETER, J. B., D. A. VERHAAG, and M. WORSFOLD. 1970. Studies of steroid myopathy examination of the possible effect of triamcinolone on mitochondria and sarcotubular vesicles of rat skeletal muscle. Biochem. Pharwacol. 19: 1627-1636. SHOJI, S., A. TAKAGI, H. SUGITA, and Y. TOYOKURA. 1974. Muscle giycogen metabolism in steroid-induced myopathy of rabbits. Exp. Neural. 45 : l-7.
1. FISKE,
C. H.,
phorus.J.
2. 3.
4. 5.
6.
7.