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Red cell membrane Ca-ATPase in cystic fibrosis: increased activation by Na
Clinica Cbimica Acta, 138 (1984) 259-265 Elsevier
259
CCA 02822
Red cell membrane Ca-ATPase in cystic fibrosis: increased activation by Na David L. Clough, a,* and Van S. Hubbard b a Department
of Physiology, Uniformed Services University, Bethesda, MD and b Pediatric Metabolism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, IUD (USA) (Received October 13th, 1983) Key worak Cystic fibrosis; Red cell; Ca -ATPase;
Sodium
Summary
In the present study, we compared the kinetics of activation by Na, of red cell membrane Ca-ATPase of cystic fibrosis (CF) patients and healthy volunteers (controls). Calmodulin (membrane-free hemolysate) was included in the assay medium to promote maximal Ca-ATPase activation. There were no significant differences between the red cell Ca-ATPase activities of the two groups, assayed either in the absence or in the presence of optimal concentrations of Na. There were also no significant differences between the apparent dissociation constants or Hill coefficients for activation of red cell Ca-ATPase by Na. On the other hand, the percent activation by Na of red cell Ca-ATPase activity of the CF patients was approximately 40% greater than that of the controls. The additional increment of Ca-ATPase activity due to the elevated percent activation was about 14% of the total red cell Ca-ATPase activity of the CF patients. Although this increment of Ca-ATPase activity is relatively small, the increased percent activation by Na does suggest an alteration in the enzyme’s response to Na. At present we do not know whether or not this alteration applies to Ca-transport or if it is of any pathophysiological importance.
Introduction
Horton et al [ 11, in 1970, observed a decrease in Ca-ATPase activity of red cells from patients with cystic fibrosis (CF) and suggested that the major defect in CF
* Send all correspondence to: David L. Clough, PhD, Department University, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. 0009-8981/84/$03.00
6 1984 Ekevier Science Publishers B.V.
of Physiology,
Uniformed
Services
260
transport.. .’ However, other might ‘... be in the Ca-ATPase portion of membrane investigators reported in 1974 that red cell Ca-ATPase activity is unaltered in CF patients [2-41. More recently, Katz [5] observed a 50% decrease in red cell Ca-ATPase activity of CF patients thus confirming the findings of Horton et al [l]; however, Foder et al [6] reported that, although they did observe a decrease in red cell Ca-ATPase activity of CF patients, the decrease was small (15%) and did not correlate with clinical score. The specific activities reported by Foder et al [6] were around 4 pmol of inorganic phosphate (Pi). mgg ’ protein * h-’ as compared to activities of less than 1 pmol Pi . mgg ’ protein * h-’ in the previous studies [l-5] and they suggested that the low specific activities might be attributable in part to suboptimal assay conditions such as the absence of monovalent cations (Na or K) or calmodulin. Monovalent cations [7] and calmodulin [8] (a soluble protein activator from red cell cytoplasm [9]) both stimulate human red cell Ca-ATPase. Katz and Emery [lo] subsequently showed that the decrease which they observed in red cell Ca-ATPase activity of CF patients was not due to an alteration in the effect of calmodulin. However, this does not rule out possible alterations in the effects of monovalent cations. Therefore, in the present study, we compared the kinetics of activation by Na, of red cell Ca-ATPase activity of CF patients and healthy control volunteers. Calmodulin (membrane-free hemolysate) and Ca were present at concentrations sufficient for maximal activation of Ca-ATPase. Methods Nine CF patients (8 male, 1 female, aged 22-32 years) and nine control subjects (8 male, 1 female, aged 21-28 years) were studied. All CF patients had a medical history and clinical evaluation indicative of the diagnosis of CF and a positive sweat test. Seven of the CF patients had exocrine pancreatic insufficiency, while two had clinically normal exocrine pancreatic function as assessed by at least three of the following studies: (a) duodenal intubation for digestive enzyme activity, (b) 72-h quantitative stool fat, (c) serum isoamylase determinations, and (d) serum carotene levels. The CF patients had an NIH clinical score [ll] ranging from 70-88 and were clinically stable at the time of blood drawing. Control subjects were volunteers without history or symptoms of the respiratory or gastrointestional tract. The studies were approved by the Institute Clinical Research Subpanel within the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases and informed consent was obtained from all subjects. For each experiment intravenous blood was collected in heparinized syringes, from one CF patient and one control subject on the same day. The blood was kept on ice until used and membranes from the two samples were prepared and assayed at the same time. Red cell membranes were prepared as described previously [9]. Briefly, this involved washing the red cells in 160 mmol/l Tris-HCl (pH 6.8), lysing the cells in 5 ~01s. of 1 mmol/l Tris-EDTA (pH 7.5) and repeated washing of the membranes with the lysing medium until they were essentially white. The membranes were given a final wash in 10 mmol/l Tris-HCl (pH 6.8), suspended in 10 mmol/l imidazole (pH 7.4), and stored on ice in a refrigerator.
