- Email: [email protected]
Further studies of membrane atpase activity in muscular dystrophy erythrocytes
ht. 3. Biochem.,
1972, 3, 33g--34+
FURTHER
STUDIES
IN MUSCULAR S. R.
[Scientechnica
(Publishers)
OF MEMB~~E DYSTROPHY
GHATTOPADHYAY
Ltd.]
ATPASE
339
ACTIVITY
ERYTHROCYTES*
AND
H. D. BROWN
Biochemistry Section, Cancer Research Center, Columbia, Missouri 65201, U.S.A. (Received 3 March,
I gp)
i\BSTRACT There is a correlation between the effect of ouabain upon membrane ATPase activity and myopathies. Au erythrocyte ghost preparation from normal donors showed a characteristic transport ATPase activity, which was inhibited by a ro-* M concentration of the cardiac glycoside, ouabain. 2. Identical preparations from myopathic patients were stimulated rather than inhibited by the same concentration of ouabain. 3. These results have been interpreted in terms of a disease-related change in membrane integrity bearing upon the function of the transport enzyme. 4. The effect of diphenv~ydantoin on the membrane ATPase has also been studied. It Hpparently alters the abnckmal response of certain patients to the cardiac glycoside, ouabain. I.
MUSCLE abnormalities have been associated with membrane transport defects by several workers (Gorsini and Gacciari, 1958; Brown, Chattopadhyay, and Patel, 1968; Brown, Rigdon, Ghattopadhyay, and Patel, 1968; Chattopadhyay, Brown, and Patel, I 969). It has been suggested that the abnormalities are themselves indications of a generalized (probably hereditary) effect and might therefore have a reflection in non-muscle cells (Gorsini and Cacciari, 1938) even in the absence of gross functional defect. This possibility led us to study the membraneassociated ATPase of red blood-cell ghosts as a possible sensitive and useful indicator of membrane defect. In experimental animals (Brown, Ghattopadhyay, and Patel, $968; Brown, Rigdon, and others, 1968; Ghattopahdyay and others, 1969) we have reported a correlation between ATPase characteristics and myopathic conditions. Similarly, in a preliminary study (Brown, Ghattopadhyay, and Patel, 1967) transport ATPase from a limited number of human dystrophic * This investigation was supported bv a grant from the ivluscular Dystrophy Assoei’ations of America Inc.
donors’ erythrocyte hosts was compared with that from normal donors. It was found that although the activity level of the membraneassociated enzyme varied from patient to patient and was not statistically different from that of the controls, there was a marked difference in the response to the cardiac glycoside, ouabain. This original observation was confirmed by the study of Peter, Worsfold, and Pearson ( I 969). These workers, however, have offered an alternative explanation for the phenomenon, in attributing the erythrocyte ATPase abnormality to an uncategorized serum factor rather than to a direct function of disease-related membrane abnormality. A recent study of erythrocyte ghost ATPase in Duchenne and other dystrophies by Araki and Mawatari (I 97 I) further confirmed the existence of this membrane-enzyme abnormality. In our present work similar measurements have been made in a larger number of the patients having varied myopathic conditions. Inferences about this relationship of enzyme activity pattern to membrane condition have been illuminated by experiments using the compounds diphenylhydantoin and hydroxyurea. The study reported here has indicated
340
CHA’M’OPADHYAY
a correlation between muscle myopathy in human patients and an abnormal response of the blood-cell ATPase to the cardiac glycoside, ouabain. In a practical sense the method may have diagnostic application and the probability exists that the observations described are indicative of a mechanism which relates to the myopathic state. MATERIALS AND METHODS Blood (5 ml.) was collected from each donor in a Vacutainer tube containing g mg. of ethylenediaminetetraacetate (EDTA) as an anticoagulant. Collected blood samples were stored at 4” C. for 24 hours before further treatment. The bIood cells were haemolysed in 2 m&f Tris buffer, pH 7.4, with 0.02 mM EDTA. After centrifugation at xo,ooog for 30 minutes, the haemolysed cells were washed three to five times with the same buffer containing 15.5 mN N&l. Between each centrifugation the ghosts were homogenized with a Teflon pestle. The preparations were either used immediately after resuspending in an equal volume of T&-sucrose buffer or were stored at -IO’ C. A temperature of 2-4’ C. was maintained throughout the preparation. In order to allow correlation with the dystrophy type, each blood sample was assayed for se&m
creatine vhosnhokinase. To accomohsh this, a separate *Vac&ainer tube without r&ticoag&nt was filled with donor blood. The serum was separated from coagulated blood by centrifugation at 6oo g for 20 minutes. The reaction mixture for the assay of creatine phosphokinase contained 0.1 ml. serum, 0.8 ml. substrate (aqueous solution of ~88 mg. Na&TP, og2g mg. Tris, oog7 mg. MgCI,, and 0*25x mg. creatine per ml. of reaction mixture), and 0.1 ml. deionized water. The reaction was ahowed to proceed for 30 minutes at 37” C. ATPase activity was measured in a reaction mixture containing 0-r ml. enzyme (0.9-x.0 mg. orotein), 0.8 ml. substrate (0.7 ma. Na,ATP per 4. of reaction mixture with ; m&I N&I, n&M KCl. and I mMMtzC1, in 0.1 M Tris-HCC sucrose buffer, pH 7.;), and 0.1 ml. of deionized water or inhrbttor in water. The samples were incubated at 40” C. for 60 minutes. Roth creatine phosphokinase CPK and ATPase reactions were stopped with cold 50 per cent trichioro-acetic acid in water (0.1 mf. per ml. reaction mixture). Inorganic phosphate was measured in sample aliauots after the precipitated protein was removed *by centrifugation _(Fiske and Subbarow. 10251. The nhenol method was used for dete&&ation of*protein in the enzyme preparation (Lowry, Rosebrough, Farr, For ~pheny~yd~to~ and Randall, rpjr)and hydroxyurea experiments preparations were made as described above; however, ghosts were
AND
BROWN
ht.
