Mechanism of depression in cardiac sarcolemmal Na+-K+-ATPase by hypochlorous acid

Kiminori Kato, Qiming Shao, Vijayan Elimban, Anton Lukas, and Naranjan S. Dhalla

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Oxidative stress during pathological conditions such as ischemia-reperfusion is known to promote the formation of hypochlorous acid (HOCl) in the heart and to result in depression of cardiac sarcolemmal (SL) Na+-K+-ATPase activity. In this study, we examined the direct effects of HOCl on SL Na+-K+-ATPase from porcine heart. HOCl decreased SL Na+-K+-ATPase activity in a concentration- and time-dependent manner. Characterization of Na+-K+-ATPase activity in the presence of different concentrations of MgATP revealed a decrease in the maximal velocity (Vmax) value, without a change in affinity for MgATP on treatment of SL membranes with 0.1 mM HOCl. The Vmax value of Na+-K+-ATPase, when determined in the presence of different concentrations of Na+, was also decreased, but affinity for Na+ was increased when treated with HOCl. Formation of acylphosphate by SL Na+-K+-ATPase was not affected by HOCl. Scatchard plot analysis of [3H]ouabain binding data indicated no significant change in the affinity or maximum binding capacity value for ouabain binding following treatment of SL membranes with HOCl. Western blot analysis of Na+-K+-ATPase subunits in HOCl-treated SL membranes showed a decrease (34 ± 9% of control) in the beta 1-subunit without any change in the alpha 1- or alpha 2-subunits. These data suggest that the HOCl-induced decrease in SL Na+-K+-ATPase activity may be due to a depression in the beta 1-subunit of the enzyme.

oxidative stress; sarcolemmal sodium-potassium-adenosinetriphosphatase; sarcolemmal ouabain binding; pig heart; sodium-potassium-adenosinetriphosphatase subunits

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ISCHEMIA-REPERFUSION IS KNOWN to produce free radicals and oxidants (9, 13, 27, 28). Previously, we examined the effect of an oxyradical-generating system (xanthine-xanthine oxidase) on the cardiac sarcolemmal (SL) Na+-K+-ATPase (26) and found that oxyradical-induced depression of Na+-K+-ATPase activity was due to a decrease in the maximal velocity of reaction (Vmax) of the enzyme, when measured in the presence of varying concentrations of Na+ and MgATP. Furthermore, the affinity of Na+-K+-ATPase for MgATP was decreased but the affinity for Na+ was increased following the treatment of SL membranes with xanthine plus xanthine oxidase (26). Some reports indicate that hypochlorous acid (HOCl) decreases Na+-K+-ATPase activity (13, 17), but the mechanism by which HOCl alters this enzyme is not clear. HOCl is one of the major oxidants formed by polymorphonuclear leukocytes in the ischemic-reperfused areas of the myocardium (13). The present study was therefore undertaken to examine the direct effect of HOCl on cardiac SL Na+-K+-ATPase to further clarify the mechanism of depression of this enzyme. Two agents, dithiothreitol (DTT) and L-methionine, which possess antioxidant properties (4, 17, 29), were employed to confirm that the changes due to HOCl were mediated through oxidative stress.

The SL Na+-K+-ATPase is an important target of ischemia-reperfusion injury, since it regulates the intracellular concentration of Na+ and contributes to the resting membrane potential in the myocardium (13, 17, 26, 27). Furthermore, a depression of Na+-K+-ATPase may contribute to Ca2+ overload in cardiomyocytes via Na+/Ca2+ exchange during ischemia-reperfusion (14, 27). The Na+-K+-ATPase consists of alpha - and beta -subunits (20, 30); the alpha -subunits determine the catalytic activity of the enzyme, whereas the beta -subunit is necessary for functional integrity of the Na+-K+-ATPase (18, 24, 30). Recently, Huang et al. (8) reported that alpha 2- and alpha 3-subunits of Na+-K+-ATPase were more sensitive than alpha 1-subunit to the effects of ischemia-reperfusion on ouabain-binding. This study tests that effect of HOCl on the alpha 1- and alpha 2-subunits of Na+-K+-ATPase in addition to investigating changes in the beta -subunit of the enzyme.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of cardiac SL membrane. Porcine hearts were obtained from a slaughterhouse or were freshly obtained in our animal holding facility. Hearts were cut into small pieces and frozen (-70°C). SL membrane was isolated according to the method of Pitts (23) as modified by Kaneko et al. (9). Marker enzyme activities (2, 9, 21) revealed a 16- to 18-fold purification of membranes with respect to Na+-K+-ATPase activity and minimal (2-4%) cross contamination with other subcellular organelles.

