Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

2003 ◽  
Vol 285 (1) ◽  
pp. C31-C38 ◽  
Author(s):  
Isabel Bize ◽  
Samara Taher ◽  
Carlo Brugnara
Keyword(s):  
Human Erythrocytes ◽  
Density Dependent ◽  
Erythrocyte Aging ◽  
Sickle Erythrocytes ◽  
Dense Fraction ◽  
Reticulocyte Maturation ◽  
Tight Correlation

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d ∼33.4 g/dl) has only about ∼5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ∼24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal ( r2 = 0.995) and sickle erythrocytes ( r2 = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity.

Cell volume, K transport, and cell density in human erythrocytes

1987 ◽  
Vol 252 (3) ◽  
pp. C269-C276 ◽  
Author(s):  
C. Brugnara ◽  
D. C. Tosteson
Keyword(s):  
Cell Volume ◽  
Cell Density ◽  
Human Erythrocytes ◽  
Ph Optimum ◽  
Cation Content ◽  
Original Volume ◽  
Dense Fraction ◽  
Normal Human ◽  
K Transport ◽  
Hypotonic Swelling

We report here studies on the regulation of cell volume and K transport in human erythrocytes separated according to density. When cell volume was increased (isosmotic swelling, nystatin technique), erythrocytes of the least dense but not of the densest fraction shrunk back toward their original volume. This process was due to a ouabain (0.1 mM) and bumetanide (0.01 mM) (OB)-resistant K loss. OB-resistant K+ efflux from the least dense fraction was stimulated by hypotonic swelling and had a bell-shaped dependence on pH (pH optimum 6.75–7.0). These pH and volume effects were not evident in the densest fraction. The swelling-induced K+ efflux from the least dense fraction was inhibited when chloride was substituted by nitrate, thiocyanate, and acetate, whereas it was stimulated by bromide. Increasing cell Mg2+ content also markedly inhibited K+ efflux from isosmotically swollen cells. N-ethylmaleimide (NEM, 1 mM) greatly increased OB-resistant K+ efflux from the least dense fraction but not from the densest fraction. These data reveal the presence, in the lease dense fraction of normal human erythrocytes, of a pathway for K+ transport that is dependent on volume, pH, and chloride, is inhibited by internal Mg2+, and possibly plays a role in determining the erythrocyte water and cation content.

scholarly journals Protein kinases and membrane protein phosphorylation in normal and abnormal human erythrocytes: variation related to mean cell age

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 850-857 ◽  
Author(s):  
G Fairbanks ◽  
J Palek ◽  
JE Dino ◽  
PA Liu
Keyword(s):  
Sickle Cell ◽  
Buoyant Density ◽  
Casein Kinase ◽  
Human Erythrocytes ◽  
Normal Limits ◽  
Cell Age ◽  
Abnormal Cells ◽  
Reticulocyte Maturation ◽  
Highly Correlated ◽  
Histone Kinase

Protein kinase activities and membrane autophosphorylation reactions of normal and abnormal human erythrocytes were analyzed. Erythrocytes from patients with high reticulocytosis due to sickle cell anemia and other disorders (n = 13) exhibited elevated activities of total and membrane- bound cAMP-independent casein kinase and cAMP-stimulated histone kinase. Relative to normal controls (n = 10), the average total activities in these abnormal cells were increased 50% and 81%, respectively. The casein and histone kinase activities of normal and abnormal erythrocytes declined significantly with increasing age and buoyant density in Stractan density gradients. Casein kinase activity was highly correlated (r = 0.88; n = 23) with the percentage of reticulocytes in the fraction, consistent with either a progressive loss of activity in mature erythrocytes or an abrupt decline during reticulocyte maturation. The cAMP-independent and cAMP-stimulated autophosphorylation activities of isolated membranes also declined with increasing erythrocyte age. On average, the initial rate of spectrin labeling was 36% lower in ghosts from Stractan gradient bottom fractions, relative to ghosts from top fractions similarly incubated with gamma-32P-ATP. Incorporation into the “band 4.5 zone” (primarily labeling bands 4.8 and 4.9, mol wt 47,800 and 44,600) was also age- dependent. In membranes of unfractionated sickle cells, spectrin autophosphorylation was within normal limits, while 4.5 zone autophosphorylation was increased. Membranes from high reticulocytosis controls (vitamin B-12 deficiency) exhibited similar autophosphorylation patterns, suggesting that the altered autophosphorylation pattern of sickle cell membranes may be attributed to the predominance of very young cells.

scholarly journals Protein kinases and membrane protein phosphorylation in normal and abnormal human erythrocytes: variation related to mean cell age