J.
~~ach~~.
resuspended in double volumes of 0. I M Tris-HClsucrose buffer, pH 7.2. The suspension was treated with 2 per cent (final volume) diphenylhydantoin or hydroxyurea. The addition of the test compound was made 20-30 minutes before beginning each experiment.
0.25 M
RESULTS
AND DISCUSSION
Several workers regard elevated CPK activity as a symptom of progressive pseudohypertrophic muscle dystrophy (Dreyms, 1960; Okinaka, Schapira, and Demos, Kumagai, Ebashi, Sugita, Momoi, Toyokura, and Fujie, r 961; von Moser, Wiesmann, Richterich and Rossi, rg66a, b) while aldolase activity is considered more characteristic of progressive muscle dystrophy (Schapira, Dreyfus, and Schapira, x953). In our study xg pseudohypertrophic, 2 limb-girdle proI Kugelberg-Welander gressive patients, disease, I Werdnig-Hoffmann disease, and 2 patients having myopathic disease of uncertain type were studied. All blood sera except that of the Kugelberg-Welander disease and one of the uncertain cases showed elevated creatine phospho~n~e activity. (Using Okinaka units, elevation varied from rg to 236 units.) The characteristic response of the membrane Na+ + Kf ATPase to ouabain is a decrease in the rate of catalytic ATP hydrolysis (Skou, 1965; Post, Merritt, Kinsolving, and Albright, 1960). The effect of ouabain upon ATPase activity in control preparations and in preparations from myopathic donors is given in Table I. The activity of red blood-cell ghost ATPase isolated from normal donors showed consistent inhibition by IO-* M ouabain. However, ghost ATPase obtained only from and limb-girdle dyspseudohypertrophic trophic donors, lysed, and washed, using precisely the same procedure, was significantly stimulated (an average of 21 patients studied showed 76 per cent stimulation; statistical evaluation given in Table I) by the same concentration of ouabain. Sodium diphenylhydantoin (Dilantin, Park Davis & Co.) and hydroxyurea (Hydrea, Squibb) have been used in this same in vitro Their use in this test system system). was based upon the po~ibility that their physiological activity might be related to
‘97%
ATPASE ACTIVITY IN MUSCULAR DYSTROPHY
3
membrane effects. have supported
The data of Korey ( I 95 I ) effect of a stabilizing
Table I.-EFFECT
diphenylhydantoin and Woodbury
34’
on membrane function, (1955) has suggested an
OF OUABAIN (IO-( M) UPON ERYTHROCYTI:( 31-10s~ATPAs.E ACTIVITY (nmol& Pi per’mg. protein per hou r)
-
ACTIVITY
-
DURATION IDENTITY
AGE
DIAGNOSIS
SEX
PERCENTAGE CHANGE
ILZs
FAMILY HISTORY
(years) Normal MR AP ::T g !%
35 28 37 32 39 30 45 49
Normal Normal Normal Normal Normal Normal Normal Normal
9 18 I5 12
E cc
I3 I7 18 16
RC UG FAH CM’H
I5 16 3 II
TAH DP
5 IO
RS
12
Js
; M
I3 9 IO
1
54.8
42.1 r43.7 40.8
2 II II 10
46.9 40.0 36.0 1~21.7
:Y:: 68.0 79’0
g
_-
I
9 I2
1
g
‘3 II 2’5 8
(?)
M M
4 7
M
IO
M
32
Limb-girdle Pseudohypertrophic
32.1 26.1 29’3 54’2
_-
Not known
+22 +3I -25
None Grandfather Sisters 5 children
+4 +52 +88 -40
None Not known None Brother
I101.7
+39 +2 +5 -8
Brother None Not known None
36.4 96.8 55.9 35’9
+34 +I4 +27 -27
None None 3 brothers 3 brothers
37’5
[3”9 47.6
,*5
3 brothers None
40.0
61.0
+52
None
+25
Not known
+I4
None
1
49’2
35’4
1136.7
I168.4
I‘59’4
L10.7 55.8 84.3 43.8 49’7
1137.6
1
.-
M
List numl r I has 8 elements. List number F-ratio DF -LI LI L2 Reg I 20 4’37 Indicates przbability to.005.