Either L-methionine (10 mM) or DTT (1 mM) was used as an antioxidant for HOCl (29). The SL membrane (0.4 mg/ml) was incubated separately with 0.1 mM HOCl in the absence and presence of antioxidant for 10 min. HOCl was prepared by vacuum distillation of sodium hypochlorite after adjusting the pH to 6.2 with dilute sulfuric acid.

Measurement of Na+-K+-ATPase and K+-dependent p-nitrophenylphosphatase activities. Estimation of Na+-K+-ATPase activity was carried out as previously described (2). Briefly, SL membranes (10 µg) were preincubated at 37°C with 1.0 mM EGTA (Tris), pH 7.4, 5 mM NaN3, 6 mM MgCl2, 100 mM NaCl, 10 mM KCl, 2.5 mM phosphoenolpyruvate (PEP), and 10 IU/ml pyruvate kinase. PEP and pyruvate were used as an ATP-regenerating system to maintain the ATP concentration in the incubation medium. The reaction was started by the addition of 0.025 ml of 80 mM Na2ATP (pH 7.4) and stopped after 10 min with 0.5 ml ice-cold 12% TCA. Liberated phosphate was measured by the method of Taussky and Shorr (31). In experiments using different concentrations of MgATP, the amounts of Mg2+ and ATP required to achieve the final concentration of MgATP were calculated using the SPECS Fortran program developed by Fabiato (7). In parallel experiments, either Na+ plus K+ or Mg2+ was omitted from the reaction medium. Na+-K+-ATPase activity was calculated as the difference between activities with and without Na+ plus K+. Mg2+-ATPase activity was estimated as the difference between activities obtained with and without Mg2+, in the absence of Na+ plus K+, in the medium.

The K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activity was determined at 37°C in 1.0 ml of reaction volume using a modified method of Pierce et al. (21, 22). The assay medium contained 30 mM imidazole-HCl, pH 7.8, 5 mM MgCl2, 1 mM EGTA, 20 mM KCl, and 10 µg SL membrane. The reaction was started by adding 5 mM p-nitrophosphate and stopped after 10 min by addition of 2 ml 1 N NaOH. The amount of p-nitrophenol formed was measured at 410 nm.

Measurement of lipid peroxidation and sulfhydryl group content. Lipid peroxidation in the SL membrane was estimated from the malondialdehyde (MDA) concentration by using the thiobarbituric acid method (28). Total sulfhydryl (SH) groups in the SL were determined with DTNB (1).

Measurement of acylphosphate. A modified method of Elmoselhi et al. (6) was used for the formation of acylphosphate. Total reaction volume was 100 µl containing 1 mg/ml SL membrane, 0.25 M sucrose, and 10 mM histidine, pH 7.0, with or without 0.1 mM HOCl, 10 mM L-methionine, and/or 1 mM DTT according to each group. Solutions were incubated at 37°C for 10 min and then immediately cooled to 4°C to stop the reaction. Fifteen microliters of this solution were added to 25 µl of a mixture containing (in mM) 10 NaCl, 30 imidazole-HCl (pH 7.0), and 15 Na-EGTA (pH 7.0). The reaction was started with the addition of [gamma -32P]ATP (8 µCi/test tube; 3,000 Ci/mmol) and incubated at 0°C for 1 min. The reaction was stopped by adding 250 µl of ice-cold TCA and 15 mM phosphoric acid (TCAP), and the tubes were placed on ice for 5 min and then centrifuged at 4°C for 10 min at 10,000 rpm. The pellets were washed once with 1,250 µl TCAP and were suspended in 70 µl of freshly prepared digestion buffer containing 10 mM MOPS (pH 5.5), 1 mM EDTA, 3% SDS, 10% sucrose, 40 mM DTT, and 0.1% methylene green. Samples were shaken vigorously on the vortex for 5-10 min and then electrophoresed immediately in 7.5% polyacrylamide gels at pH 4. Electrophoresis was carried out at 10-12°C using a current of 20 mA/gel for 4 h. The gels were dried, autoradiographed, and exposed overnight at -70°C to Kodak XAR-5 film backed by DuPont lighting screen.

