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

1998 ◽  
Vol 275 (3) ◽  
pp. C826-C831 ◽  
Author(s):  
Kiminori Kato ◽  
Qiming Shao ◽  
Vijayan Elimban ◽  
Anton Lukas ◽  
Naranjan S. Dhalla
Keyword(s):  
Atpase Activity ◽  
Hypochlorous Acid ◽  
Binding Capacity ◽  
Ischemia Reperfusion ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Ouabain Binding ◽  
Plot Analysis ◽  
Binding Data ◽  
Atpase Subunits

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 ( V max) value, without a change in affinity for MgATP on treatment of SL membranes with 0.1 mM HOCl. The V max 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 β1-subunit without any change in the α1- or α2-subunits. These data suggest that the HOCl-induced decrease in SL Na+-K+-ATPase activity may be due to a depression in the β1-subunit of the enzyme.

scholarly journals Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

2013 ◽  
Vol 304 (1) ◽  
pp. H94-H103 ◽  
Author(s):  
Aude Belliard ◽  
Yoann Sottejeau ◽  
Qiming Duan ◽  
Jessa L. Karabin ◽  
Sandrine V. Pierre
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Ion Transport ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Resistant Mutant ◽  
Surface Expression ◽  
Live Cells ◽  
Mutant Form ◽  
Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

Temperature-dependent binding of cyclosporine to an erythrocyte protein.

1987 ◽  
Vol 33 (4) ◽  
pp. 481-485 ◽  
Author(s):  
R P Agarwal ◽  
G A Threatte ◽  
R A McPherson
Keyword(s):  
Binding Constant ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Temperature Dependent ◽  
Fixed Amount ◽  
Competitive Binding Assay ◽  
Plot Analysis ◽  
Binding Data ◽  
Saturation Capacity ◽  
Increasing Temperature

Abstract In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.

Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum

1998 ◽  
Vol 84 (2) ◽  
pp. 425-430 ◽  
Author(s):  
Terence G. Favero ◽  
David Colter ◽  
Paul F. Hooper ◽  
Jonathan J. Abramson
Keyword(s):  
Reactive Oxygen Species ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Structural Damage ◽  
Hypochlorous Acid ◽  
Reactive Oxygen ◽  
Sulfhydryl Groups ◽  
Dependent Manner ◽  
Oxygen Species

Favero, Terence G., David Colter, Paul F. Hooper, and Jonathan J. Abramson. Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 84(2): 425–430, 1998.—Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 μM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (ATPase), inhibiting transport function. HOCl inhibits sarcoplasmic reticulum Ca2+-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 μM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca2+-handling kinetics in muscle cells.

Activation of proteases and changes in Na+-K+-ATPase subunits in hearts subjected to ischemia-reperfusion

2013 ◽  
Vol 114 (3) ◽  
pp. 351-360 ◽  
Author(s):  
Alison L. Müller ◽  
Darren Freed ◽  
Naranjan S. Dhalla
Keyword(s):  
Atpase Activity ◽  
Protein Content ◽  
Cardiac Dysfunction ◽  
Ischemia Reperfusion ◽  
Global Ischemia ◽  
Calpain Inhibitor ◽  
Mmp Inhibitor ◽  
Rat Hearts ◽  
Atpase Subunits

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and changes in sarcolemmal Na+-K+-ATPase subunits and activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, as well as reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 30–60 min of global ischemia were accompanied by augmented activities of both calpain and matrix metalloproteinases (MMPs) and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. Compared with control values, the activities of both calpain and MMP-2 were increased, whereas the activity and protein content for all subunits of Na+-K+-ATPase were decreased upon reperfusion for 5–40 min, except that α1- and α2-subunit content was not depressed in 5 min I/R hearts. MDL28170, a calpain inhibitor, was more effective in attenuating the I/R-induced alterations in cardiac contracture, Na+-K+-ATPase activity, and α2-subunit than doxycycline, an MMP inhibitor. Incubation of control sarcolemma preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1-, α2-, and β2-subunits, without changes in the β1-subunit. These results support the view that activation of both calpain and MMP-2 are involved in depressing Na+-K+-ATPase activity and degradation of its subunits directly or indirectly in hearts subjected to I/R injury.

Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

1988 ◽  
Vol 255 (6) ◽  
pp. E912-E919 ◽  
Author(s):  
R. S. Haber ◽  
J. N. Loeb
Keyword(s):  
Thyroid Hormone ◽  
Atpase Activity ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Ouabain Binding ◽  
High Affinity ◽  
Mammalian Tissues ◽  
Dissociation Constant Kd ◽  
Crude Membrane Fraction

The administration of thyroid hormone is known to result in an induction of the Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na+-K+-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain, we have studied the effects of 3,3',5-triiodo-L-thyronine (T3) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na+-K+-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (KIs) for ouabain of approximately 10(-7) and 10(-4) M, respectively. Treatment of hypothyroid rats with T3 (50 micrograms/100 g body wt on 3 alternate days) nearly tripled that portion of the Na+-K+-ATPase activity corresponding to the high-ouabain-affinity form (increased by 178 +/- 24%), whereas the enzyme activity corresponding to the low-ouabain-affinity form was only slightly changed (increased by 20 +/- 5%). Measurement of the specific binding of [3H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (Kd) of slightly less than 10(-7) M, whose maximal binding capacity was increased by T3 treatment by 185%. The calculated catalytic turnover associated with the high-affinity site was 70-80 molecules ATP hydrolyzed.site-1.s-1 and was unchanged by T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Sodium Transport in Chronic Renal Failure