5
_-
Normal Normal Normal Normal Normal Normal Normal Normal
+24
’ 34’2
.-
3:”.51.: 54”
-49
-54 -34 -31 -32
I[II’1
34’5 rog.2
I3 II
;
63.3 57’4 44’4 79’1 44.6 32’9 56.9 75’9
89.3
M M M M
i;;
45 6
12
:
type
PJ
M
? M
18 I5 ‘3
_-
L
1
-
_-
-
M
DysIZphic PseudoI4 hypertrophic
LW
_-
83.7 30.2
1105.2 _-
34’4
-_
_-
2 has 21 elements. t between I and 2:DF-L2 DifMeans Student t General t -52’030 7 4’754 6.399
TDF 27
CHATTOPADHYAY AND BROWN
342
enhancement by this drug of ’ sodium pump ’ action. Our rest&s and those of others (Pincus and Giarman, I 967 ; Festoff and Appel, I 968) can be interpreted to support the possibility that the effectiveness of diphenylhydantoin is related to its action upon membrane ATPase. Sodium diphenylhydantoin has been used therapeutically in neurological disease (Park
bt. J. &o&em.
the abnormal response of erythrocyte ATPase to ouabain in certain patients (T&e II) (Sperelakis and Henn (1970) have recently reported a negative result of diphenylhydantoin upon ATPase activity in cultured chick heart. This result is in accord with this report for human erythrocyte preparations where activity per se was the function
Table II-EFISCT OF OUABAIN (IO-~ M) UPON DIPHENYLHMANTOIN PRETREATED ERYTHROCYTE GHOST (PSEIJDOHYPERTROPHIC MUSCULARDY.STROPHY)ATPME ACTIVITY
IDBNT~Y
CJv RL LW :;
CC
OUABAIN (IO-* M) EFFECT(percentage change)
AGE
Ghost ATPase (not treated with diphenylhyd~toin)
‘5 12
i
Pseudohypertrophic
Ia 13 1a 9
E DA
17
+39 +5 +4
-8 +x4
13 IO
-25 +26 j-2 f52 i24 -27 -i-27 -4
18 I4 II 3 5
+ Stimulation;
+a8 -40 i-22
16
16
-
DUGNOSIS
(years)
-
_-
Ghost ATPase (pretreated with diph~y~yd~toin)
ia3 -55 -7 f77 i-
I
-55 -25 i-2 -20 -45 f40 -25 + I -20 I$!
Inhibition.
Davis & Co., r g67), particularly epilepsy and other convulsive states, in Parkinson’s syndrome, and in cardiomyopathy. Although the physiological activity of the compound has not been established, the subcell membrane is a probable site of action. Overall, our results (Tuble II and Brown, Chattopadhyay, and Patef, x967), and those of Peter and others (1969) and Araki and Mawatari (1971) establish a relationship between muscular dystrophy and the ouabain response of ATPase isolated from erythrocytes. The thesis that membrane abnormality is involved in this abnormal characteristic of the enzyme appears to be given further substance by the effect of diphenylhydantoin upon this system. Diphenylhydantoin alters
studied.) Sixteen pseudohypertrophic erythrocyte ghost samples were considered in this report. Normally, in I I out of 16 blood samples (i.e., 6g per cent) ATPase activity was stimulated by IO-* M ouabain; but when these I I samples were pretreated with diphenylhydantoin before the ouabain effect was measured, ATPase activity in 45 per cent of the cases was altered from ouabain stimulation to ouabain inhibition. Although the scatter of this data indicates the existence of one or more unrecognized variables, possibly in the uniformity of the patient classification, we tentatively consider that a response to the drug occurs. Diphenylhydantoin has been used ciinically in the treatment of cardiac dysrhythmias
1972, 3
ATPASE ACTIVITY IN MUSCULARDYSTROPHY
1969) and experi(Gibson and Harris, mentally to otherwise reverse myopathic states (Gruener and Stern, 1972). It is possible that the sporadic success of this drug as a muscle therapeutic agent may relate the non-uniformity of our in t.itm results. Although the data reported in Table II are not significant, if the entire group of subjects is considered to be a homogenous group (indeed the patients have all been diagnosed as having pseudomuscular dystrophy), then the phenomenon does not occur at all in blood derived from normal donors. Treated in this sense, the effect has reality, although admittedly we cannot offer an explanation for the difference in character between the dystrophv samples which responded and those which did not. We suggest that the ATPase-response alteration reported in muscle of selperal experimental animals (Brown, Rigdon and others, 1968; Brown, Chattopadhyay, and Patel, 1968; Chattopadhyay and others, 1969) and in red blood-cells of human patients (Brown, Chattopadhyay, and Patel, 1967 ; Peter and others, 1969; Arnki and Mawatari, 1971) is of possible clinical value in diagnosis and as a technique for the screening of compounds potentially useful as muscle drugs. The phenomenon described lends insight into a probable siteofmembranefunction abnormality. ACKNOWLEDGEMENTS The authors acknowledge with thanks the cooperation of Dr. Lloyd A. McLaughlin and the Crippled Children’s Hospital of New Orleans.
REFERENCES ARAKI, S.: and MA~ATARI, S. (lg71), ‘ Ouabain and erythrocyte-ghost adenosine triphosphate. Effects in human muscular dystrophies ‘, Archs Neural., 24, 187-190.