Determination of [3H]ouabain binding. Experiments were carried out according to a method previously described (2). The SL membrane (1.0 mg/ml) with or without 0.1 mM HOCl treatment was resuspended in 10 mM Tris · HCl (pH 7.5), and 50 µl of the suspension were transferred to the reaction mixture (0.5 ml final vol). The reaction mixture contained 1.5 mM MgCl2, 1.0 mM phosphate, 10 mM Tris · HCl (pH 7.5, at 37°C), and 0.5-500 nM [3H]ouabain (18.0 Ci/mmol, New England Nuclear) in the absence or presence of 2.0 mM ouabain, a concentration sufficient to inhibit >95% of specific [3H]ouabain binding. SDS (9 µg/ml) was added to the incubation medium to permeabilize the SL vesicles to ouabain. After 1 h at 37°C, the reaction was terminated by filtration (pore size, 0.45 µm; Millipore, Bedford, MA). Filters were washed three times with 2.5 ml ice-cold buffered washing solution containing 50 mM Tris · HCl, pH 5.0, 0.1 mM ouabain, and 15.0 mM KCl, and the radioactivity of the filters was counted. The nonspecific [3H]ouabain binding (in the presence of excess unlabeled ouabain) was subtracted from the total binding (in the absence of unlabeled ouabain) to obtain the specific binding of [3H]ouabain.

Western blot analysis. Relative Na+-K+-ATPase content was measured by SDS-PAGE (15). The SL proteins were then electroblotted to Immobilon-P transfer membrane (Millipore). Monoclonal anti-alpha 1-subunit of Na+-K+-ATPase mouse IgG (1:10,000), polyclonal anti-alpha 2-subunit rabbit IgG (1:2,000), or polyclonal anti-beta 1-subunit rabbit IgG (1:2,000) from Upstate Biotechnology (Lake Placid, NY) were used to detect subunits. The membranes were subsequently incubated for 1 h with biotinylated anti-mouse IgG (1:1,000) for alpha 1-subunit and biotinylated anti-rabbit IgG antibody (1:3,000) for alpha 2- and beta 1-subunits (Amersham, Arlington Heights, IL). Finally, the membranes were incubated with strepdavidin-conjugated horseradish peroxidase (1:5,000; Amersham) and processed for chemiluminescence (ECL kit, Amersham) using Hyperfilm-ECL (Amersham). Na+-K+-ATPase bands were analyzed using a model GS-670 imaging densitometer (Bio-Rad, Mississauga, ON, Canada) with Image Analysis software version 1.0 and expressed in relation to control values.

Statistical analysis. All values are presented as means ± SE. Statistical analyses were conducted using Student's t-test, one-way ANOVA, and Scheffe's F test where appropriate. A value of P < 0.05 was considered significant. The Michaelis-Menten constant (Km), apparent rate constant (Ka), and Vmax were calculated using Lineweaver-Burk plots for the data of Na+-K+-ATPase activity in the presence of different concentrations of MgATP or Na+. Estimates of kinetic parameters [dissociation constant (Kd) and maximal density (Bmax)] were obtained from Scatchard plot analysis in a [3H]ouabain binding study. The data on [3H]ouabain binding were analyzed according to the LIGAND computer program of McPherson (19), which uses the F test for the best fit.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Catalytic activity of Na+-K+-ATPase. Incubation of cardiac SL membranes with HOCl diminished the Na+-K+-ATPase activity with respect to concentration and time (Fig. 1). The data in Fig. 2 show the kinetic characteristics of Na+-K+-ATPase for MgATP, whereas Fig. 3 shows the characteristics for Na+. The results summarized in Table 1 indicate that Vmax, when determined in the presence of varying concentrations of MgATP, was decreased by HOCl, but Km was not changed significantly between control and HOCl-treated preparations. In the kinetic study employing different concentrations of Na+, both Vmax and Ka were significantly decreased by HOCl. The data in Table 2 show that the HOCl-induced depression in Na+-K+-ATPase activity was partially prevented by 10 mM L-methionine or 1 mM DTT. On the other hand, the decrease in K+-pNPPase activity by HOCl was completely prevented by either 10 mM L-methionine or 1 mM DTT. L-Methionine or DTT alone did not exert any effect on the Na+-K+-ATPase or K+-pNPPase activities (data not shown). HOCl increased the SL MDA formation; both L-methionine and DTT prevented this effect (Table 2). HOCl decreased the SL SH group content and L-methionine prevented this effect (Table 2); DTT was not used in this experiment because it interferes with the SH group content estimation.


