Pergamon Press
Life Sciences Vol . 22, pp . 127-132 Printed in the U .S .A .
(NA+ +K+ )-ATPASE OF DUCHENNE MUSCULAR DYSTROPHY ERYTHROCYTE GHOSTS Terry W . Pearson l Department of Human Physiology, University of California School of Medicine, Davis, California 95616 Received in final form November 18, 1977) SUMMARY Adenosine triphosphatase (ATPase) activity of erythrocyte ghosts from Duchenne muscular dystrophy (DMD) patients and carriers was stimulated by ouabain while nonrt~yopathic donor preparations were inhibited . Ethacrynic acid was an effective inhibitor in both DMD patients and carriers as well as in controls . However, responsiveness of the enzyme to suramin and harmaline generally differed in DMD groups from that of nonmyopathic donors . These data are consistent with the hypothesis that modified affinity of +Na ~inding sites) may account for some properties of the (Na +K )-ATPase activitiy of D~~iD erythrocytes . The relationship between adenosine triphosphatase activity of erythrocytes from individuals with Duchenne muscular dystrophy and the conductance of specific ions is largely unknown . Consequently, transport phenomena in red blond cells of dystrophic animals are poorly understood . An abnormal membrane (Na + K )-ATPase in DMD erythrocyte ghosts has been suggested based on reports that the enzyme activities of human DMD ,erythrocyte ghosts (1,2,3) and those of the white Pekin duck (4) are stimulated by the cardiac glycoside ouabain . Furthermore, a plasma factor has been hypothesized to be responsible for the altered erythrocyte properties (2) . It is difficult to interpret these findings, however, since the differing categories of muscular dystrophy studied have frequently utilized controls poorly matched by both age and sex to that of dystrophic individuals . These observations implied the need for further investigation+with carefully selected controls . Therefore, the responsiveness of the (Na +K )-ATPase activity to ouabain has been examined in erythrocyte ghosts of : 1) children with Duchenne muscular dystrophy, 2) carriers of Duchenne dystrophy (5), and 3) consistently matched controls . Other pharmacological agents studied include suramin, ethacrynic acid and harmaline . Suramin was chosen because of its r~ported inhibitory effects on cation fluxes in intact red cells and on (Na +K )ATPase in red cell ghosts (6) . Ethacrynic acid and harmaline were also used because they affect sodium dependent transport systems (7,8) or the ATPases of other systems (9,10,11) .
1 Present address : Cell Physiology Laboratory, Biology Dept ., Michigan Cancer Foundation, 110 E . Warren Ave ., Detroit, Michigan 48201
127
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Erythrocyte ATPase in Muscular Dystrophy
Vol . 22, No . 1, 1978
METHODS Samples of blood were donated at the University of California-Sacramento Medical Center Muscle Clinic by Duchenne dystrophy patients, their mothers (genetic carriers), and normal subjects (controls) . In all instances, DMD donors had been previously diagnosed (clinical features, muscle biopsy, serum creative phosphokinase activity) . Controls were matched by age and sex to that of corresponding patients and mothers . The methods of Brown et aZ . (1) and Peter et at . (2) were modified for preparation of red cell ghosts . All steps were carried out at 2 to 3°C . Blood (10 to 30 ml) was drawn into heparinized tubes and red blood cells sedimented at 2000 x g for 10 minutes . After removal of the huffy layer, the red cells were hemolyzed by dilution for 30 minutes in 5 volumes of 0 .02 mM EDTA and 2 mM Tris-HC1 (Sigma Chemical Co .), pH 7 .4 . The ghosts were sedimented at 48,000 x g for 10 minutes, the hemolysis procedure repeated, and the ghosts then spun at 48,000 x g for 30 minutes . The membranes were then washed three times with a solution containing 2 mM Tris-HC1, 0 .02 mM EDTA, and 15 .5 mM NaCI at pH 7 .4, sedimented at 48,000 x g for 30 minutes each time, and resuspended by gentle homogenization with a Teflon pestle in a Potter-Elvehjem tissue mill . Finally, the ghosts were suspended in approximately 1 ml of 250 mM sucrose in 100 mM Tris-HC1, pH 7 .2, and frozen at -10°C overnight or until use . The ATPase activity persisted for approximately 2 weeks ; longer storage in the frozen state resulted in a reduction in enzyme activity as well as loss of its ability to be stimulated . ATPase activity was assayed by the method outlined by Matsui and Schwartz (12) . The reaction mixture contained, in a total volume