BROWN. H. D.. CHATTOPADHYAY.S. K.. and PATE;, A. B. (x967), ‘ Erythrocvte abnormality in human myopathy ‘) Science, &.Y., 157, 15771j78.
BROWN, H. D., CHATTOPADHYAY,S. K., and PATEL, A. B. (x968). ‘ Diazacholesterol effect upon membrane ATPase ‘, Metabolism, 17, jjjj59.
343
in the myopathic duck ‘, Enzymol. Biol. Clin., 9, 433-446.
CHATTOPADHYAY,S. K., BROWN, H. D., and PATEL, A. B. (ig6g), ‘ Enzyme studies in the dystrophic mouse ‘, Acta biol. med. ger., 22, 1-6. CORSINI,F., and CACCIARI,E. (Ig58), ‘ Glicolisi e l’aldolasi eritrocitarie nella distrofias muscolare (Erythrocyte glycolysis and aldolase in muscular dystrophy) ‘, Clin. Pediat., Bologna, 40, 743. DREYFUS,.I. C., SCHAPIRA,G., and DEMOS, 1. (1960); ’ Study of serum crkatine kinase- h mvonathic oatients and their families ‘. Rev. fr.‘&ud.
clin.*biol., 5, 384-386.
’
FESTOFF, B. W., and APPEL, S. H. ( 1968): ’ Effect of diphenylhydantoin on synaptosome sodium;;ta$sium ATPase ‘, 3. clin. Invest., 47, 2752FISKE, C. H., and SUBBAROW,Y. (igq), ‘ The calorimetric determination of phosphorus ‘,
3.
biol. Chem., 66, 37j-400.
GIBSON,K., and HARRIS, P. (ig6g), ‘ Diphenylhydantoin and human myocardial microsomal (Na+, K+)-ATPase ‘, Biochem. Biophys. Res. Commun., 35, 75-78.
GRUENER, R. P., and STERN, L. Z. (ig72), ‘ Diphenylhydantoin reverses membrane effects in steroid myopathy ‘, h’ature New Biol., Land., 235,54-j5.
-
KOREY, S. R. (Iqsi), -_ ,- ’ Effect of Dilantin and Mesantoin on the giant axon of the squid ‘, Proc. Sot. exp. Biol. Med.,
76, 297-299.
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL,R. J. (1g51), ‘ Protein measurement with the Folin phenol reagent ‘, 3. biol. Chem., x93,265-275. VON MOSER,H., WIESMANN, U., RICHTERICH,R.,
and ROSSI. E. CIo66a). ‘ Promessive Muskeldystrophie: VIII. Haufigke&, Klinik und Genetick der Tvnen I und II ‘. Schweir. med. Wschr., ~6, I 6q- I 74. VOX Mo~E~,-H. ,-W~E~MANN, U., RICHTERICH,R., and Rossr. E. fIo66b). ‘ Proszressive Muskeldystrophie: VIII. Haufigkeyt, Klinik und Genetick der Typen I und II ‘, Schweit. med. Wschr.) g6, 205-2 I I. OKINAKA.J., KUMAGAI,H., EBASHI,S., SUGITA, H., MOMOI,A. H., TOYOKURA,Y., and FUJIE,Y. ( I g6 I ) , ‘ Serum creatine phosphokinase ‘, Archs II
Neural., 4, j20-j2
j.
PARK DAVIS & COMPANY.nublication M-r?A. ” z Detroit, Michigan, Sept.: 1967. PETER,J. B., WORSFOLD,M., and PEARSON, C. M. (ig6g)> ‘ Erythrocyte ghost adenosine triphosphatase (ATPase) in Duchenne dystrophy ‘, 3. lab. clin. Med., 74, 103-108. PXNCUS, J. H:, and GIARMAN,N. J. (Ig67), ‘ The effect of dlphenylhydantoin on sodium-, potassium-, magnesium-stimulated adenosine triphosphatase activity of rat brain ‘) Biochem. Pharmac.:
16, 600-603.
C. R., BROWX.H. D., RIGDOX,R. H., CHATTOPADHYAY, POST,R. L., MERRITT,C. R., KINSOLVING, and .~LBRIGHT, C. D. (Ig6o)> ‘ Membrane S. I(.: and PATEL, A. (1968). ‘ Enzyme studies
344
CHATTOPADHYAY
adenosine triphosphatase as a participant active transport of sodium and potassium !mt~an erythrocyte ‘, 3. biol. Chem., 235,
in the in the 17g6-
G., DRBYFUS, J. C., and SCHAPIRA, F. ‘ L’elevation du taux de l’aldolase (‘953), serique ; test biochimeque des myopathies ‘, Sem. H&. Paris, 2~. 1917. SKOU, J. ‘C. (1g65):~’ Enzymatic basis for active transport of Na+ and K+ across cell membrane I, Physiol. Rev., 4, 596-617. SPERBLAKIS, N., and )IENN, F. A. (Ig7o), ‘ Effect of diphenylhydantoin on membrane potentials SCHAPI~,
AND BROWN and Na- K-ATPase of cultured chick heart cells ‘, Am. 3. Physiol., 218, 1224-1227. WOODBURY, D. M. (Ig55), ‘ Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats ‘, 3. Pharmac. e.rp. %., xx5s 74-95.