261
Membrane-free hemolysate was prepared from outdated red cells obtained from a blood bank. The cells were washed and lysed as described previously [9]. The membranes were removed by centrifugation and the hemolysate was dialized for 3 days against three changes of about 25 ~01s. of distilled water at 4°C. Each pair of membrane preparations (cystic fibrosis and control) was assayed with a different hemolysate; however, both membrane preparations of each pair were assayed with the same hemolysate. Hemolysates were tested on individual membrane preparations to ensure maximal activation of Ca-ATPase activity. In the final incubation medium, the concentrations of contaminant Na and K, contributed by the hemolysate, were less than 50 pmol/l (flame photometry) and contaminant Ca was less than 10 pmol/l (atomic absorption spectrometry). Ca-ATPase activity was assayed as described previously [9], except that the free Ca concentration was buffered with Ca-EGTA buffers calculated to give a free Ca concentration of 8 X lop5 mol/l [12], and the Na concentration was varied to determine the kinetics of activation. Total ATPase activity was assayed by measuring the amount of Pi released from ATP during a l-h incubation of membranes at 37°C in 2 ml of a medium containing (mmol/l): 2 Tris-ATP (Sigma Chemical Company, St. Louis, MO, USA), 2.5 MgCl,, 0.18 CaCl,, 0.1 EGTA, 40 Tris-HCl (pH 7.5 at 37”(Z), 0.5 ouabain and a sufficient amount of membrane-free hemolysate (containing calmodulin) to maximally activate the Ca-ATPase. Mg-ATPase was assayed under similar conditions except that Ca was omitted and the EGTA concentration was 0.5 mmol/l. Ca-ATPase activity was calculated by subtracting the Mg-ATPase activity from the total ATPase activity and specific activities were expressed as prnol Pi * mgg ’ * protein h -I. Pi was assayed by the method of Fisk and SubbaRow [13] and membrane protein was assayed by the method of Lowry et al [14]. Activation of Ca-ATPase by Na was expressed as percent activation (PA). PA was calculated by dividing the increment of Ca-ATPase activity due to the presence of Na (total Ca-ATPase activity at each Na concentration minus Ca-ATPase activity in the absence of Na) by the activity in the absence of Na, and multiplying the ratio by 100%. Maximal percent activation (MA) was expressed as the greatest PA occurring on each individual Na activation curve. The Na concentration at which MA occurred was not always the same for different membrane preparations. To allow a comparison of the pattern of activation by Na, Ca-ATPase activities were also expressed as percent maximal activation (PMA). PMA was calculated as (PA/MA) X 100%. MA was, therefore, converted to 100%. Hill plots of PMA’s were used to determine the apparent dissociation constants for Na (K,, the antilog of the intercept on the abscissa) and Hill coefficients (n, the negative of the slope). Students t test was used for all statistical comparisons and differences were considered significant at p < 0.05.
Results Neither the sex of the subject, clinical function appeared to affect the results.
score, nor the status of exocrine
pancreatic
262
OCONTROL ICF .B>P>.7
.-
2! :
r;
3.0 -
“t FpLizzLm~
.3>p>.2
F 2.0B z =
p<.O25
.3>p>.2
><.05
Q CONTROL
1.0-
Na ABSENT
Na PRESENT
DIFFERENCE [Na] mmd/l
Fig. 1. Specific Ca-ATPase activity of red cell membranes from CF patients (CF) and healthy volunteers (control). Activities are shown in the absence of Na and in the presence of Na concentrations which were found to produce maximal activity (determined from individual Na activation curves). Difference equals Ca-ATPase activity in the presence of Na minus Ca-ATPase. activity in the absence of Na. Values are means f SEM, n = 9 for both groups. Fig. 2. Percent activation (PA) by Na (l-80 mmol/l) and maximal PA (MA) of red cell membrane Ca-ATPase from CF patients (CF) and healthy volunteers (control). Values are means f SEM, n = 7 for both groups.