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Fig. 1.   Effect of hypochlorous acid (HOCl) on porcine heart sarcolemmal Na+-K+-ATPase (open circle ) and Mg2+-ATPase (bullet ) activities at different times of treatment with 0.1 mM HOCl (A) and different concentrations of HOCl (B). Each value is mean ± SE of 5 experiments. A: * P < 0.05 compared with value in 0 min incubation time. B: * P < 0.05 compared with value in 0 mM HOCl.


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Fig. 2.   Effect of 0.1 mM HOCl on porcine heart sarcolemmal Na+-K+-ATPase activities at different concentrations of MgATP. Each value is mean ± SE of 5 experiments. Inset: Lineweaver-Burk plot of a representative experiment. open circle , Control; bullet , 0.1 mM HOCl. * P < 0.05 compared with control.


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Fig. 3.   Effect of 0.1 mM HOCl on porcine heart sarcolemmal Na+-K+-ATPase activities at different concentrations of Na+. Each value is mean ± SE of 5 experiments. Inset: Lineweaver-Burk plot of a representative experiment. open circle , Control; bullet , 0.1 mM HOCl. * P < 0.05 compared with control.

                              
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Table 1.   Kinetic characteristics of porcine cardiac sarcolemmal Na+-K+-ATPase treated with or without 0.1 mM HOCl

                              
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Table 2.   Effect of 0.1 mM HOCl on porcine heart sarcolemmal Na+-K+-ATPase, K+-pNPPase, MDA content, and SH group content

Acylphosphates and ouabain binding. Figure 4 illustrates the results of treatment with 0.1 mM HOCl on acylphosphate formation. These bands were located at ~110 kDa. Treatment of SL membranes with 0.1 mM HOCl in the absence and presence of 10 mM L-methionine or 1 mM DTT did not produce any significant effect on the formation of acylphosphate. Figure 5 shows the effect of HOCl on ouabain binding. As indicated, there are no changes in either Bmax or Kd values between control and 0.1 mM HOCl-treated preparation.


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Fig. 4.   Effect of HOCl (0.1 mM), L-methionine (LM; 10 mM), and dithiothreitol (DTT; 1 mM) on acylphosphate intermediate of porcine heart sarcolemmal Na+-K+-ATPase. A representative autoradiograph is shown at top.


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Fig. 5.   Scatchard plot analysis of specific [3H]ouabain binding to porcine heart sarcolemmal preparation. Values are taken from a typical experiment in which 3 control and 3 HOCl treatment heart preparations were employed. Inset: results of dissociation constant (Kd) and maximum binding capacity (Bmax). Each value is a mean of 3 experiments. open circle , Control; bullet , 0.1 mM HOCl.

Western blot analysis of Na+-K+-ATPase subunits. The bands of both the alpha 1- and alpha 2-subunits of the SL Na+-K+-ATPase in our study are located at ~110 kDa (Fig. 6), which agrees with previous reports (see review in Ref. 30). On the other hand, the beta -subunit band was located at ~55 kDa rather than at 35 kDa as reported by others (30). However, Pedemonte and Kaplan (20) reported the band of the Na+-K+-ATPase beta -subunit to be at 55 kDa, which agrees with our results. Such a difference was explained by the fact that the electrophoretic mobility of the beta -subunit was influenced largely by N-linked carbohydrate groups (30). Figure 7 shows that HOCl decreased the density of beta 1-subunit of Na+-K+-ATPase and that L-methionine or DTT significantly prevented this effect. On the other hand, HOCl had no effect on both alpha 1- and alpha 2-subunits of the SL Na+-K+-ATPase.


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Fig. 6.   Representative Western blots depicting the effect of treatment with HOCl on expression of the alpha 1-, alpha 2-, and beta 1-subunits of porcine heart sarcolemmal Na+-K+-ATPase. Lane 1, molecular mass markers (kDa); lane 2, control; lane 3, 0.1 mM HOCl; lane 4, HOCl + 10 mM LM; lane 5, HOCl + 1 mM DTT. A band of nonspecific binding was seen at ~75 kDa in all blots.


