scholarly journals Extracellular Protons Regulate the Extracellular Cation Selectivity of the Sodium Pump

2002 ◽  
Vol 120 (4) ◽  
pp. 497-508 ◽  
Author(s):  
Mark A. Milanick ◽  
Krista L. Arnett
Keyword(s):  
Sodium Pump ◽  
Human Erythrocytes ◽  
Red Cells ◽  
Ouabain Binding ◽  
Proton Binding ◽  
Cation Selectivity ◽  
Rubidium Influx ◽  
Sodium Binding ◽  
Extracellular Transport ◽  
Transport Site

The effects of 0.3–10 nM extracellular protons (pH 9.5–8.0) on ouabain-sensitive rubidium influx were determined in 4,4′-diisocyanostilbene-2, 2′-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (∼5–10 mM). The Kd for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the Kd for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

1985 ◽  
Vol 63 (11) ◽  
pp. 1454-1459
Author(s):  
M. W. Wolowyk ◽  
J. C. Ellory
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Sodium Pump ◽  
Red Cells ◽  
Ouabain Binding ◽  
High Potassium ◽  
Fetal Sheep ◽  
Sodium Pumps ◽  
Low Potassium ◽  
Pump Activity

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from −30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.

Effects of Synthetic Atrial Natriuretic Peptides on Sodium-Potassium Transport in Human Erythrocytes

1985 ◽  
Vol 69 (2) ◽  
pp. 223-226 ◽  
Author(s):  
G. A. Sagnella ◽  
D. A. Nolan ◽  
A. C. Shore ◽  
G. A. MacGregor
Keyword(s):  
Loop Diuretic ◽  
Sodium Pump ◽  
Human Erythrocytes ◽  
Atrial Natriuretic Peptides ◽  
Sodium Potassium ◽  
Potassium Ion Transport ◽  
Wide Range ◽  
Natriuretic Effect ◽  
Cotransport System ◽  
Sodium And Potassium

1. The effects of synthetic human and rat atrial peptides on sodium and potassium ion transport has been investigated in intact human erythrocytes. 2. The effects of these peptides have been tested on the active, sodium pump-dependent (ouabain-sensitive) and on the sodium-potassium cotransport system (bumetanide-sensitive) with 86Rb used as a tracer. 3. Human (α-ANP, 28 amino acids) or rat (atriopeptin III) atrial peptides, over a wide range of concentrations, did not influence the uptake of 86Rb in either the ouabain-sensitive or the bumetanide-sensitive transport system. 4. These results suggest that the natriuretic effect of the atrial peptides is not mediated through inhibition of the sodium pump or the loop-diuretic-sensitive Na-K cotransport.

scholarly journals Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

1996 ◽  
Vol 107 (2) ◽  
pp. 271-291 ◽  
Author(s):  
S Q Liu ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Rate Constant ◽  
Band 3 ◽  
Complex Model ◽  
Amino Acid Residues ◽  
Ping Pong ◽  
Extracellular Transport ◽  
Charged Form ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

scholarly journals Red cell diphosphoglycerate mutase. Immunochemical studies in vertebrate red cells, including a human variant lacking 2,3-DPG

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 953-958 ◽  
Author(s):  
LL Peterson
Keyword(s):  
Enzyme Activity ◽  
Genetic Variant ◽  
Neonatal Period ◽  
Human Erythrocytes ◽  
Red Cells ◽  
The Other ◽  
Radial Immunodiffusion ◽  
Red Cell ◽  
Human Adult ◽  
2,3 Dpg

Abstract Diphosphoglycerate mutase (DPGM) was purified to homogeneity from human erythrocytes. The enzyme and Freund adjuvant were injected into chickens and yielded a monospecific precipitating antibody. Radial immunodiffusion with this antibody was used to measure the amount of DPGM in hemolysates from human adult and cord red cells. Dog, rabbit, rat, chicken, and goat red cells all had DPGM during the neonatal period, but goat adult red cells had no detectable enzyme. Single bands with no spurs were present on Ouchterlony plates in which human hemolysate was placed adjacent to hemolysates from the other species tested. The amount of human red cell DPGM did not differ between young and old cells separated by centrifugation. Red cells from a patient with a DPGM genetic variant who had erythrocytosis and no detectable enzyme activity contained a reduced amount of DPGM as determined by radial immunodiffusion. The abnormal DPGM differed from normal by immunoelectrophoresis and in stability as measured by the amount of crossreacting material in young versus old erythrocytes.

scholarly journals Haemolytic Effects of Hypo-osmotic Salt Solutions on Human Erythrocytes

2012 ◽  
Vol 9 (2) ◽  
pp. 35-39 ◽  
Author(s):  
O Nepal ◽  
J P Rao
Keyword(s):  
Sodium Chloride ◽  
Morphological Changes ◽  
Nickel Chloride ◽  
Human Erythrocytes ◽  
Red Cells ◽  
Salt Solutions ◽  
Potassium Salts ◽  
Blood Samples ◽  
Chloride Salts ◽  
Human Red Cells