Key Word Index: Ouabain, ATPase, diphenylhydantoin, muscular dystrophy, red blood cells.
1972, 3, 33g--34+
FURTHER
STUDIES
IN MUSCULAR S. R.
[Scientechnica
(Publishers)
OF MEMB~~E DYSTROPHY
GHATTOPADHYAY
Ltd.]
ATPASE
339
ACTIVITY
ERYTHROCYTES*
AND
H. D. BROWN
Biochemistry Section, Cancer Research Center, Columbia, Missouri 65201, U.S.A. (Received 3 March,
I gp)
i\BSTRACT There is a correlation between the effect of ouabain upon membrane ATPase activity and myopathies. Au erythrocyte ghost preparation from normal donors showed a characteristic transport ATPase activity, which was inhibited by a ro-* M concentration of the cardiac glycoside, ouabain. 2. Identical preparations from myopathic patients were stimulated rather than inhibited by the same concentration of ouabain. 3. These results have been interpreted in terms of a disease-related change in membrane integrity bearing upon the function of the transport enzyme. 4. The effect of diphenv~ydantoin on the membrane ATPase has also been studied. It Hpparently alters the abnckmal response of certain patients to the cardiac glycoside, ouabain. I.
MUSCLE abnormalities have been associated with membrane transport defects by several workers (Gorsini and Gacciari, 1958; Brown, Chattopadhyay, and Patel, 1968; Brown, Rigdon, Ghattopadhyay, and Patel, 1968; Chattopadhyay, Brown, and Patel, I 969). It has been suggested that the abnormalities are themselves indications of a generalized (probably hereditary) effect and might therefore have a reflection in non-muscle cells (Gorsini and Cacciari, 1938) even in the absence of gross functional defect. This possibility led us to study the membraneassociated ATPase of red blood-cell ghosts as a possible sensitive and useful indicator of membrane defect. In experimental animals (Brown, Ghattopadhyay, and Patel, $968; Brown, Rigdon, and others, 1968; Ghattopahdyay and others, 1969) we have reported a correlation between ATPase characteristics and myopathic conditions. Similarly, in a preliminary study (Brown, Ghattopadhyay, and Patel, 1967) transport ATPase from a limited number of human dystrophic * This investigation was supported bv a grant from the ivluscular Dystrophy Assoei’ations of America Inc.
donors’ erythrocyte hosts was compared with that from normal donors. It was found that although the activity level of the membraneassociated enzyme varied from patient to patient and was not statistically different from that of the controls, there was a marked difference in the response to the cardiac glycoside, ouabain. This original observation was confirmed by the study of Peter, Worsfold, and Pearson ( I 969). These workers, however, have offered an alternative explanation for the phenomenon, in attributing the erythrocyte ATPase abnormality to an uncategorized serum factor rather than to a direct function of disease-related membrane abnormality. A recent study of erythrocyte ghost ATPase in Duchenne and other dystrophies by Araki and Mawatari (I 97 I) further confirmed the existence of this membrane-enzyme abnormality. In our present work similar measurements have been made in a larger number of the patients having varied myopathic conditions. Inferences about this relationship of enzyme activity pattern to membrane condition have been illuminated by experiments using the compounds diphenylhydantoin and hydroxyurea. The study reported here has indicated
340
CHA’M’OPADHYAY
a correlation between muscle myopathy in human patients and an abnormal response of the blood-cell ATPase to the cardiac glycoside, ouabain. In a practical sense the method may have diagnostic application and the probability exists that the observations described are indicative of a mechanism which relates to the myopathic state. MATERIALS AND METHODS Blood (5 ml.) was collected from each donor in a Vacutainer tube containing g mg. of ethylenediaminetetraacetate (EDTA) as an anticoagulant. Collected blood samples were stored at 4” C. for 24 hours before further treatment. The bIood cells were haemolysed in 2 m&f Tris buffer, pH 7.4, with 0.02 mM EDTA. After centrifugation at xo,ooog for 30 minutes, the haemolysed cells were washed three to five times with the same buffer containing 15.5 mN N&l. Between each centrifugation the ghosts were homogenized with a Teflon pestle. The preparations were either used immediately after resuspending in an equal volume of T&-sucrose buffer or were stored at -IO’ C. A temperature of 2-4’ C. was maintained throughout the preparation. In order to allow correlation with the dystrophy type, each blood sample was assayed for se&m
creatine vhosnhokinase. To accomohsh this, a separate *Vac&ainer tube without r&ticoag&nt was filled with donor blood. The serum was separated from coagulated blood by centrifugation at 6oo g for 20 minutes. The reaction mixture for the assay of creatine phosphokinase contained 0.1 ml. serum, 0.8 ml. substrate (aqueous solution of ~88 mg. Na&TP, og2g mg. Tris, oog7 mg. MgCI,, and 0*25x mg. creatine per ml. of reaction mixture), and 0.1 ml. deionized water. The reaction was ahowed to proceed for 30 minutes at 37” C. ATPase activity was measured in a reaction mixture containing 0-r ml. enzyme (0.9-x.0 mg. orotein), 0.8 ml. substrate (0.7 ma. Na,ATP per 4. of reaction mixture with ; m&I N&I, n&M KCl. and I mMMtzC1, in 0.1 M Tris-HCC sucrose buffer, pH 7.;), and 0.1 ml. of deionized water or inhrbttor in water. The samples were incubated at 40” C. for 60 minutes. Roth creatine phosphokinase CPK and ATPase reactions were stopped with cold 50 per cent trichioro-acetic acid in water (0.1 mf. per ml. reaction mixture). Inorganic phosphate was measured in sample aliauots after the precipitated protein was removed *by centrifugation _(Fiske and Subbarow. 10251. The nhenol method was used for dete&&ation of*protein in the enzyme preparation (Lowry, Rosebrough, Farr, For ~pheny~yd~to~ and Randall, rpjr)and hydroxyurea experiments preparations were made as described above; however, ghosts were
AND
BROWN
ht.
