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.

Reductions of myocardial Na-K-ATPase activity and ouabain binding sites in heart failure: prevention by nadolol

1993 ◽  
Vol 265 (6) ◽  
pp. H2086-H2093 ◽  
Author(s):  
T. H. Fan ◽  
R. P. Frantz ◽  
H. Elam ◽  
S. Sakamoto ◽  
N. Imai ◽  
...  
Keyword(s):  
Heart Failure ◽  
Atpase Activity ◽  
Binding Sites ◽  
Early Stage ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Adrenergic Stimulation ◽  
Ouabain Binding ◽  
Systemic Hemodynamic

To study the changes in myocardial digitalis binding sites in heart failure, we measured myocardial ouabain binding sites, Na-K-adenosinetriphosphatase (ATPase) activity, and ventricular muscle mechanical responses to acetylstrophanthidin in dogs with right-heart failure (RHF) produced by tricuspid avulsion and pulmonary artery constriction. Sham-operated dogs were studied as the control. RHF produced a significant decrease in ouabain binding sites in the right and left ventricular myocardium, which was accompanied by a proportional decrease in Na-K-ATPase activity. However, RHF and sham-operated dogs did not differ in systemic hemodynamic or right ventricular trabeculate muscle isometric contractile responses to acetylstrophanthidin. To determine whether chronic beta-adrenergic stimulation contributed to the development of Na-K-ATPase downregulation, we administered nadolol (40 mg/day) to a separate group of dogs during an early stage of RHF development. Nadolol effectively prevented the reduction of myocardial ouabain binding sites that occurred in RHF. Thus we conclude that myocardial ouabain binding sites and Na-K-ATPase activity are reduced in dogs with experimental heart failure and that these changes probably occur as a result of the attendant heightened sympathetic activity.

Sarcolemmal Na(+)-K(+)-ATPase: inactivation by neutrophil-derived free radicals and oxidants

1990 ◽  
Vol 259 (5) ◽  
pp. H1330-H1336 ◽  
Author(s):  
R. C. Kukreja ◽  
A. B. Weaver ◽  
M. L. Hess
Keyword(s):  
Free Radicals ◽  
Atpase Activity ◽  
Reperfusion Injury ◽  
Binding Sites ◽  
Human Neutrophils ◽  
Fenton’S Reagent ◽  
Inhibitory Effects ◽  
Ouabain Binding ◽  
Fenton's Reagent ◽  
Per Se

One of the targets of free radicals and neutrophil-derived oxidants that is known to be generated during ischemic-reperfusion injury of the myocardium is the sarcolemma. We therefore examined the susceptibility of sarcolemmal Na(+)-K(+)-ATPase and ouabain binding sites to O2-., H2O2,.OH, HOCl, NH2Cl, and stimulated neutrophils. O2-. generated from xanthine oxidase action on xanthine had no significant effect on Na(+)-K(+)-ATPase activity. The inhibition of Na(+)-K(+)-ATPase activity and ouabain binding by H2O2 was dependent on concentration and the time of incubation. H2O2 (10 mM) inhibited 80% of Na(+)-K(+)-ATPase activity at 90 min..OH generated by Fenton's reagent (200 microM Fe2+ + 5 mM H2O2) significantly decreased maximum binding of ouabain (43.06 +/- 1.45 to 31.96 +/- 2.37 pmol/mg) and was significantly protected by 5 mM mannitol (P less than 0.05). The dissociation constant of ouabain binding was unaffected by Fenton's reagent or H2O2. In contrast, lower concentrations of HOCl, NH2Cl, or PMA-stimulated human neutrophils (4 X 10(6) cells/ml) had significant inhibitory effects on Na(+)-K(+)-ATPase activity. We conclude that O-2. per se is not damaging to sarcolemmal Na(+)-K(+)-ATPase activity. The formation of H2O2 and the more destructive .OH or HOCl and NH2Cl disrupt sarcolemmal function by inhibiting Na(+)-K(+)-ATPase activity and destroying ouabain binding sites.

Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

2005 ◽  
Vol 289 (1) ◽  
pp. R266-R274 ◽  
Author(s):  
A. C. Petersen ◽  
K. T. Murphy ◽  
R. J. Snow ◽  
J. A. Leppik ◽  
R. J. Aughey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Atpase Activity ◽  
Mrna Expression ◽  
Binding Sites ◽  
Time Course ◽  
Acute Exercise ◽  
Vastus Lateralis ◽  
Vastus Lateralis Muscle ◽  
Ouabain Binding ◽  
Atpase Gene

We investigated whether depressed muscle Na+-K+-ATPase activity with exercise reflected a loss of Na+-K+-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na+-K+-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at ∼40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na+-K+-ATPase activity via 3- O-methylfluorescein phosphatase (3- O-MFPase) activity, Na+-K+-ATPase content via [3H]ouabain binding sites, and Na+-K+-ATPase α1-, α2-, α3-, β1-, β2- and β3-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [3H]ouabain binding sites but decreased 3- O-MFPase activity by 10.7 (SD 8)% ( P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated α1-isoform mRNA by 1.5-fold at fatigue ( P < 0.05). This increase was inversely correlated with the percent change in 3- O-MFPase activity from rest to fatigue (%Δ3- O-MFPaserest-fatigue) ( r = −0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) α1-isoform mRNA was increased 1.4-fold ( P < 0.05) and approached a significant inverse correlation with %Δ3- O-MFPaserest-fatigue ( r = −0.56, P = 0.08). Exercise elevated α2-isoform mRNA at fatigue 2.5-fold ( P < 0.05), which was inversely correlated with %Δ3- O-MFPaserest-fatigue ( r = −0.60, P = 0.05). The average postexercise α2-isoform mRNA was increased 2.2-fold ( P < 0.05) and was inversely correlated with the %Δ3- O-MFPaserest-fatigue ( r = −0.68, P < 0.05). Nonsignificant correlations were found between %Δ3- O-MFPaserest-fatigue and other isoforms. Thus acute exercise transiently decreased Na+-K+-ATPase activity, which was correlated with increased Na+-K+-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na+-K+-ATPase activity with exercise.

