Activation by N-ethylmaleimide of a latent K+-Cl- flux in human red blood cells

1984 ◽  
Vol 246 (5) ◽  
pp. C385-C390 ◽  
Author(s):  
P. K. Lauf ◽  
N. C. Adragna ◽  
R. P. Garay
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Coupled Transport ◽  
Human Red Blood Cells ◽  
Low Concentrations ◽  
Reactive Groups ◽  
High Concentrations ◽  
K Transport

Twenty to fifty percent of the ouabain-insensitive Na+ and K+ fluxes in human red blood cells are mediated by Cl(-) -dependent coupled transport (cotransport). In this paper we report on the effect of the sulfhydryl group reagent N-ethylmaleimide (NEM) on Cl(-) -dependent ouabain-insensitive Na+ and K+ fluxes in human red blood cells. We found that NEM altered Na+ -K+ cotransport and activated a latent Cl(-) -dependent K+ transport mode normally apparently silent. This conclusion was based on the following observations. 1) At low concentrations (0.25 mM) NEM abolished the bumetanide-sensitive Na+ efflux and had no effect, even at a 10-fold higher concentration, on the bumetanide-sensitive K+ efflux. 2) At concentrations above 0.1 mM, NEM stimulated Cl(-) -dependent K+ efflux that was only partially inhibited by high concentrations of bumetanide or furosemide. In experiments using Rb+ as a K+ analogue, NEM activated Rb+ influx by stimulating the maximum velocity and lowering the apparent external cation affinity. The data suggest the presence of chemically reactive groups in human red blood cells for both Cl(-) -dependent K+ transport activated by NEM and Cl(-) -dependent coupled Na+-K+ movements.

scholarly journals Detection of acyl-CoA-binding protein in human red blood cells and investigation of its role in membrane phospholipid renewal

1995 ◽  
Vol 306 (3) ◽  
pp. 793-799 ◽  
Author(s):  
H Fyrst ◽  
J Knudsen ◽  
M A Schott ◽  
B H Lubin ◽  
F A Kuypers
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Human Liver ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Human Red Blood Cells ◽  
Low Concentrations ◽  
Membrane Bound ◽  
High Concentrations

Acyl-CoA-binding protein (ACBP) has been identified in a number of tissues and shown to affect the intracellular distribution and utilization of acyl-CoA. We have detected ACBP in the cytosol but not the membrane of human red blood cells and, using an e.l.i.s.a. with antibodies prepared against human liver ACBP, found that its concentration was 0.5 microM. To investigate the role of ACBP in human red blood cells, we added purified human liver ACBP and radiolabelled acyl-CoA to isolated membranes from these cells. ACBP prevented high concentrations of acyl-CoA from binding to the membrane but could not keep the acyl-CoA in the aqueous phase at low concentrations. This suggested the presence of a pool in the membrane with a binding affinity for acyl-CoA that was greater than that of ACBP for acyl-CoA. In the presence of lysophospholipid, this membrane-bound pool of acyl-CoA was rapidly used as a substrate by acyl-CoA:lysophospholipid acyltransferase (LAT) to generate phospholipid from lysophospholipid. We also found that ACBP-bound acyl-CoA was preferred over free acyl-CoA as a substrate by LAT. These results are the first documentation that human red blood cells contain ACBP and that this protein can affect the utilization of acyl-CoA in plasma membranes of these cells. The interactions between acyl-CoA, ACBP and the membrane suggest that there are several pools of acyl-CoA in the human red blood cell and that ACBP may have a role in regulating their distribution and fate.

Loop diuretic and anion modification of NEM-induced K transport in human red blood cells

1990 ◽  
Vol 258 (4) ◽  
pp. C622-C629 ◽  
Author(s):  
L. R. Berkowitz
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Loop Diuretic ◽  
Sulfhydryl Group ◽  
Loop Diuretics ◽  
Human Red Blood Cells ◽  
Anion Substitution ◽  
Cellular Target ◽  
K Transport ◽  
Permeant Anion

The thioalkylating agent N-ethylmaleimide (NEM) causes ouabain-insensitive K loss from human red blood cells. This K loss is inhibited when intracellular Cl is replaced by another permeant anion or when loop diuretics are placed in the incubation medium after NEM exposure. In this report, we have tested the possibility that Cl replacement or loop diuretics not only influence the transport of K induced by NEM but also the interaction of NEM with its target sulfhydryl group. This possibility was examined by replacing intracellular Cl or exposing the cells to loop diuretics before NEM exposure, then measuring K loss in a Cl medium free of loop diuretics. We found that such pretreatment with either Cl substitution or loop diuretics stimulated, rather than inhibited, NEM-induced K loss. This enhancement was not additive in that the increase in K loss induced by anion substitution was not increased further when loop diuretics were also present. These data suggest that anion substitution and loop diuretics enhance the interaction of NEM with its cellular target but inhibit the K loss induced by NEM.