Specific Ca-ATPase activities of red cell membranes from CF patients and healthy control volunteers are shown in Fig. 1. There was no significant difference between the Ca-ATPase activities of the two groups either in the absence of Na or in the presence of Na concentrations which produced maximal activation. Fig. 2 shows Na activation curves for Ca-ATPase from CF patients and controls plotted as PA vs. the Na concentration. The activation curve for the CF patients was elevated relative to that of the controls and the mean of the MA’s by Na (the greatest PA occurring on each individual curve before averaging) was significantly
0 CONTROL .CF
b
loa_. INal.
I 20
40
so
50
‘.
-
Fig. 3. Percent of maximal activation by Na of red cell membrane Ca-ATPase from CF patients (CF) and healthy volunteers (control). Inset: Hill plot of data of Fig. 3 drawn by linear regression. Values are means f SEM. No values were significantly different at p < 0.05, n = 7 for both groups.
263
greater (42.8%) for the CF patients than for the controls (CF MA = 85.7 + 8.2’%, n = 7; control MA = 60 k7.18, n = 7; p -c 0.05). The increase in PA for the cystic fibrosis patients relative to the controls, averaged for all Na concentrations from 5 to 80 mmol/l, was 45.9 f 3.6%. Therefore, the mean PA of the Ca-ATPase of the CF patients over this range of Na concentrations was around 46% greater than that of the controls. In Fig. 3 PMA has been plotted against the Na concentration to allow a comparison of the patterns of activation by Na for the CF patients and the controls. The pattern of activation appears to be essentially the same for the two groups and there was no significant difference between the Ko’s (CF K, = 10.5 f 2.1 mmol/l, n = 7; control K, = 10.1 + 1.8 mmol/l, n = 7, NS) or the Hill coefficients (CF n = 1.23 k 0.06, n = 7; control n = 1.30 k 0.07, n = 7, NS). Discussion No significant difference was observed between red cell membrane Ca-ATPase activities of CF patients and controls assayed either in the presence or absence of Na. The specific activities observed in the presence of calmodulin (membrane-free hemolysate) and Na were in the range of those reported by Bond and Clough [9] and more recently by Foder et al [6]. In these two studies and also in the present study, monovalent cations and calmodulin were included in the assay medium to promote maximal activation of red cell Ca-ATPase. As pointed out by Foder et al, the lower specific red cell Ca-ATPase activities reported in earlier CF studies may have been due in part to suboptimal concentrations of substrate or activators (see [6]). In the present study, the percent activation by Na of red cell Ca-ATPase of the CF patients was about 40% greater than that of the controls. This increase in PA should exist at all Na concentrations because the K, and Hill coefficient for activation by Na were essentially the same for the two groups. However, it would have a greater effect on total specific Ca-ATPase activities assayed in the presence of relatively high Na concentrations (around 60 mmol/l) because at higher Na concentrations the Na-dependent component of total Ca-ATPase activity would be proportionally larger. In our experiments 60 mmol/l Na appeared to be optimal because the mean PMA for both groups was around 94% and increasing the Na concentration above 60 mmol/l resulted in a progressive decrease in Ca-ATPase activity of some membrane preparations. At 60 mmol/l Na the specific Ca-ATPase activity of the CF patients was increased by 80.7 k 7.1% whereas that of the control was increased by 56.3 + 6.4%. It should be pointed out that, although the increase in PA by Na, of red cell Ca-ATPase activity of CF patients was relatively large, its contribution to total Ca-ATPase activity was relatively small, averaging 0.46 pmol Pi smg-’ protein. h-’ or about 14% of the total Ca-ATPase activity. On the other hand, this increase does suggest an alteration in the enzyme’s response to monovalent cations. This altered response might be a compensatory response secondary to the decreased red cell Ca-ATPase activity of CF patients observed by others [1,5,6], although we failed to observe a significant decrease in the present study.