J.
~~ach~~.
resuspended in double volumes of 0. I M Tris-HClsucrose buffer, pH 7.2. The suspension was treated with 2 per cent (final volume) diphenylhydantoin or hydroxyurea. The addition of the test compound was made 20-30 minutes before beginning each experiment.
0.25 M
RESULTS
AND DISCUSSION
Several workers regard elevated CPK activity as a symptom of progressive pseudohypertrophic muscle dystrophy (Dreyms, 1960; Okinaka, Schapira, and Demos, Kumagai, Ebashi, Sugita, Momoi, Toyokura, and Fujie, r 961; von Moser, Wiesmann, Richterich and Rossi, rg66a, b) while aldolase activity is considered more characteristic of progressive muscle dystrophy (Schapira, Dreyfus, and Schapira, x953). In our study xg pseudohypertrophic, 2 limb-girdle proI Kugelberg-Welander gressive patients, disease, I Werdnig-Hoffmann disease, and 2 patients having myopathic disease of uncertain type were studied. All blood sera except that of the Kugelberg-Welander disease and one of the uncertain cases showed elevated creatine phospho~n~e activity. (Using Okinaka units, elevation varied from rg to 236 units.) The characteristic response of the membrane Na+ + Kf ATPase to ouabain is a decrease in the rate of catalytic ATP hydrolysis (Skou, 1965; Post, Merritt, Kinsolving, and Albright, 1960). The effect of ouabain upon ATPase activity in control preparations and in preparations from myopathic donors is given in Table I. The activity of red blood-cell ghost ATPase isolated from normal donors showed consistent inhibition by IO-* M ouabain. However, ghost ATPase obtained only from and limb-girdle dyspseudohypertrophic trophic donors, lysed, and washed, using precisely the same procedure, was significantly stimulated (an average of 21 patients studied showed 76 per cent stimulation; statistical evaluation given in Table I) by the same concentration of ouabain. Sodium diphenylhydantoin (Dilantin, Park Davis & Co.) and hydroxyurea (Hydrea, Squibb) have been used in this same in vitro Their use in this test system system). was based upon the po~ibility that their physiological activity might be related to
‘97%
ATPASE ACTIVITY IN MUSCULAR DYSTROPHY
3
membrane effects. have supported
The data of Korey ( I 95 I ) effect of a stabilizing
Table I.-EFFECT
diphenylhydantoin and Woodbury
34’
on membrane function, (1955) has suggested an
OF OUABAIN (IO-( M) UPON ERYTHROCYTI:( 31-10s~ATPAs.E ACTIVITY (nmol& Pi per’mg. protein per hou r)
-
ACTIVITY
-
DURATION IDENTITY
AGE
DIAGNOSIS
SEX
PERCENTAGE CHANGE
ILZs
FAMILY HISTORY
(years) Normal MR AP ::T g !%
35 28 37 32 39 30 45 49
Normal Normal Normal Normal Normal Normal Normal Normal
9 18 I5 12
E cc
I3 I7 18 16
RC UG FAH CM’H
I5 16 3 II
TAH DP
5 IO
RS
12
Js
; M
I3 9 IO
1
54.8
42.1 r43.7 40.8
2 II II 10
46.9 40.0 36.0 1~21.7
:Y:: 68.0 79’0
g
_-
I
9 I2
1
g
‘3 II 2’5 8
(?)
M M
4 7
M
IO
M
32
Limb-girdle Pseudohypertrophic
32.1 26.1 29’3 54’2
_-
Not known
+22 +3I -25
None Grandfather Sisters 5 children
+4 +52 +88 -40
None Not known None Brother
I101.7
+39 +2 +5 -8
Brother None Not known None
36.4 96.8 55.9 35’9
+34 +I4 +27 -27
None None 3 brothers 3 brothers
37’5
[3”9 47.6
,*5
3 brothers None
40.0
61.0
+52
None
+25
Not known
+I4
None
1
49’2
35’4
1136.7
I168.4
I‘59’4
L10.7 55.8 84.3 43.8 49’7
1137.6
1
.-
M
List numl r I has 8 elements. List number F-ratio DF -LI LI L2 Reg I 20 4’37 Indicates przbability to.005.