1982 ◽  
Vol 62 (5) ◽  
pp. 489-494 ◽  
Author(s):  
R. Swaminathan ◽  
G. Clegg ◽  
M. Cumberbatch ◽  
Z. Zareian ◽  
F. McKenna
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Atpase Activity ◽  
Healthy Subjects ◽  
Binding Capacity ◽  
Sodium Content ◽  
Ouabain Binding ◽  
Sodium Pumps ◽  
Group A ◽  
Erythrocyte Sodium

1. Erythrocyte sodium, sodium transport (ouabain-sensitive efflux rate of sodium, oMosNa, and ouabain-sensitive efflux rate constant of sodium, oMosNa), sodium-potassium activated ouabain-sensitive adenosine triphosphatase (Na+, K+-ATPase) activity and [3H]ouabain-binding capacity were measured in 15 patients with chronic renal failure and in 10 healthy subjects. 2. As a group, patients with chronic renal failure had a lower erythrocyte sodium and oMosNa compared with healthy subjects. 3. When patients were divided according to their erythrocyte sodium (greater or less than 4 mmol/kg of cells), in the group of patients whose erythrocyte sodium was less than 4 mmol/kg of cells (group A) the oMosNa was higher than that in healthy subjects and the oMosNa, Na+, K+-ATPase activity and [3H]ouabain-binding capacity were the same as those in healthy subjects. In the subgroup of patients with renal failure whose erythrocyte sodium content was greater than 4 mmol/kg of cells (group B) the oMosNa was less and plasma urea concentration higher than in group A and Na+, K+-ATPase activity, [3H]ouabain-binding capacity and oMosNa were lower than in healthy subjects. 4. These results suggest that in early chronic renal failure there is stimulation of ‘sodium pumps’ (without alteration in their number), which causes a lowering of erythrocyte sodium content, and that as the disease progresses there is inhibition of the ‘sodium pumps’ as well as a reduction in membrane permeability so that erythrocyte sodium is near normal.

Enhancement of airway epithelial Na+,K(+)-ATPase activity by NO2 and protective role of nordihydroguaiaretic acid

1996 ◽  
Vol 270 (2) ◽  
pp. L266-L272 ◽  
Author(s):  
T. W. Robison ◽  
K. J. Kim
Keyword(s):  
Atpase Activity ◽  
Binding Capacity ◽  
Binding Constants ◽  
Nordihydroguaiaretic Acid ◽  
Protective Role ◽  
Airway Epithelial ◽  
Ouabain Binding ◽  
Control Value ◽  
Active Ion Transport ◽  
Air Control

We examined the effects of nitrogen dioxide (NO2) on guinea pig tracheobronchial (GPTE) ouabain-sensitive 86Rb uptake, as an index of Na+,K(+)-ATPase activity, and specific [3H]ouabain binding. A 1-h exposure of GPTE monolayers to 5 ppm NO2 increased ouabain-sensitive 86Rb uptake (nmol.mg protein-1.30 min-1) to 512 +/- 39 compared with an air-control value of 278 +/- 20. Similarly, 1 ppm NO2 increased 86Rb uptake to 336 +/- 19 from an air control of 219 +/- 31. The specific [3H]ouabain-binding capacity (Bmax) for monolayers exposed to 5 ppm NO2 was increased to 23.2 +/- 1.2 pmol/mg protein in comparison with an air-control value of 18.4 +/- 0.4; however, there was no change at 1 ppm NO2. Binding constants (Kd) for 1 or 5 ppm NO2 were increased to 0.64 +/- 0.02 and 0.79 +/- 0.08 microM, respectively, in comparison with an air-control value of 0.53 +/- 0.02 microM. Changes of Bmax and Kd may be consistent with a recruitment of latent pumps to the basolateral cell plasma membrane and/or increased turnover of the sodium pump. However, the increase of Bmax was no more than 126% of the air control, while 86Rb uptake increased to 184%, suggesting that an increased turnover is the more predominant effect. Incubation of GPTE monolayers during NO2 exposure with nordihydroguaiaretic acid, an antioxidant, blocked the increase of ouabain-sensitive 86Rb uptake almost completely and partially protected transepithetial resistance, suggesting that lipid peroxidation processes may play a role in alterations of airway epithelial barrier and active ion transport properties.

Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes

1987 ◽  
Vol 65 (2) ◽  
pp. 95-104 ◽  
Author(s):  
Alberto Severini ◽  
K. V. S. Prasad ◽  
Anthony F. Almeida ◽  
J. Gordin Kaplan
Keyword(s):  
Protein Synthesis ◽  
Atpase Activity ◽  
Binding Sites ◽  
Resting Cells ◽  
Ouabain Binding ◽  
Activated Lymphocytes ◽  
Permeabilized Cells ◽  
Atpase Subunits ◽  
The Difference ◽  
Resting Lymphocytes

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 × 103 ± 12 × 103/cell in resting to 60 × 103 ± 27 × 103/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: (i) there was an increase in the Vmax of ouabain binding, without variation in the Km; (ii) the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; (iii) there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.

scholarly journals Insulin stimulates the translocation of Na+/K+-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle

1990 ◽  
Vol 272 (3) ◽  
pp. 727-733 ◽  
Author(s):  
M Omatsu-Kanbe ◽  
H Kitasato
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Binding Capacity ◽  
Marker Enzyme ◽  
Frog Skeletal Muscle ◽  
Ouabain Binding ◽  
Significant Difference

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular ‘light fraction’ (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.

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