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Fig. 7.   Summary of Western blot analysis of subunit expression of porcine heart sarcolemmal Na+-K+-ATPase. Bars, data obtained from 4 Western blots for each subunit. I, control; II, 0.1 mM HOCl; III, HOCl + 10 mM LM; IV, HOCl + 1 mM DTT. * P < 0.05 compared with control.

    DISCUSSION
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Introduction
Methods
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This study demonstrates that treatment of SL membranes with HOCl causes a decrease in Na+-K+-ATPase activity and SH group content but an increase in MDA content. Other studies (13, 17) also report a depression in cardiac Na+-K+-ATPase by HOCl, which is a highly reactive oxidant (5, 25). Oxyradical generation under in vitro conditions also decreases Na+-K+-ATPase activity and SH group content of SL membranes but increases formation of MDA (12, 26). It is likely that the observed depression in the SL Na+-K+-ATPase activity by HOCl may be due to a decrease in the SH group content of the enzyme, since SH groups are important for the activity of Na+-K+-ATPase (12, 25). On the other hand, deleterious effects of different oxidants and oxyradicals may reflect increased formation of lipid peroxides (8, 9, 12, 17, 25, 26), and thus it is possible that the HOCl-induced depression may be due to the observed increase in the MDA content of the SL membranes. Treatment of SL membranes with L-methionine, which prevented the HOCl-induced changes in MDA and SH group content due to its antioxidant property, partially prevented (~70% protection) the HOCl-induced depression in Na+-K+-ATPase activity. Furthermore, DTT, which prevents the oxidation of SH groups, completely attenuated the HOCl-induced increase in MDA content and partially prevented the HOCl-induced decrease in Na+-K+-ATPase activity (~80% protection). Thus both elevated levels of MDA and depressed content of SH groups may be involved in the decreased Na+-K+-ATPase activity in HOCl-treated membranes. Inactivation of Na+-K+-ATPase by HOCl in cardiac membrane (13) and uncoupling of Na+ pump by HOCl in coronary artery (6) are also postulated mechanisms for the HOCl-induced changes in Na+-K+-ATPase.

The depression in SL Na+-K+-ATPase activity by HOCl was associated with a decrease in Vmax of the enzymatic reaction when determined in the presence of different concentrations of MgATP or Na+. Because HOCl-treated membranes exhibited a decrease in the activity of K+-pNPPase and this effect was prevented by L-methionine or DTT, it is possible that the observed depression in Na+-K+-ATPase activity reflects a depressant effect of HOCl on the catalytic sites of the enzyme. However, this may not be the case because the affinity for MgATP (1/Km) was unaltered and the affinity for Na+ (1/Ka) was in fact increased on treatment of membranes with HOCl. Furthermore, neither the binding of ouabain nor the formation of acylphosphate was affected by HOCl. It should be mentioned that Na+, K+, ATP, and ouabain binding occur at the catalytic sites represented by alpha -subunits of Na+-K+-ATPase (20, 30). Because no change in the content of the alpha 1- and alpha 2-subunits of the enzyme was seen on HOCl treatment, the observed depression in Na+-K+-ATPase activity by HOCl may not be due to its effect on the catalytic sites of the enzyme.

Unlike the alpha 1- and alpha 2-subunits of SL Na+-K+-ATPase, the content of beta -subunit was decreased by HOCl treatment. This observation suggests that the beta -subunit of Na+-K+-ATPase may be more sensitive to HOCl than the alpha 1- and alpha 2-subunits of the enzyme. Differential effects of various oxidants on alpha 1-, alpha 2- and alpha 3-subunits of Na+-K+-ATPase have been observed by other investigators (8, 16, 32). Our lack of effect of HOCl on the alpha 1- and alpha 2-subunits of the porcine heart Na+-K+-ATPase may be due to species differences (3) or to the techniques used in this study. Nonetheless, it should be pointed out that the depressed activity of porcine kidney Na+-K+-ATPase in hypothyroidism is associated with a decrease in the abundance of beta -subunit without any change in the alpha -subunit (18). Furthermore, inactivation of Na+-K+-ATPase by a high concentration of 2-mercaptoethanol at high temperature is also associated with deterioration of the beta -subunit without any effect on the alpha -subunits (10). The observed effect of HOCl on the beta -subunit of Na+-K+-ATPase, unlike the alpha -subunits, may be due to the presence of SH groups and disulfide bonds in the beta -subunit (11). Earlier studies have shown that the beta -subunit is required for the enzymatic activity of Na+-K+-ATPase because it cannot be separated from the alpha -subunits without an irreversible loss of enzyme function (18, 30). It should also be noted that SL Na+-K+-ATPase activity is decreased on incubation with antibody for the beta -subunit (24). Thus it appears that the observed depression in SL Na+-K+-ATPase activity by HOCl may be mainly due to the malfunction of beta -subunit of the enzyme.