of 1 ml, 2 mM TrisATP (Sigma Chemical Co .), 5 mM MgC1 2 , 1 mM EDTA, 50 mM Tris-HC1 (pH 7 .4), 100 mM NaCI, 20 mM KC1, 0 .2 ml of ghost suspension (approximately 300 ug+ of+ enzyme protein), a~ 0 .1 ml of either water or inhibitor . To calculate (Na +K )-ATPase, the Mg -ATPase activity (assayed in the absence of NaCI and KC1) wad s~bstracted from the total ATPase activity (assayed in the presence of Na and K ). Incubations were performed in triplicates at 37°C and parallel control tubes (without enzyme) were incubated simultaneously . After pre-incubation for 3 minutes, the reaction was started by addition of ATP and incubated for 10 minutes at 37°C . The reaction was stopped by addition of ice-cold trichloroacetic acid in a final concentration of 5X (w/v) . ATP utilization (release of inorganic phosphate) was determined by the method of Fiske and Subbarow (13) and protein concentration estimated by the method of Lowry et aZ . (14) . The following inhibitors were employed : 0 .1 mM ouabain (Calbiochem), 0 .1 mM ethacrynic acid (Merck, Sharp and Dohme Research Labs .), 71 uM suramin (gift of Imperial Chemical Industries, Ltd . Pharmaceuticals Division), and 0 .1 mM harmaline (Sigma Chemical Co .) . Concentrations refer to final concentrations present in incubation media . The (Na++K+ )-ATPase specific activity is expressed as umoles of Pi reData are presented as the man + leased per minute per milligram of protein . the standard error and differences between the means of various groups analyzed by the Student t test . In the presence of inhibitors, the percent of stimulation or inhibition was determined and the mean of the percentage change calculated .
Vol . 22, No . 1, 1978
12 9
Erythrocyte ATPase in Muscular Dystrophy
RESULTS Two experimental results presented i~ TABLE 1 are consistent with previously reported data (1,2) : 1) the Na +K )-ATPase activity of the control donors (nonmyopathic) is greater than that of DMD patients . This difference in activity is statistically significant (p
Life Sciences Vol . 22, pp . 127-132 Printed in the U .S .A .
(NA+ +K+ )-ATPASE OF DUCHENNE MUSCULAR DYSTROPHY ERYTHROCYTE GHOSTS Terry W . Pearson l Department of Human Physiology, University of California School of Medicine, Davis, California 95616 Received in final form November 18, 1977) SUMMARY Adenosine triphosphatase (ATPase) activity of erythrocyte ghosts from Duchenne muscular dystrophy (DMD) patients and carriers was stimulated by ouabain while nonrt~yopathic donor preparations were inhibited . Ethacrynic acid was an effective inhibitor in both DMD patients and carriers as well as in controls . However, responsiveness of the enzyme to suramin and harmaline generally differed in DMD groups from that of nonmyopathic donors . These data are consistent with the hypothesis that modified affinity of +Na ~inding sites) may account for some properties of the (Na +K )-ATPase activitiy of D~~iD erythrocytes . The relationship between adenosine triphosphatase activity of erythrocytes from individuals with Duchenne muscular dystrophy and the conductance of specific ions is largely unknown . Consequently, transport phenomena in red blond cells of dystrophic animals are poorly understood . An abnormal membrane (Na + K )-ATPase in DMD erythrocyte ghosts has been suggested based on reports that the enzyme activities of human DMD ,erythrocyte ghosts (1,2,3) and those of the white Pekin duck (4) are stimulated by the cardiac glycoside ouabain . Furthermore, a plasma factor has been hypothesized to be responsible for the altered erythrocyte properties (2) . It is difficult to interpret these findings, however, since the differing categories of muscular dystrophy studied have frequently utilized controls poorly matched by both age and sex to that of dystrophic individuals . These observations implied the need for further investigation+with carefully selected controls . Therefore, the responsiveness of the (Na +K )-ATPase activity to ouabain has been examined in erythrocyte ghosts of : 1) children with Duchenne muscular dystrophy, 2) carriers of Duchenne dystrophy (5), and 3) consistently matched controls . Other pharmacological agents studied include suramin, ethacrynic acid and harmaline . Suramin was chosen because of its r~ported inhibitory effects on cation fluxes in intact red cells and on (Na +K )ATPase in red cell ghosts (6) . Ethacrynic acid and harmaline were also used because they affect sodium dependent transport systems (7,8) or the ATPases of other systems (9,10,11) .