scholarly journals Interaction of HK and LK Goat Red Blood Cells with Ouabain

1974 ◽  
Vol 64 (5) ◽  
pp. 536-550 ◽  
Author(s):  
John R. Sachs ◽  
Philip B. Dunham ◽  
Donna L. Kropp ◽  
J. Clive Ellory ◽  
Joseph F. Hoffman
Keyword(s):  
Red Blood Cells ◽  
Binding Sites ◽  
Blood Cells ◽  
Red Cells ◽  
Ouabain Binding ◽  
High Potassium ◽  
Considerable Heterogeneity ◽  
Low Potassium ◽  
The Difference ◽  
Calculated Number

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (Kc) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low Kc cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal Kc, it does not alter the apparent number of sites in LK cells when Kc has been reduced.

scholarly journals Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes

1990 ◽  
Vol 269 (3) ◽  
pp. 757-766 ◽  
Author(s):  
G Schmalzing ◽  
S Kröner
Keyword(s):  
Binding Sites ◽  
Rank Order ◽  
Secretory Cells ◽  
Free Calcium ◽  
Membrane Insertion ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Permeabilized Cells ◽  
Sodium Pumps ◽  
Maximal Stimulation

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5′-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5′-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5′-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.

(Na+-K+)-ATPase Activity and Ouabain-Binding Sites in the Cerebral Cortex of Young and Aged Fischer-344 Rats

Gerontology ◽  
1983 ◽  
Vol 29 (4) ◽  
pp. 242-247 ◽  
Author(s):  
Joseph C. LaManna ◽  
Gregory Doull ◽  
Kimberly McCracken ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Atpase Activity ◽  
Binding Sites ◽  
Fischer 344 Rats ◽  
Ouabain Binding ◽  
Fischer 344

Ischemia-induced alteration of myocardial Na+-K+-ATPase activity and ouabain binding sites in hypercholesterolemic rabbits

1996 ◽  
Vol 127 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Wen-Jone Chen ◽  
Shoei-Yn Lin-Shiau ◽  
Huei-Chen Huang ◽  
Yuan-Teh Lee
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Ouabain Binding

scholarly journals Active Cation Transport and Ouabain Binding in High Potassium and Low Potassium Red Blood Cells of Sheep

1971 ◽  
Vol 58 (1) ◽  
pp. 94-116 ◽  
Author(s):  
Philip B. Dunham ◽  
Joseph F. Hoffman
Keyword(s):  
Binding Sites ◽  
High Specificity ◽  
Cation Transport ◽  
Nonspecific Binding ◽  
Ouabain Binding ◽  
High Potassium ◽  
High K ◽  
Low Potassium ◽  
The Difference ◽  
Specificity Of Binding

Red cells from high K sheep contained 82 mM K/liter cells and had a pump flux of 0.86 mM K/liter cells x hr; similarly, LK cells had 16.5 mM K/liter cells and a pump flux of 0.12 mM K/liter cells x hr. Using [3H]-ouabain, the relation between the number of ouabain molecules bound per cell and the concomitant per cent inhibition of the pump was found to be approximately linear for both HK and LK cells. The number of glycoside molecules necessary for 100 % inhibition of the pump was 42 for HK cells and 7.6 for LK cells, after correction for six nonspecific binding sites for each type of cell. The ratio of ouabain molecules/cell at 100 % inhibition was 5.5, HK to LK, and the ratio of the normal K pump fluxes was 7.2, HK to LK. The similarity of these ratios suggests that an important difference between HK and LK cells, determining the difference in pump fluxes, is the number of pump sites. The turnover times (ions/site x min) are 6000 and 4800 for HK and LK cells, respectively. The results also indicate a high specificity of binding of ouabain to pump sites.

scholarly journals The binding of ouabain to normal and chronic lymphocytic leukemic lymphocytes

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 994-1000
Author(s):  
JS Wiley ◽  
N Kraft ◽  
IA Cooper
Keyword(s):  
Peripheral Blood ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphocytic Leukemia ◽  
Ouabain Binding ◽  
Normal Peripheral Blood ◽  
Binding Characteristics ◽  
Specific Binding Sites ◽  
Equilibrium Binding ◽  
The Difference

The binding of the cardiac glycoside, ouabain, to cells had been used to quantify the number of active cation pumps. In this study, lymphocytes were incubated with 3H-ouabain and the equilibrium binding analyzed for the maximal number of specific binding sites. Lymphocytes from normal peripheral blood bound 44,200 +/- 9920 molecules/cell, compared with 29,200 +/- 8370 molecules/cell for the lymphocytes of chronic lymphocytic leukemia (CLL) subjects. This difference was significant (p less than 0.01) and did not reflect a lower number of sites on B cells than T cells, since B-cell-enriched lymphocytes from normal peripheral blood showed the same ouabain binding characteristics as the standard T-cell-rich preparation. Although monocytes bind threefold more ouabain than lymphocytes, the small monocyte contamination (3.0%) in normal lymphocyte preparations could not account for the difference between normal and CLL. The fewer ouabain binding sites on CLL lymphocytes may reflect both their smaller size (by 10%) and lower mitotic activity compared with lymphocytes from normal peripheral blood.

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