    ACKNOWLEDGEMENTS

This work was supported by a grant from the Medical Research Council (MRC) of Canada (MRC Group in Experimental Cardiology).

    FOOTNOTES

K. Kato was a postdoctoral fellow of the Heart and Stroke Foundation of Canada, and A. Lukas was the Myles Robinson Heart Scholar during the tenure of this study.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6.

Received 19 March 1998; accepted in final form 8 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Boyne, A. A methodology for analysis of tissue sulfhydryl components. Anal. Biochem. 46: 639-653, 1972[Medline].

2.   Dixon, I. M. C., T. Hata, and N. S. Dhalla. Sarcolemmal Na+-K+-ATPase activity in congestive heart failure due to myocardial infarction. Am. J. Physiol. 262 (Cell Physiol. 31): C664-C671, 1992[Abstract/Free Full Text].

3.   Eakle, K. A., K. S. Kim, M. A. Kabalin, and R. A. Farley. High-affinity ouabain binding by yeast cells expression Na+,K+-ATPase subunits and the gastric H+,K+-ATPase subunit. Proc. Natl. Acad. Sci. USA 89: 2834-2838, 1992[Abstract].

4.   Eley, D. W., B. Korecky, and H. Fliss. Dithiothreitol restores contractile function to oxidant-injured cardiac muscle. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1321-H1325, 1989[Abstract/Free Full Text].

5.   Eley, D. W., B. Korecky, H. Fliss, and M. Desilets. Calcium homeostasis in rabbit ventricular myocytes. Disruption by hypochlorous acid and restoration by dithiothreitol. Circ. Res. 69: 1132-1138, 1991[Abstract].

6.   Elmoselhi, A. B., A. Butcher, S. E. Samson, and A. K. Grover. Free radicals uncouple the sodium pump in pig coronary artery. Am. J. Physiol. 266 (Cell Physiol. 35): C720-C728, 1994[Abstract/Free Full Text].

7.   Fabiato, A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. In: Methods in Enzymology, edited by S. Fleischer, and B. Fleischer. New York: Academic, 1988, vol. 157, p. 378-417.

8.   Huang, W. H., Y. Wang, A. Askari, N. Zolotarjova, and M. Ganjeizadeh. Different sensitivities of the Na+/K+-ATPase isoforms to oxidants. Biochim. Biophys. Acta 1190: 108-114, 1994[Medline].

9.   Kaneko, M., V. Elimban, and N. S. Dhalla. Mechanism for depression of heart sarcolemmal Ca2+ pump by oxygen free radicals. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H804-H811, 1989[Abstract/Free Full Text].

10.   Kawamura, M., K. Ohmizo, M. Morohashi, and K. Nagano. Protective effect of Na+ and K+ against inactivation of (Na+ + K+)-ATPase by high concentrations of 2-mercaptoethanol at high temperatures. Biochim. Biophys. Acta 821: 115-120, 1985[Medline].

11.   Kirley, T. L. Determination of three disulfide bonds and one free sulfhydryl in the subunit of (Na,K)-ATPase. J. Biol. Chem. 264: 7185-7192, 1989[Abstract/Free Full Text].

12.   Kramer, J. H., I. T. Mak, and W. B. Weglicki. Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. Circ. Res. 55: 120-124, 1984[Abstract].

13.   Kukreja, R. C., A. B. Weaver, and M. L. Hess. Sarcolemmal Na+-K+-ATPase: inactivation by neutrophil-derived free radicals and oxidants. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1330-H1336, 1990[Abstract/Free Full Text].

14.   Lazdunski, M., C. Frelin, and P. Vigne. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J. Mol. Cell. Cardiol. 17: 1029-1042, 1985[Medline].