264
Katz [5] observed a 50% decrease in red cell Ca-ATPase activity of CF patients. In this study monovalent cations (Na or K) were not included in the assay medium. Foder et al [6] included 60 mmol/l K in their assay medium and observed only a 15% decrease in Ca-ATPase activity. The quantitative difference between these results might be explained in part by an increase in PA, by monovalent cations, of red cell Ca-ATPase from CF patients. This effect would tend to reduce any difference between red cell Ca-ATPase activities of the two groups, observed in the absence of monovalent cations. On the other hand, some of the previous investigators [2-41 who failed to observe a decrease in red cell Ca-ATPase activity of CF patients, did not include monovalent cations in their incubation media. The results of these studies are, therefore, independent of any alterations in the effects of monovalent cations. Like Ca-ATPase, red cell Ca-transport is also activated by monovalent cations [15]. An alteration in the effects of monovalent cations on Ca-ATPase activity might, therefore, be expected to apply to Ca-transport as well. If the increase in PA by Na observed in the present study does pertain to Ca-transport, it would seem unlikely that an increase of only 14% would have any substantial effect on the overall capacity for Ca-transport in red cells from CF patients. However, it would also seem unlikely that this alteration is due to a second class of Ca-ATPase unrelated to Ca transport, because there was no significant difference between either the K,‘s or the Hill coefficients of the CF patients and controls. Ansah and Katz [16] observed a decrease in Ca-uptake by inside-out red cell vesicles from CF patients, and in these transport studies calmodulin and 40 mmol/l Na were included in the incubation medium. Red cell Ca-transport, therefore, appears to be reduced in CF patients, even when assayed in the presence of monovalent cations. We are, therefore, unable to speculate at this time on the possible pathophysiological role of the increased PA by Na, of red cell Ca-ATPase of CF patients. Whether or not this effect of monovalent cations applies to red cell Ca-transport in CF patients remains to be determined. Acknowledgements We thank Karen Knoble for her excellent technical assistance and Patricia Prather for her expert assistance in the preparation of this manuscript. This study was supported by Uniformed Services University Grant 818 CO 7604. References 1 Horton CR, Cole WQ, Bader H. Depressed (Ca)-transport ATPase in cystic fibrosis erythrocytes. Biochem Biophys Res Commun 1970; 40: 505-509. 2 Duffy MJ, Cohn EV, Schwartz V. Ca-uptake and binding by isolated erythrocyte membranes from cystic fibrosis and control subjects. Chn Chim Acta 1974; 50: 97-101. 3 Feig SA, Segel GB, Kern KA, Osher AB, Schwartz RH. Erythrocyte transport function in cystic fibrosis. Pediatr Res 1974; 8: 594-597. 4 McEvoy FA, Davies RJ, Goodchild MC, Anderson CM. Erythrocyte membrane properties in cystic fibrosis. Clin Chim Acta 1974: 54: 195-204.
265 5 Katz S. Calcium and sodium transport processes in patients with cystic fibrosis. I. A specific decrease in Mg-dependent, Ca-adenosine triphosphatase activity in erythrocyte membranes from cystic fibrosis patients. Pediatr Res 1978; 12: 1033-1038. 6 Foder B, Scharff 0, Tonnesen P. Activator associated Ca-ATPase in erythrocyte membranes from cystic fibrosis patients. Chn Chim Acta 1980; 104: 187-193. 7 Bond GH, Green JW. Effects of monovalent cations on the (Mg + Ca)-dependent ATPase of the red cell membrane. Biochim Biophys Acta 1971; 241: 393-398. 8 Gopinath RM, Vincenzi FE. Phosphodiesterase protein activation mimics red blood cell cytoplasmic activation of (Ca-Mg)ATPase. Biochem Biophys Res Commun 1977; 77: 1203-1209. 9 Bond GH, Clot@ DL. A soluble protein activator of (Mg + Ca)-dependent ATPase in human red cell membranes. Biochim Biophys Acta 1973; 323: 592-599. 10 Katz S, Emery DL. The role of calmodulin in the regulation of (Mg+Ca)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls. Cell Calcium 1981; 2: 545-552. 11 Taussig LM, Kattwinkel J, Friedewald WT, di Sant’ Agnese PA. A new prognostic score and clinical evaluation system for cystic fibrosis. J Pediat 1973; 82: 380-390. 12 Portzehl H, Caldwell PC, Ruegg JC. The dependence of relaxation of muscle fibers from the crab Maia Squinado on the internal concentration of free calcium ions. Biochim Biophys Acta 1964; 79: 581-591. 13 Fiske CH, SubbaRow Y. The calorimetric determination of phosphorous. J Biol Chem 1925; 66: 375-400. 14 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 15 Wierichs R, Bader H. Influence of monovalent ions on the activity of the (Ca+Mg)-ATPase and Ca-transport of human red blood cells. Biochim Biophys Acta 1980; 596: 325-328. 16 Ansah T, Katz S. Evidence for a Ca-transport deficiency in patients with cystic fibrosis. Cell Calcium 1980; 1: 195-203.