5
_-
Normal Normal Normal Normal Normal Normal Normal Normal
+24
’ 34’2
.-
3:”.51.: 54”
-49
-54 -34 -31 -32
I[II’1
34’5 rog.2
I3 II
;
63.3 57’4 44’4 79’1 44.6 32’9 56.9 75’9
89.3
M M M M
i;;
45 6
12
:
type
PJ
M
? M
18 I5 ‘3
_-
L
1
-
_-
-
M
DysIZphic PseudoI4 hypertrophic
LW
_-
83.7 30.2
1105.2 _-
34’4
-_
_-
2 has 21 elements. t between I and 2:DF-L2 DifMeans Student t General t -52’030 7 4’754 6.399
TDF 27
CHATTOPADHYAY AND BROWN
342
enhancement by this drug of ’ sodium pump ’ action. Our rest&s and those of others (Pincus and Giarman, I 967 ; Festoff and Appel, I 968) can be interpreted to support the possibility that the effectiveness of diphenylhydantoin is related to its action upon membrane ATPase. Sodium diphenylhydantoin has been used therapeutically in neurological disease (Park
bt. J. &o&em.
the abnormal response of erythrocyte ATPase to ouabain in certain patients (T&e II) (Sperelakis and Henn (1970) have recently reported a negative result of diphenylhydantoin upon ATPase activity in cultured chick heart. This result is in accord with this report for human erythrocyte preparations where activity per se was the function
Table II-EFISCT OF OUABAIN (IO-~ M) UPON DIPHENYLHMANTOIN PRETREATED ERYTHROCYTE GHOST (PSEIJDOHYPERTROPHIC MUSCULARDY.STROPHY)ATPME ACTIVITY
IDBNT~Y
CJv RL LW :;
CC
OUABAIN (IO-* M) EFFECT(percentage change)
AGE
Ghost ATPase (not treated with diphenylhyd~toin)
‘5 12
i
Pseudohypertrophic
Ia 13 1a 9
E DA
17
+39 +5 +4
-8 +x4
13 IO
-25 +26 j-2 f52 i24 -27 -i-27 -4
18 I4 II 3 5
+ Stimulation;
+a8 -40 i-22
16
16
-
DUGNOSIS
(years)
-
_-
Ghost ATPase (pretreated with diph~y~yd~toin)
ia3 -55 -7 f77 i-
I
-55 -25 i-2 -20 -45 f40 -25 + I -20 I$!
Inhibition.
Davis & Co., r g67), particularly epilepsy and other convulsive states, in Parkinson’s syndrome, and in cardiomyopathy. Although the physiological activity of the compound has not been established, the subcell membrane is a probable site of action. Overall, our results (Tuble II and Brown, Chattopadhyay, and Patef, x967), and those of Peter and others (1969) and Araki and Mawatari (1971) establish a relationship between muscular dystrophy and the ouabain response of ATPase isolated from erythrocytes. The thesis that membrane abnormality is involved in this abnormal characteristic of the enzyme appears to be given further substance by the effect of diphenylhydantoin upon this system. Diphenylhydantoin alters
studied.) Sixteen pseudohypertrophic erythrocyte ghost samples were considered in this report. Normally, in I I out of 16 blood samples (i.e., 6g per cent) ATPase activity was stimulated by IO-* M ouabain; but when these I I samples were pretreated with diphenylhydantoin before the ouabain effect was measured, ATPase activity in 45 per cent of the cases was altered from ouabain stimulation to ouabain inhibition. Although the scatter of this data indicates the existence of one or more unrecognized variables, possibly in the uniformity of the patient classification, we tentatively consider that a response to the drug occurs. Diphenylhydantoin has been used ciinically in the treatment of cardiac dysrhythmias
1972, 3
ATPASE ACTIVITY IN MUSCULARDYSTROPHY
1969) and experi(Gibson and Harris, mentally to otherwise reverse myopathic states (Gruener and Stern, 1972). It is possible that the sporadic success of this drug as a muscle therapeutic agent may relate the non-uniformity of our in t.itm results. Although the data reported in Table II are not significant, if the entire group of subjects is considered to be a homogenous group (indeed the patients have all been diagnosed as having pseudomuscular dystrophy), then the phenomenon does not occur at all in blood derived from normal donors. Treated in this sense, the effect has reality, although admittedly we cannot offer an explanation for the difference in character between the dystrophv samples which responded and those which did not. We suggest that the ATPase-response alteration reported in muscle of selperal experimental animals (Brown, Rigdon and others, 1968; Brown, Chattopadhyay, and Patel, 1968; Chattopadhyay and others, 1969) and in red blood-cells of human patients (Brown, Chattopadhyay, and Patel, 1967 ; Peter and others, 1969; Arnki and Mawatari, 1971) is of possible clinical value in diagnosis and as a technique for the screening of compounds potentially useful as muscle drugs. The phenomenon described lends insight into a probable siteofmembranefunction abnormality. ACKNOWLEDGEMENTS The authors acknowledge with thanks the cooperation of Dr. Lloyd A. McLaughlin and the Crippled Children’s Hospital of New Orleans.
REFERENCES ARAKI, S.: and MA~ATARI, S. (lg71), ‘ Ouabain and erythrocyte-ghost adenosine triphosphate. Effects in human muscular dystrophies ‘, Archs Neural., 24, 187-190.
BROWN. H. D.. CHATTOPADHYAY.S. K.. and PATE;, A. B. (x967), ‘ Erythrocvte abnormality in human myopathy ‘) Science, &.Y., 157, 15771j78.