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 850-857 ◽  
Author(s):  
G Fairbanks ◽  
J Palek ◽  
JE Dino ◽  
PA Liu
Keyword(s):  
Sickle Cell ◽  
Buoyant Density ◽  
Casein Kinase ◽  
Human Erythrocytes ◽  
Normal Limits ◽  
Cell Age ◽  
Abnormal Cells ◽  
Reticulocyte Maturation ◽  
Highly Correlated ◽  
Histone Kinase

Abstract Protein kinase activities and membrane autophosphorylation reactions of normal and abnormal human erythrocytes were analyzed. Erythrocytes from patients with high reticulocytosis due to sickle cell anemia and other disorders (n = 13) exhibited elevated activities of total and membrane- bound cAMP-independent casein kinase and cAMP-stimulated histone kinase. Relative to normal controls (n = 10), the average total activities in these abnormal cells were increased 50% and 81%, respectively. The casein and histone kinase activities of normal and abnormal erythrocytes declined significantly with increasing age and buoyant density in Stractan density gradients. Casein kinase activity was highly correlated (r = 0.88; n = 23) with the percentage of reticulocytes in the fraction, consistent with either a progressive loss of activity in mature erythrocytes or an abrupt decline during reticulocyte maturation. The cAMP-independent and cAMP-stimulated autophosphorylation activities of isolated membranes also declined with increasing erythrocyte age. On average, the initial rate of spectrin labeling was 36% lower in ghosts from Stractan gradient bottom fractions, relative to ghosts from top fractions similarly incubated with gamma-32P-ATP. Incorporation into the “band 4.5 zone” (primarily labeling bands 4.8 and 4.9, mol wt 47,800 and 44,600) was also age- dependent. In membranes of unfractionated sickle cells, spectrin autophosphorylation was within normal limits, while 4.5 zone autophosphorylation was increased. Membranes from high reticulocytosis controls (vitamin B-12 deficiency) exhibited similar autophosphorylation patterns, suggesting that the altered autophosphorylation pattern of sickle cell membranes may be attributed to the predominance of very young cells.

scholarly journals Acid pH induces formation of dense cells in sickle erythrocytes

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 487-495 ◽  
Author(s):  
C Brugnara ◽  
T Van Ha ◽  
DC Tosteson
Keyword(s):  
Carbon Monoxide ◽  
Cell Shrinkage ◽  
Density Fractions ◽  
Kcl Cotransport ◽  
Acid Ph ◽  
Sickle Erythrocytes ◽  
Dense Fraction ◽  
Cotransport System ◽  
Constant Cell ◽  
Ph Dependent

When erythrocytes from patients homozygous for hemoglobin S (SS) are swollen or exposed to pH less than 7.40, they lose K, Cl, and water through a volume and pH-dependent KCl cotransport system. We report that carbon monoxide-treated SS cells become progressively denser when incubated for eight to 12 hours in media with pH less than 7.40 (7.3 to 7.0) at constant cell 2,3-diphosphoglycerate (DPG). This phenomenon is maximal in fresh SS cells from the top and middle density fractions, and is absent in cells from the densest fraction. When AA cells are separated according to density, acid pH induces cell shrinkage in the least dense fraction of AA cells, which has considerable KCl cotransport, but produces no change in cell density in the densest fractions of AA erythrocyte, which have no KCl cotransport. These data suggest that dense cells can form in oxygenated SS erythrocytes when the KCl cotransport system is activated by acidification.

scholarly journals Acid pH induces formation of dense cells in sickle erythrocytes

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 487-495 ◽  
Author(s):  
C Brugnara ◽  
T Van Ha ◽  
DC Tosteson
Keyword(s):  
Carbon Monoxide ◽  
Cell Shrinkage ◽  
Density Fractions ◽  
Kcl Cotransport ◽  
Acid Ph ◽  
Sickle Erythrocytes ◽  
Dense Fraction ◽  
Cotransport System ◽  
Constant Cell ◽  
Ph Dependent

Abstract When erythrocytes from patients homozygous for hemoglobin S (SS) are swollen or exposed to pH less than 7.40, they lose K, Cl, and water through a volume and pH-dependent KCl cotransport system. We report that carbon monoxide-treated SS cells become progressively denser when incubated for eight to 12 hours in media with pH less than 7.40 (7.3 to 7.0) at constant cell 2,3-diphosphoglycerate (DPG). This phenomenon is maximal in fresh SS cells from the top and middle density fractions, and is absent in cells from the densest fraction. When AA cells are separated according to density, acid pH induces cell shrinkage in the least dense fraction of AA cells, which has considerable KCl cotransport, but produces no change in cell density in the densest fractions of AA erythrocyte, which have no KCl cotransport. These data suggest that dense cells can form in oxygenated SS erythrocytes when the KCl cotransport system is activated by acidification.