259
CCA 02822
Red cell membrane Ca-ATPase in cystic fibrosis: increased activation by Na David L. Clough, a,* and Van S. Hubbard b a Department
of Physiology, Uniformed Services University, Bethesda, MD and b Pediatric Metabolism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, IUD (USA) (Received October 13th, 1983) Key worak Cystic fibrosis; Red cell; Ca -ATPase;
Sodium
Summary
In the present study, we compared the kinetics of activation by Na, of red cell membrane Ca-ATPase of cystic fibrosis (CF) patients and healthy volunteers (controls). Calmodulin (membrane-free hemolysate) was included in the assay medium to promote maximal Ca-ATPase activation. There were no significant differences between the red cell Ca-ATPase activities of the two groups, assayed either in the absence or in the presence of optimal concentrations of Na. There were also no significant differences between the apparent dissociation constants or Hill coefficients for activation of red cell Ca-ATPase by Na. On the other hand, the percent activation by Na of red cell Ca-ATPase activity of the CF patients was approximately 40% greater than that of the controls. The additional increment of Ca-ATPase activity due to the elevated percent activation was about 14% of the total red cell Ca-ATPase activity of the CF patients. Although this increment of Ca-ATPase activity is relatively small, the increased percent activation by Na does suggest an alteration in the enzyme’s response to Na. At present we do not know whether or not this alteration applies to Ca-transport or if it is of any pathophysiological importance.
Introduction
Horton et al [ 11, in 1970, observed a decrease in Ca-ATPase activity of red cells from patients with cystic fibrosis (CF) and suggested that the major defect in CF
* Send all correspondence to: David L. Clough, PhD, Department University, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. 0009-8981/84/$03.00
6 1984 Ekevier Science Publishers B.V.
of Physiology,
Uniformed
Services
260
transport.. .’ However, other might ‘... be in the Ca-ATPase portion of membrane investigators reported in 1974 that red cell Ca-ATPase activity is unaltered in CF patients [2-41. More recently, Katz [5] observed a 50% decrease in red cell Ca-ATPase activity of CF patients thus confirming the findings of Horton et al [l]; however, Foder et al [6] reported that, although they did observe a decrease in red cell Ca-ATPase activity of CF patients, the decrease was small (15%) and did not correlate with clinical score. The specific activities reported by Foder et al [6] were around 4 pmol of inorganic phosphate (Pi). mgg ’ protein * h-’ as compared to activities of less than 1 pmol Pi . mgg ’ protein * h-’ in the previous studies [l-5] and they suggested that the low specific activities might be attributable in part to suboptimal assay conditions such as the absence of monovalent cations (Na or K) or calmodulin. Monovalent cations [7] and calmodulin [8] (a soluble protein activator from red cell cytoplasm [9]) both stimulate human red cell Ca-ATPase. Katz and Emery [lo] subsequently showed that the decrease which they observed in red cell Ca-ATPase activity of CF patients was not due to an alteration in the effect of calmodulin. However, this does not rule out possible alterations in the effects of monovalent cations. Therefore, in the present study, we compared the kinetics of activation by Na, of red cell Ca-ATPase activity of CF patients and healthy control volunteers. Calmodulin (membrane-free hemolysate) and Ca were present at concentrations sufficient for maximal activation of Ca-ATPase. Methods Nine CF patients (8 male, 1 female, aged 22-32 years) and nine control subjects (8 male, 1 female, aged 21-28 years) were studied. All CF patients had a medical history and clinical evaluation indicative of the diagnosis of CF and a positive sweat test. Seven of the CF patients had exocrine pancreatic insufficiency, while two had clinically normal exocrine pancreatic function as assessed by at least three of the following studies: (a) duodenal intubation for digestive enzyme activity, (b) 72-h quantitative stool fat, (c) serum isoamylase determinations, and (d) serum carotene levels. The CF patients had an NIH clinical score [ll] ranging from 70-88 and were clinically stable at the time of blood drawing. Control subjects were volunteers without history or symptoms of the respiratory or gastrointestional tract. The studies were approved by the Institute Clinical Research Subpanel within the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases and informed consent was obtained from all subjects. For each experiment intravenous blood was collected in heparinized syringes, from one CF patient and one control subject on the same day. The blood was kept on ice until used and membranes from the two samples were prepared and assayed at the same time. Red cell membranes were prepared as described previously [9]. Briefly, this involved washing the red cells in 160 mmol/l Tris-HCl (pH 6.8), lysing the cells in 5 ~01s. of 1 mmol/l Tris-EDTA (pH 7.5) and repeated washing of the membranes with the lysing medium until they were essentially white. The membranes were given a final wash in 10 mmol/l Tris-HCl (pH 6.8), suspended in 10 mmol/l imidazole (pH 7.4), and stored on ice in a refrigerator.