1 Present address : Cell Physiology Laboratory, Biology Dept ., Michigan Cancer Foundation, 110 E . Warren Ave ., Detroit, Michigan 48201
127
12 8
Erythrocyte ATPase in Muscular Dystrophy
Vol . 22, No . 1, 1978
METHODS Samples of blood were donated at the University of California-Sacramento Medical Center Muscle Clinic by Duchenne dystrophy patients, their mothers (genetic carriers), and normal subjects (controls) . In all instances, DMD donors had been previously diagnosed (clinical features, muscle biopsy, serum creative phosphokinase activity) . Controls were matched by age and sex to that of corresponding patients and mothers . The methods of Brown et aZ . (1) and Peter et at . (2) were modified for preparation of red cell ghosts . All steps were carried out at 2 to 3°C . Blood (10 to 30 ml) was drawn into heparinized tubes and red blood cells sedimented at 2000 x g for 10 minutes . After removal of the huffy layer, the red cells were hemolyzed by dilution for 30 minutes in 5 volumes of 0 .02 mM EDTA and 2 mM Tris-HC1 (Sigma Chemical Co .), pH 7 .4 . The ghosts were sedimented at 48,000 x g for 10 minutes, the hemolysis procedure repeated, and the ghosts then spun at 48,000 x g for 30 minutes . The membranes were then washed three times with a solution containing 2 mM Tris-HC1, 0 .02 mM EDTA, and 15 .5 mM NaCI at pH 7 .4, sedimented at 48,000 x g for 30 minutes each time, and resuspended by gentle homogenization with a Teflon pestle in a Potter-Elvehjem tissue mill . Finally, the ghosts were suspended in approximately 1 ml of 250 mM sucrose in 100 mM Tris-HC1, pH 7 .2, and frozen at -10°C overnight or until use . The ATPase activity persisted for approximately 2 weeks ; longer storage in the frozen state resulted in a reduction in enzyme activity as well as loss of its ability to be stimulated . ATPase activity was assayed by the method outlined by Matsui and Schwartz (12) . The reaction mixture contained, in a total volume of 1 ml, 2 mM TrisATP (Sigma Chemical Co .), 5 mM MgC1 2 , 1 mM EDTA, 50 mM Tris-HC1 (pH 7 .4), 100 mM NaCI, 20 mM KC1, 0 .2 ml of ghost suspension (approximately 300 ug+ of+ enzyme protein), a~ 0 .1 ml of either water or inhibitor . To calculate (Na +K )-ATPase, the Mg -ATPase activity (assayed in the absence of NaCI and KC1) wad s~bstracted from the total ATPase activity (assayed in the presence of Na and K ). Incubations were performed in triplicates at 37°C and parallel control tubes (without enzyme) were incubated simultaneously . After pre-incubation for 3 minutes, the reaction was started by addition of ATP and incubated for 10 minutes at 37°C . The reaction was stopped by addition of ice-cold trichloroacetic acid in a final concentration of 5X (w/v) . ATP utilization (release of inorganic phosphate) was determined by the method of Fiske and Subbarow (13) and protein concentration estimated by the method of Lowry et aZ . (14) . The following inhibitors were employed : 0 .1 mM ouabain (Calbiochem), 0 .1 mM ethacrynic acid (Merck, Sharp and Dohme Research Labs .), 71 uM suramin (gift of Imperial Chemical Industries, Ltd . Pharmaceuticals Division), and 0 .1 mM harmaline (Sigma Chemical Co .) . Concentrations refer to final concentrations present in incubation media . The (Na++K+ )-ATPase specific activity is expressed as umoles of Pi reData are presented as the man + leased per minute per milligram of protein . the standard error and differences between the means of various groups analyzed by the Student t test . In the presence of inhibitors, the percent of stimulation or inhibition was determined and the mean of the percentage change calculated .
Vol . 22, No . 1, 1978
12 9
Erythrocyte ATPase in Muscular Dystrophy
RESULTS Two experimental results presented i~ TABLE 1 are consistent with previously reported data (1,2) : 1) the Na +K )-ATPase activity of the control donors (nonmyopathic) is greater than that of DMD patients . This difference in activity is statistically significant (p