Elemental analysis of human erythrocytes

1989 ◽  
Vol 47 ◽  
pp. 242-243
Author(s):  
S. A. Livesey ◽  
A. A. del Campo ◽  
E. S. Griffey ◽  
D. Ohlmer ◽  
T. Schifani ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Elemental Analysis ◽  
Human Erythrocyte ◽  
Spectroscopic Analysis ◽  
Model System ◽  
Human Erythrocytes ◽  
List Type ◽  
Chemical Fixation ◽  
Ethanol Dehydration ◽  
Lowicryl K4m

The aim of this study is to compare methods of sample preparation for elemental analysis. The model system which is used is the human erythrocyte. Energy dispersive spectroscopic analysis has been previously reported for cryofixed and cryosectioned erythrocytes. Such work represents the reference point for this study. The use of plastic embedded samples for elemental analysis has also been documented. The work which is presented here is based on human erythrocytes which have been either chemically fixed and embedded or cryofixed and subsequently processed by a variety of techniques which culminated in plastic embedded samples.Heparinized and washed erythrocytes were prepared by the following methods for this study :(1). Chemical fixation in 4% paraformaldehyde/0.25% glutaraldehyde/0.2 M sucrose in 0.1 M Na cacodylate, pH 7.3 for 30 min, followed by ethanol dehydration, infiltration and embedding in Lowicryl K4M at -20° C.

Urbanization impacts on production and recruitment of Fundulus heteroclitus in salt marsh creeks

2020 ◽  
Vol 645 ◽  
pp. 187-204
Author(s):  
PJ Rudershausen ◽  
JA Buckel
Keyword(s):  
Salt Marsh ◽  
Salt Marshes ◽  
Fundulus Heteroclitus ◽  
Multiple Factor Analysis ◽  
Southeastern Usa ◽  
Density Dependent ◽  
Dependent Relationship ◽  
Juvenile Abundance ◽  
The Relationship ◽  
Throw Trap

It is unclear how urbanization affects secondary biological production in estuaries in the southeastern USA. We estimated production of larval/juvenile Fundulus heteroclitus in salt marsh areas of North Carolina tidal creeks and tested for factors influencing production. F. heteroclitus were collected with a throw trap in salt marshes of 5 creeks subjected to a range of urbanization intensities. Multiple factor analysis (MFA) was used to reduce dimensionality of habitat and urbanization effects in the creeks and their watersheds. Production was then related to the first 2 dimensions of the MFA, month, and year. Lastly, we determined the relationship between creek-wide larval/juvenile production and abundance from spring and abundance of adults from autumn of the same year. Production in marsh (g m-2 d-1) varied between years and was negatively related to the MFA dimension that indexed salt marsh; higher rates of production were related to creeks with higher percentages of marsh. An asymptotic relationship was found between abundance of adults and creek-wide production of larvae/juveniles and an even stronger density-dependent relationship was found between abundance of adults and creek-wide larval/juvenile abundance. Results demonstrate (1) the ability of F. heteroclitus to maintain production within salt marsh in creeks with a lesser percentage of marsh as long as this habitat is not removed altogether and (2) a density-dependent link between age-0 production/abundance and subsequent adult recruitment. Given the relationship between production and marsh area, natural resource agencies should consider impacts of development on production when permitting construction in the southeastern USA.

IN VITRO UPTAKE OF RADIOACTIVE 131I LABELLED L-TRIIODOTHYRONINE BY HUMAN ERYTHROCYTES AS AN INDEX OF THYROID FUNCTION

1960 ◽  
Vol XXXIV (II) ◽  
pp. 305-311 ◽  
Author(s):  
M. G. Woldring ◽  
A. Bakker ◽  
H. Doorenbos
Keyword(s):  
Thyroid Function ◽  
Incubation Time ◽  
Clinical Results ◽  
Human Erythrocytes ◽  
Red Cell ◽  
Clinical Value ◽  
Blood Samples ◽  
In Vitro Uptake

ABSTRACT The red cell triiodothyronine uptake technique as used in our hospital is described. Incubation time is of almost no importance. The temperature during incubation should be 37° C. Further improvement of the technique is obtained when all blood samples are brought up to 40 % haematocrit prior to incubation. Clinical results are discussed. It is yet too early to give a definite assessment of its clinical value, but it is definitely superior to the measurement of the BMR.

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