261
Membrane-free hemolysate was prepared from outdated red cells obtained from a blood bank. The cells were washed and lysed as described previously [9]. The membranes were removed by centrifugation and the hemolysate was dialized for 3 days against three changes of about 25 ~01s. of distilled water at 4°C. Each pair of membrane preparations (cystic fibrosis and control) was assayed with a different hemolysate; however, both membrane preparations of each pair were assayed with the same hemolysate. Hemolysates were tested on individual membrane preparations to ensure maximal activation of Ca-ATPase activity. In the final incubation medium, the concentrations of contaminant Na and K, contributed by the hemolysate, were less than 50 pmol/l (flame photometry) and contaminant Ca was less than 10 pmol/l (atomic absorption spectrometry). Ca-ATPase activity was assayed as described previously [9], except that the free Ca concentration was buffered with Ca-EGTA buffers calculated to give a free Ca concentration of 8 X lop5 mol/l [12], and the Na concentration was varied to determine the kinetics of activation. Total ATPase activity was assayed by measuring the amount of Pi released from ATP during a l-h incubation of membranes at 37°C in 2 ml of a medium containing (mmol/l): 2 Tris-ATP (Sigma Chemical Company, St. Louis, MO, USA), 2.5 MgCl,, 0.18 CaCl,, 0.1 EGTA, 40 Tris-HCl (pH 7.5 at 37”(Z), 0.5 ouabain and a sufficient amount of membrane-free hemolysate (containing calmodulin) to maximally activate the Ca-ATPase. Mg-ATPase was assayed under similar conditions except that Ca was omitted and the EGTA concentration was 0.5 mmol/l. Ca-ATPase activity was calculated by subtracting the Mg-ATPase activity from the total ATPase activity and specific activities were expressed as prnol Pi * mgg ’ * protein h -I. Pi was assayed by the method of Fisk and SubbaRow [13] and membrane protein was assayed by the method of Lowry et al [14]. Activation of Ca-ATPase by Na was expressed as percent activation (PA). PA was calculated by dividing the increment of Ca-ATPase activity due to the presence of Na (total Ca-ATPase activity at each Na concentration minus Ca-ATPase activity in the absence of Na) by the activity in the absence of Na, and multiplying the ratio by 100%. Maximal percent activation (MA) was expressed as the greatest PA occurring on each individual Na activation curve. The Na concentration at which MA occurred was not always the same for different membrane preparations. To allow a comparison of the pattern of activation by Na, Ca-ATPase activities were also expressed as percent maximal activation (PMA). PMA was calculated as (PA/MA) X 100%. MA was, therefore, converted to 100%. Hill plots of PMA’s were used to determine the apparent dissociation constants for Na (K,, the antilog of the intercept on the abscissa) and Hill coefficients (n, the negative of the slope). Students t test was used for all statistical comparisons and differences were considered significant at p < 0.05.
Results Neither the sex of the subject, clinical function appeared to affect the results.
score, nor the status of exocrine
pancreatic
262
OCONTROL ICF .B>P>.7
.-
2! :
r;
3.0 -
“t FpLizzLm~
.3>p>.2
F 2.0B z =
p<.O25
.3>p>.2
><.05
Q CONTROL
1.0-
Na ABSENT
Na PRESENT
DIFFERENCE [Na] mmd/l
Fig. 1. Specific Ca-ATPase activity of red cell membranes from CF patients (CF) and healthy volunteers (control). Activities are shown in the absence of Na and in the presence of Na concentrations which were found to produce maximal activity (determined from individual Na activation curves). Difference equals Ca-ATPase activity in the presence of Na minus Ca-ATPase. activity in the absence of Na. Values are means f SEM, n = 9 for both groups. Fig. 2. Percent activation (PA) by Na (l-80 mmol/l) and maximal PA (MA) of red cell membrane Ca-ATPase from CF patients (CF) and healthy volunteers (control). Values are means f SEM, n = 7 for both groups.