BROWN, H. D., CHATTOPADHYAY,S. K., and PATEL, A. B. (x968). ‘ Diazacholesterol effect upon membrane ATPase ‘, Metabolism, 17, jjjj59.
343
in the myopathic duck ‘, Enzymol. Biol. Clin., 9, 433-446.
CHATTOPADHYAY,S. K., BROWN, H. D., and PATEL, A. B. (ig6g), ‘ Enzyme studies in the dystrophic mouse ‘, Acta biol. med. ger., 22, 1-6. CORSINI,F., and CACCIARI,E. (Ig58), ‘ Glicolisi e l’aldolasi eritrocitarie nella distrofias muscolare (Erythrocyte glycolysis and aldolase in muscular dystrophy) ‘, Clin. Pediat., Bologna, 40, 743. DREYFUS,.I. C., SCHAPIRA,G., and DEMOS, 1. (1960); ’ Study of serum crkatine kinase- h mvonathic oatients and their families ‘. Rev. fr.‘&ud.
clin.*biol., 5, 384-386.
’
FESTOFF, B. W., and APPEL, S. H. ( 1968): ’ Effect of diphenylhydantoin on synaptosome sodium;;ta$sium ATPase ‘, 3. clin. Invest., 47, 2752FISKE, C. H., and SUBBAROW,Y. (igq), ‘ The calorimetric determination of phosphorus ‘,
3.
biol. Chem., 66, 37j-400.
GIBSON,K., and HARRIS, P. (ig6g), ‘ Diphenylhydantoin and human myocardial microsomal (Na+, K+)-ATPase ‘, Biochem. Biophys. Res. Commun., 35, 75-78.
GRUENER, R. P., and STERN, L. Z. (ig72), ‘ Diphenylhydantoin reverses membrane effects in steroid myopathy ‘, h’ature New Biol., Land., 235,54-j5.
-
KOREY, S. R. (Iqsi), -_ ,- ’ Effect of Dilantin and Mesantoin on the giant axon of the squid ‘, Proc. Sot. exp. Biol. Med.,
76, 297-299.
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL,R. J. (1g51), ‘ Protein measurement with the Folin phenol reagent ‘, 3. biol. Chem., x93,265-275. VON MOSER,H., WIESMANN, U., RICHTERICH,R.,
and ROSSI. E. CIo66a). ‘ Promessive Muskeldystrophie: VIII. Haufigke&, Klinik und Genetick der Tvnen I und II ‘. Schweir. med. Wschr., ~6, I 6q- I 74. VOX Mo~E~,-H. ,-W~E~MANN, U., RICHTERICH,R., and Rossr. E. fIo66b). ‘ Proszressive Muskeldystrophie: VIII. Haufigkeyt, Klinik und Genetick der Typen I und II ‘, Schweit. med. Wschr.) g6, 205-2 I I. OKINAKA.J., KUMAGAI,H., EBASHI,S., SUGITA, H., MOMOI,A. H., TOYOKURA,Y., and FUJIE,Y. ( I g6 I ) , ‘ Serum creatine phosphokinase ‘, Archs II
Neural., 4, j20-j2
j.
PARK DAVIS & COMPANY.nublication M-r?A. ” z Detroit, Michigan, Sept.: 1967. PETER,J. B., WORSFOLD,M., and PEARSON, C. M. (ig6g)> ‘ Erythrocyte ghost adenosine triphosphatase (ATPase) in Duchenne dystrophy ‘, 3. lab. clin. Med., 74, 103-108. PXNCUS, J. H:, and GIARMAN,N. J. (Ig67), ‘ The effect of dlphenylhydantoin on sodium-, potassium-, magnesium-stimulated adenosine triphosphatase activity of rat brain ‘) Biochem. Pharmac.:
16, 600-603.
C. R., BROWX.H. D., RIGDOX,R. H., CHATTOPADHYAY, POST,R. L., MERRITT,C. R., KINSOLVING, and .~LBRIGHT, C. D. (Ig6o)> ‘ Membrane S. I(.: and PATEL, A. (1968). ‘ Enzyme studies
344
CHATTOPADHYAY
adenosine triphosphatase as a participant active transport of sodium and potassium !mt~an erythrocyte ‘, 3. biol. Chem., 235,
in the in the 17g6-
G., DRBYFUS, J. C., and SCHAPIRA, F. ‘ L’elevation du taux de l’aldolase (‘953), serique ; test biochimeque des myopathies ‘, Sem. H&. Paris, 2~. 1917. SKOU, J. ‘C. (1g65):~’ Enzymatic basis for active transport of Na+ and K+ across cell membrane I, Physiol. Rev., 4, 596-617. SPERBLAKIS, N., and )IENN, F. A. (Ig7o), ‘ Effect of diphenylhydantoin on membrane potentials SCHAPI~,
AND BROWN and Na- K-ATPase of cultured chick heart cells ‘, Am. 3. Physiol., 218, 1224-1227. WOODBURY, D. M. (Ig55), ‘ Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats ‘, 3. Pharmac. e.rp. %., xx5s 74-95.
Key Word Index: Ouabain, ATPase, diphenylhydantoin, muscular dystrophy, red blood cells.