15.   Liu, X., Q. Shao, and N. S. Dhalla. Myosin light chain phosphorylation in cardiac hypertrophy and failure due to myocardial infarction. J. Mol. Cell. Cardiol. 27: 2613-2621, 1995[Medline].

16.   Maixent, J. M., and L. G. Lelievre. Differential inactivation of inotropic and toxic digitalis receptors in ischemic dog heart. Molecular basis of the deleterious effects of digitalis. J. Biol. Chem. 262: 12458-12462, 1987[Abstract/Free Full Text].

17.   Matsuoka, T., M. Kato, and K. J. Kako. Effect of oxidants on Na,K,ATPase and its reversal. Basic Res. Cardiol. 85: 330-341, 1990[Medline].

18.   McDonough, A. A., K. Geering, and R. A. Farley. The sodium pump needs its subunit. FASEB J. 4: 1598-1605, 1990[Abstract/Free Full Text].

19.   McPherson, G. Analysis of radioligand binding experiments. A collection of computer programs for the IBM-PC. J. Pharmacol. Methods 14: 213-228, 1985[Medline].

20.   Pedemonte, C. H., and J. H. Kaplan. Chemical modification as an approach to elucidation of sodium pump structure-function relations. Am. J. Physiol. 258 (Cell Physiol. 27): C1-C23, 1990[Abstract/Free Full Text].

21.   Pierce, G. N., and N. S. Dhalla. Sarcolemmal Na+-K+-ATPase activity in diabetic rat heart. Am. J. Physiol. 245 (Cell Physiol. 14): C241-C247, 1983[Abstract].

22.   Pierce, G. N., P. S. Sekhon, H. P. Meng, and T. G. Maddaford. Effects of chronic swimming training on cardiac sarcolemmal function and composition. J. Appl. Physiol. 66: 1715-1721, 1989[Abstract/Free Full Text].

23.   Pitts, B. J. R. Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. J. Biol. Chem. 254: 6232-6235, 1979[Abstract].

24.   Rhee, H., and L. Hokin. Inhibition of the purified sodium-potassium activated adenosinetriphosphatase from the rectal gland of squalus acanthias by antibody against the glycoprotein subunit. Biochem. Biophys. Res. Commun. 63: 1139-1145, 1975[Medline].

25.   Schraufstatter, I. U., K. Browne, A. Harris, P. A. Hyslop, J. H. Jackson, O. Quehenberger, and C. G. Cochrane. Mechanisms of hypochlorite injury of target cells. J. Clin. Invest. 85: 554-562, 1990[Medline].

26.   Shao, Q., T. Matsubara, S. K. Bhatt, and N. S. Dhalla. Inhibition of cardiac sarcolemmal Na+-K+-ATPase by oxyradical generating systems. Mol. Cell. Biochem. 147: 139-144, 1995[Medline].

27.   Shattock, M. J., and H. Matsuura. Measurement of Na+-K+ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress. Circ. Res. 72: 91-101, 1993[Abstract].

28.   Singal, P. K., R. E. Beamish, and N. S. Dhalla. Potential oxidative pathways of catecholamines in the formation of lipid peroxides and genesis of heart disease. Adv. Exp. Med. Biol. 161: 391-401, 1983[Medline].

29.   Suzuki, S., M. Kaneko, D. C. Chapman, and N. S. Dhalla. Alterations in cardiac contractile proteins due to oxygen free radicals. Biochim. Biophys. Acta 1074: 95-100, 1991[Medline].

30.   Sweadner, K. J. Isozymes of the Na+/K+-ATPase. Biochim. Biophys. Acta 988: 185-220, 1989[Medline].

31.   Taussky, H., and E. Schorr. A microcaloric method for the estimation of inorganic phosphorous. J. Biol. Chem. 202: 678-685, 1953.

32.   Xie, Z., M. Jack-Hays, Y. Wang, S. M. Periyasamy, G. Blanco, W. H. Huang, and A. Askari. Different oxidant sensitivities of the alpha 1 and alpha 2 isoforms of Na+/K+-ATPase expressed in baculovirus-infected insect cells. Biochem. Biophys. Res. Commun. 207: 155-159, 1995[Medline].


Am J Physiol Cell Physiol 275(3):C826-C831
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society