Specific Ca-ATPase activities of red cell membranes from CF patients and healthy control volunteers are shown in Fig. 1. There was no significant difference between the Ca-ATPase activities of the two groups either in the absence of Na or in the presence of Na concentrations which produced maximal activation. Fig. 2 shows Na activation curves for Ca-ATPase from CF patients and controls plotted as PA vs. the Na concentration. The activation curve for the CF patients was elevated relative to that of the controls and the mean of the MA’s by Na (the greatest PA occurring on each individual curve before averaging) was significantly
0 CONTROL .CF
b
loa_. INal.
I 20
40
so
50
‘.
-
Fig. 3. Percent of maximal activation by Na of red cell membrane Ca-ATPase from CF patients (CF) and healthy volunteers (control). Inset: Hill plot of data of Fig. 3 drawn by linear regression. Values are means f SEM. No values were significantly different at p < 0.05, n = 7 for both groups.
263
greater (42.8%) for the CF patients than for the controls (CF MA = 85.7 + 8.2’%, n = 7; control MA = 60 k7.18, n = 7; p -c 0.05). The increase in PA for the cystic fibrosis patients relative to the controls, averaged for all Na concentrations from 5 to 80 mmol/l, was 45.9 f 3.6%. Therefore, the mean PA of the Ca-ATPase of the CF patients over this range of Na concentrations was around 46% greater than that of the controls. In Fig. 3 PMA has been plotted against the Na concentration to allow a comparison of the patterns of activation by Na for the CF patients and the controls. The pattern of activation appears to be essentially the same for the two groups and there was no significant difference between the Ko’s (CF K, = 10.5 f 2.1 mmol/l, n = 7; control K, = 10.1 + 1.8 mmol/l, n = 7, NS) or the Hill coefficients (CF n = 1.23 k 0.06, n = 7; control n = 1.30 k 0.07, n = 7, NS). Discussion No significant difference was observed between red cell membrane Ca-ATPase activities of CF patients and controls assayed either in the presence or absence of Na. The specific activities observed in the presence of calmodulin (membrane-free hemolysate) and Na were in the range of those reported by Bond and Clough [9] and more recently by Foder et al [6]. In these two studies and also in the present study, monovalent cations and calmodulin were included in the assay medium to promote maximal activation of red cell Ca-ATPase. As pointed out by Foder et al, the lower specific red cell Ca-ATPase activities reported in earlier CF studies may have been due in part to suboptimal concentrations of substrate or activators (see [6]). In the present study, the percent activation by Na of red cell Ca-ATPase of the CF patients was about 40% greater than that of the controls. This increase in PA should exist at all Na concentrations because the K, and Hill coefficient for activation by Na were essentially the same for the two groups. However, it would have a greater effect on total specific Ca-ATPase activities assayed in the presence of relatively high Na concentrations (around 60 mmol/l) because at higher Na concentrations the Na-dependent component of total Ca-ATPase activity would be proportionally larger. In our experiments 60 mmol/l Na appeared to be optimal because the mean PMA for both groups was around 94% and increasing the Na concentration above 60 mmol/l resulted in a progressive decrease in Ca-ATPase activity of some membrane preparations. At 60 mmol/l Na the specific Ca-ATPase activity of the CF patients was increased by 80.7 k 7.1% whereas that of the control was increased by 56.3 + 6.4%. It should be pointed out that, although the increase in PA by Na, of red cell Ca-ATPase activity of CF patients was relatively large, its contribution to total Ca-ATPase activity was relatively small, averaging 0.46 pmol Pi smg-’ protein. h-’ or about 14% of the total Ca-ATPase activity. On the other hand, this increase does suggest an alteration in the enzyme’s response to monovalent cations. This altered response might be a compensatory response secondary to the decreased red cell Ca-ATPase activity of CF patients observed by others [1,5,6], although we failed to observe a significant decrease in the present study.