Background While it is well known that hypotonic solutions of sodium chloride induce hemolysis, the effects of other salt solutions on human erythrocytes have not been well documented. Objective The study is to compare the effects of other salt solutions on human red cells. Methods Iso-osmotic and hypo-osmotic solutions of various salts were prepared after taking into account their molecular weight and osmotic pressure. Five healthy volunteers between the age of 22-30 years were randomly selected and ten blood samples were collected from them. The study was conducted from January 2009 to February 2009. Blood was collected from subjects by venepuncture into heparinised tubes. 20 ?l of blood was pipetted into 1 ml of each solution and incubated for one hour at 37ºC in a water bath. The solutions were centrifuged and the colour of the supernatant was read in a spectrophotometer. Supernatant from blood added to distilled water was considered 100% hemolysed. Results Iso-osmotic salt solutions were free of hemolysis. Among chloride salts, sodium chloride showed the least hemolysis and potassium chloride and nickel chloride resulted into greater hemolysis. Among potassium salts, potassium bromate caused highest amount of hemolysis whereas potassium sulphate showed the least. Conclusion The significant differences in hemolytic pattern in hypo-osmotic salts solutions suggest that the hypo-osmotic stress causes morphological changes in red cells that alter their permeability to various ions leading to hemolysis. This probably occurs through opening of volume sensitive channels. DOI: http://dx.doi.org/10.3126/kumj.v9i2.6285Kathmandu Univ Med J 2011;9(2):35-9

Glucocorticoids increase sodium pump α2- and β1-subunit abundance and mRNA in rat skeletal muscle

2001 ◽  
Vol 280 (3) ◽  
pp. C509-C516 ◽  
Author(s):  
Curtis B. Thompson ◽  
Inge Dorup ◽  
Julie Ahn ◽  
Patrick K. K. Leong ◽  
Alicia A. McDonough
Keyword(s):  
Sodium Pump ◽  
Mrna Level ◽  
Chronic Obstructive Lung Disease ◽  
Specific Pattern ◽  
Chronic Obstructive ◽  
Mrna Levels ◽  
Ouabain Binding ◽  
Adrenal Steroid ◽  
Control Value ◽  
Hindlimb Muscle

Fourteen-day adrenal steroid treatment increases [3H]ouabain binding sites 22–48% in muscle biopsies from patients treated with adrenal steroids for chronic obstructive lung disease and in rats treated with dexamethasone (Dex). Ouabain binding measures plasma membrane sodium pumps (Na+-K+-ATPase) with isoform-dependent affinity. In this study we have established the specific pattern of Dex regulation of sodium pump isoform protein and mRNA levels in muscle. Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days. Abundance of sodium pump catalytic α1- and α2-subunits and glycoprotein β1- and β2-subunits was determined by immunoblot in soleus, extensor digitorum longus, whole gastrocnemius, and diaphragm and was normalized to the mean vehicle control value. Dex increased α2 and β1 protein in all muscle types by 53–78% and ∼50%, respectively. Dex increased α1protein only in diaphragm (65 ± 7%). At the mRNA level in whole hindlimb muscle, Dex increased α2 (6.4 ± 0.5-fold) and β1 (1.54 ± 0.15-fold) and decreased β2 (to 0.36 ± 0.6 of control). In summary, α2β1 is the Dex-responsive pump in all skeletal muscles, and changes in α2 and β1mRNA levels can drive the 50% change in α2β1-subunits, which can account for the reported increase in [3H]ouabain binding.

scholarly journals Relative contributions of the slippage and tunneling mechanisms to anion net efflux from human erythrocytes.

1984 ◽  
Vol 84 (6) ◽  
pp. 877-893 ◽  
Author(s):  
O Fröhlich
Keyword(s):  
Membrane Potential ◽  
Conformational Change ◽  
Chemical Nature ◽  
Human Erythrocytes ◽  
Alternative Process ◽  
Anion Transporter ◽  
Transport Site ◽  
Cl Conductance ◽  
Channel Type

The rates of anion net efflux from gramicidin-treated erythrocytes in the presence of a K gradient were measured at 25 degrees C, pH 7.8, as rates of loss of Ki. The experiments served to estimate the relative contributions of two hypothetical mechanisms to Cl net efflux at low extracellular Cl concentrations. Cl, Br, and NO3 net effluxes were measured into media of different Cl, Br, or NO3 concentrations, respectively, to determine and compare the relative rates of the extracellular anion-inhibitable components. They were 48, 160, and 230 mmol/(kg Hb X min), respectively, at a membrane potential of about -90 mV. This indicates that the anion-inhibitable efflux is not due solely to the return translocation of the empty transport site ("slippage") because slippage should be independent of the chemical nature of the anion. Cl net efflux was also measured as a function of the intracellular Cl concentration into media containing either 0 or 50 mM Cl. Under both conditions, net efflux was linearly dependent on Cli between 30 and 300 mM Cli and was 0 when back-extrapolated to 0 Cli. This observation is not compatible with the slippage process, which under these conditions would have been expected to be independent of Cli above 15 mM Cli. It was concluded that slippage contributes negligibly to Cl net efflux even at low extracellular anion concentrations and that the alternative process of "tunneling"--that is, movement of the anion through the anion transporter without a conformational change in a channel-type behavior--is the major, if not the sole, mechanism underlying Cl conductance.

Mechanism of X-ray Inhibition of the Sodium Pump in Human Erythrocytes: Adenotriphosphatase Activity of Stromata

Nature ◽  
1962 ◽  
Vol 196 (4850) ◽  
pp. 186-187 ◽  
Author(s):  
FRANCESCO BRESCIANI ◽  
FERDINANDO AURICCHIO ◽  
CESARE FIORE
Keyword(s):  
Sodium Pump ◽  
Human Erythrocytes ◽  
X Ray
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