Volume-dependent K+ transport in rabbit red blood cells comparison with oxygenated human SS cells

1989 ◽  
Vol 257 (1) ◽  
pp. C114-C121 ◽  
Author(s):  
N. al-Rohil ◽  
M. L. Jennings
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Cell Volume Regulation ◽  
Sulfhydryl Group ◽  
Cell Swelling ◽  
Inhibitory Potency ◽  
Ph Optimum ◽  
The Relationship ◽  
Low K ◽  
K Transport

In this study the volume-dependent or N-ethylmaleimide (NEM)-stimulated, ouabain-insensitive K+ influx and efflux were measured with the tracer 86Rb+ in rabbit red blood cells. The purpose of the work was to examine the rabbit as a potential model for cell volume regulation in human SS red blood cells and also to investigate the relationship between the NEM-reactive sulfhydryl group(s) and the signal by which cell swelling activates the transport. Ouabain-resistant K+ efflux and influx increase nearly threefold in cells swollen hypotonically by 15%. Pretreatment with 2 mM NEM stimulates efflux 5-fold and influx 10-fold (each measured in an isotonic medium). The ouabain-resistant K+ efflux was dependent on the major anion in the medium. The anion dependence of K+ efflux in swollen or NEM-stimulated cells was as follows: Br- greater than Cl- much greater than NO3- = acetate. The magnitudes of both the swelling- and the NEM-stimulated fluxes are much higher in young cells (density separated but excluding reticulocytes) than in older cells. Swelling- or NEM-stimulated K+ efflux in rabbit red blood cells was inhibited 50% by 1 mM furosemide, and the inhibitory potency of furosemide was enhanced by extracellular K+, as is known to be true for human AA and low-K+ sheep red blood cells. The swelling-stimulated flux in both rabbit and human SS cells has a pH optimum at approximately 7.4. We conclude that rabbit red blood cells are a good model for swelling-stimulated K+ transport in human SS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell volume and metabolic dependence of NEM-activated K+-Cl- flux in human red blood cells

1985 ◽  
Vol 249 (1) ◽  
pp. C124-C128 ◽  
Author(s):  
P. K. Lauf ◽  
C. M. Perkins ◽  
N. C. Adragna
Keyword(s):  
Red Blood Cells ◽  
Cell Volume ◽  
Blood Cells ◽  
Time Course ◽  
Transport Activity ◽  
Human Red Blood Cells ◽  
Atp Breakdown ◽  
The Difference ◽  
Metabolic Dependence ◽  
K Transport

The effects of incubation in anisosmotic media and of metabolic depletion on ouabain-resistant (OR) Cl--dependent K+ influxes stimulated by N-ethylmaleimide (NEM) were studied in human red blood cells using Rb+ as K+ analogue. The NEM-stimulated but not the basal Rb+-Cl- influx measured in phosphate-buffered anisosmotic media was found to be cell volume dependent. When cellular ATP, [ATP]c, was lowered to less than 0.10 of its initial level by exposure to nonmetabolizable 2-deoxy-D-glucose, the NEM-stimulated but not the basal Cl--dependent Rb+ influxes were abolished. Metabolically depleted red blood cells subsequently repleted by incubation in glucose plus inosine regained the NEM-inducible Rb+ (K+) transport activity. The difference in the time course of ATP breakdown and Rb+ influx inhibition suggests that energization of the NEM-stimulated Rb+ flux by metabolism may involve factors additional to ATP.

Effects of cadmium and zinc on calcium uptake in human red blood cells

1984 ◽  
Vol 247 (3) ◽  
pp. C143-C149 ◽  
Author(s):  
G. A. Plishker
Keyword(s):  
Red Blood Cells ◽  
Disulfide Bond ◽  
Blood Cells ◽  
Calcium Uptake ◽  
Sodium Potassium ◽  
Human Red Blood Cells ◽  
Low Concentrations ◽  
Influx Pathways ◽  
Extracellular Sodium ◽  
Passive Influx