264
Katz [5] observed a 50% decrease in red cell Ca-ATPase activity of CF patients. In this study monovalent cations (Na or K) were not included in the assay medium. Foder et al [6] included 60 mmol/l K in their assay medium and observed only a 15% decrease in Ca-ATPase activity. The quantitative difference between these results might be explained in part by an increase in PA, by monovalent cations, of red cell Ca-ATPase from CF patients. This effect would tend to reduce any difference between red cell Ca-ATPase activities of the two groups, observed in the absence of monovalent cations. On the other hand, some of the previous investigators [2-41 who failed to observe a decrease in red cell Ca-ATPase activity of CF patients, did not include monovalent cations in their incubation media. The results of these studies are, therefore, independent of any alterations in the effects of monovalent cations. Like Ca-ATPase, red cell Ca-transport is also activated by monovalent cations [15]. An alteration in the effects of monovalent cations on Ca-ATPase activity might, therefore, be expected to apply to Ca-transport as well. If the increase in PA by Na observed in the present study does pertain to Ca-transport, it would seem unlikely that an increase of only 14% would have any substantial effect on the overall capacity for Ca-transport in red cells from CF patients. However, it would also seem unlikely that this alteration is due to a second class of Ca-ATPase unrelated to Ca transport, because there was no significant difference between either the K,‘s or the Hill coefficients of the CF patients and controls. Ansah and Katz [16] observed a decrease in Ca-uptake by inside-out red cell vesicles from CF patients, and in these transport studies calmodulin and 40 mmol/l Na were included in the incubation medium. Red cell Ca-transport, therefore, appears to be reduced in CF patients, even when assayed in the presence of monovalent cations. We are, therefore, unable to speculate at this time on the possible pathophysiological role of the increased PA by Na, of red cell Ca-ATPase of CF patients. Whether or not this effect of monovalent cations applies to red cell Ca-transport in CF patients remains to be determined. Acknowledgements We thank Karen Knoble for her excellent technical assistance and Patricia Prather for her expert assistance in the preparation of this manuscript. This study was supported by Uniformed Services University Grant 818 CO 7604. References 1 Horton CR, Cole WQ, Bader H. Depressed (Ca)-transport ATPase in cystic fibrosis erythrocytes. Biochem Biophys Res Commun 1970; 40: 505-509. 2 Duffy MJ, Cohn EV, Schwartz V. Ca-uptake and binding by isolated erythrocyte membranes from cystic fibrosis and control subjects. Chn Chim Acta 1974; 50: 97-101. 3 Feig SA, Segel GB, Kern KA, Osher AB, Schwartz RH. Erythrocyte transport function in cystic fibrosis. Pediatr Res 1974; 8: 594-597. 4 McEvoy FA, Davies RJ, Goodchild MC, Anderson CM. Erythrocyte membrane properties in cystic fibrosis. Clin Chim Acta 1974: 54: 195-204.
265 5 Katz S. Calcium and sodium transport processes in patients with cystic fibrosis. I. A specific decrease in Mg-dependent, Ca-adenosine triphosphatase activity in erythrocyte membranes from cystic fibrosis patients. Pediatr Res 1978; 12: 1033-1038. 6 Foder B, Scharff 0, Tonnesen P. Activator associated Ca-ATPase in erythrocyte membranes from cystic fibrosis patients. Chn Chim Acta 1980; 104: 187-193. 7 Bond GH, Green JW. Effects of monovalent cations on the (Mg + Ca)-dependent ATPase of the red cell membrane. Biochim Biophys Acta 1971; 241: 393-398. 8 Gopinath RM, Vincenzi FE. Phosphodiesterase protein activation mimics red blood cell cytoplasmic activation of (Ca-Mg)ATPase. Biochem Biophys Res Commun 1977; 77: 1203-1209. 9 Bond GH, Clot@ DL. A soluble protein activator of (Mg + Ca)-dependent ATPase in human red cell membranes. Biochim Biophys Acta 1973; 323: 592-599. 10 Katz S, Emery DL. The role of calmodulin in the regulation of (Mg+Ca)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls. Cell Calcium 1981; 2: 545-552. 11 Taussig LM, Kattwinkel J, Friedewald WT, di Sant’ Agnese PA. A new prognostic score and clinical evaluation system for cystic fibrosis. J Pediat 1973; 82: 380-390. 12 Portzehl H, Caldwell PC, Ruegg JC. The dependence of relaxation of muscle fibers from the crab Maia Squinado on the internal concentration of free calcium ions. Biochim Biophys Acta 1964; 79: 581-591. 13 Fiske CH, SubbaRow Y. The calorimetric determination of phosphorous. J Biol Chem 1925; 66: 375-400. 14 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 15 Wierichs R, Bader H. Influence of monovalent ions on the activity of the (Ca+Mg)-ATPase and Ca-transport of human red blood cells. Biochim Biophys Acta 1980; 596: 325-328. 16 Ansah T, Katz S. Evidence for a Ca-transport deficiency in patients with cystic fibrosis. Cell Calcium 1980; 1: 195-203.