Cadmium and zinc increased the accumulation of calcium in human red blood cells by increasing passive influx without enhancing the permeability to other ions. The effect of cadmium and zinc appeared specific to these metals, because barium, magnesium, cobalt, strontium, manganese, and nickel had no effect. Changes in calcium uptake by extracellular sodium, potassium, and pH were not altered by zinc and cadmium. Inhibition of calcium uptake by quinine, oligomycin, and iodoacetate was not affected by cadmium or zinc. These results suggest that cadmium and zinc increase calcium movement through normal influx pathways. Cadmium and zinc acted synergistically apparently by different mechanisms. Zinc and cadmium differentially affected calcium uptake in different extracellular calcium concentrations. The cadmium effect was increased by low concentrations of 2-mercaptoethanol and above pH 8.0, while the zinc effect was less sensitive to these factors. These findings suggest that the cadmium effect involves a disulfide bond between cysteinyl residues and the zinc effect involves a different site.

Observed differences in dextran and polyvinylpyrrolidone as rouleaux-inducing agents

1980 ◽  
Vol 58 (3) ◽  
pp. 271-274 ◽  
Author(s):  
Lionel S. Sewchand ◽  
Dieter Bruckschwaiger
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Animal Species ◽  
Critical Concentration ◽  
Rbc Membrane ◽  
Low Concentrations ◽  
High Concentrations ◽  
Induced Aggregation ◽  
Do So

The effectiveness of dextran fractions (Dx-500, Dx-100, Dx-70) and polyvinylpyrrolidone (PVP-360, PVP-40) in inducing aggregation of red blood cells (RBC) was studied in a nonflowing environment. The Dx fractions, at low concentrations, induced aggregation of human RBC but failed to do so at high concentrations (concentrations greater than 70 g/L). The effect was different on RBC from animal species (cat and rabbit); aggregation increased steadily with the Dx concentration and there was no critical concentration beyond which Dx failed to induce aggregation. The PVP was found to be very effective, at all concentrations, in inducing aggregation of RBC from both human and the animal species. These results have a twofold significance: (1) they suggest that Dx and PVP, both neutral polymers, interact differently with the human RBC membrane; and (2) the association of Dx with the human RBC membrane is different from that with cat and rabbit RBC membranes.

K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood cells

1996 ◽  
Vol 271 (4) ◽  
pp. C1111-C1121 ◽  
Author(s):  
Z. Etzion ◽  
V. L. Lew ◽  
R. M. Bookchin
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Highly Nonlinear ◽  
Heterogeneous Expression ◽  
Transport Path ◽  
High Concentrations ◽  
K Transport ◽  
Slow Turnover

Previous studies have suggested ion transport heterogeneity among sickle cell anemia (SS) reticulocytes that could influence their dehydration susceptibility. We examined Ca2(+)-independent K transport in the lowest density (F1), reticulocyte-rich SS cells, measuring the effects of acidification, ouabain, and bumetanide on their unidirectional K(86Rb) fluxes. Unlike those of normal red blood cells and SS discocytes, the SS-F1 K(86Rb) fluxes were highly nonlinear, with large 5-min flux components (previously unobserved) and a more gradual decline over 60 min. Analysis revealed two distinct K pools: a rapid-turnover pool in a small fraction of cells, whose major ouabain-resistant K(86Rb) transport path showed distinctive properties including inhibition by high concentrations of bumetanide (> or = 1 mM) and stimulation at pH 7.0, and another heterogeneous, relatively slow-turnover pool, in most of the F1 cells, whose main ouabain-resistant K(86Rb) path was insensitive to bumetanide but was stimulated at pH 7.0, which is consistent with heterogeneous expression of the acid-sensitive K-Cl cotransport and with both rapid and slower generation of dehydrated SS cells.

Oxidants and regulation of K+-Cl−cotransport in equine red blood cells

2000 ◽  
Vol 279 (4) ◽  
pp. C981-C989 ◽  
Author(s):  
M. C. Muzyamba ◽  
P. F. Speake ◽  
J. S. Gibson
Keyword(s):  
Carbon Monoxide ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Prolonged Exposure ◽  
Reduced Glutathione ◽  
Low Concentrations ◽  
High Concentrations

The effect of oxidants on K+-Cl−cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O2. The substituted benzaldehyde, 12C79 (5 mM), markedly increased O2affinity. In N2, however, O2saturation was low (<10%) but KCC remained active. Nitrite (NO2−) oxidized heme to methemoglobin (metHb). High concentrations of NO2−(1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O2independent but remained sensitive to other stimuli. 1-Chloro-2,4-dinitrobenzene (1–3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60–120 min, 1 mM) or high concentrations (3 mM) stimulated an O2-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.

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