Urea-stimulated K-Cl cotransport in equine red blood cells

Speake, P. F.; Gibson, JS

doi:10.1007/s004240050369

Cited by 25 publications

(28 citation statements)

References 23 publications

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“…Even though KCC activity is reduced in the hypertonic conditions found in this organ, in equine red cells urea stimulation of KCC is not abolished by hypertonic shrinkage (29). Stimulation of KCC by urea is inhibited by okadaic acid (27) and by calyculin A (21,29), potent inhibitors of PP1 and PP2A (18), indicating that urea stimulation is mediated by phosphorylation/dephosphorylation. Stimulation of KCC by urea could be mediated by inhibition of the KCC inhibitory kinase (12,29).…”

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confidence: 83%

“…Urea activates KCC in dog, human, sheep, and horse erythrocytes (12,21,27,29). It has been postulated that urea may influence dehydration of SS cells pass-ing through the kidney (11,21).…”

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confidence: 99%

“…It has been postulated that urea may influence dehydration of SS cells pass-ing through the kidney (11,21). Even though KCC activity is reduced in the hypertonic conditions found in this organ, in equine red cells urea stimulation of KCC is not abolished by hypertonic shrinkage (29). Stimulation of KCC by urea is inhibited by okadaic acid (27) and by calyculin A (21,29), potent inhibitors of PP1 and PP2A (18), indicating that urea stimulation is mediated by phosphorylation/dephosphorylation.…”

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confidence: 99%

“…Stimulation of KCC by urea is inhibited by okadaic acid (27) and by calyculin A (21,29), potent inhibitors of PP1 and PP2A (18), indicating that urea stimulation is mediated by phosphorylation/dephosphorylation. Stimulation of KCC by urea could be mediated by inhibition of the KCC inhibitory kinase (12,29). However, the absence of a lag time in urea-induced KCC stimulation in SS cells (11) suggests that urea may also stimulate an activating reaction, possibly a phosphatase (19).…”

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

Bize

Taher

Brugnara

2003

American Journal of Physiology-Cell Physiology

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Bize, Isabel, Samara Taher, and Carlo Brugnara. Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes. Am J Physiol Cell Physiol 285: C31-C38, 2003. First published February 26, 2002 10.1152/ajpcell.00447.2002The 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 ϳ33.4 g/dl) has only about ϳ5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ϳ24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 Ϯ 0.4 vs. 27.2 Ϯ 0.2 g/dl, respectively), whereas those for KCC activity were not (d50% 31.4 Ϯ 0.9 vs. 26.8 Ϯ 0.3 g/dl, respectively, P ϭ 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r 2 ϭ 0.995) and sickle erythrocytes (r 2 ϭ 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. phosphorylation; protein phosphatase; urea; cell size; density IT HAS BEEN POSTULATED that the elevated activity of K-Cl cotransport (KCC) in sickle erythrocytes contributes to their dehydration, their short life-span, and the pathology associated with this disease (7). Reticulocyte maturation is associated with a decrease in cell volume mediated by KCC (23), followed by a sharp decrease in KCC activity (2,7,8,15,16,24). The functional inactivation of KCC in denser cells can be reversed by a variety of stimuli except in the 5% densest cells, indicating that in mature erythrocytes a significant amount of KCC-protein remains in the membrane in a latent state (9,14). In normal (AA) and sickle (SS) erythrocytes of increasing densities, a slight decrease in membrane-associated KCC-protein has been reported (31). Thus it appears that the large decrease in KCC activity with increasing cell densities is due almost exclusively to functional inactivation. The functional state of KCC is determined by phosphorylation/ dephosphorylation, either directly or via an associated protein. In mature erythrocytes, the low activity of KCC in isosmotic media appears to be due to high levels of phosphorylation of the transporter (or an associated protein) and is thought to be the result of the high activity of a kinase relative to the activity of a phosphatase, the substrate of which is the KCC transporter (19,13,17), although direct phosphorylation of the KCC-protein has yet to be demonstrated.In erythrocytes, KCC dephosphorylation appears to be mediated by membrane-associated protein phosphatase(s) (4, 28). The activating ph...

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confidence: 83%

“…Urea activates KCC in dog, human, sheep, and horse erythrocytes (12,21,27,29). It has been postulated that urea may influence dehydration of SS cells pass-ing through the kidney (11,21).…”

mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

Bize

Taher

Brugnara

2003

American Journal of Physiology-Cell Physiology

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“…18 Activation is dependent on urea concentration up to 1 M 13,15,16 and is reversible, 13,15 suggesting that protein denaturation is not involved. Gibson et al 14 and Culliford et al 15 first reported stimulation by urea of KCC in SS RBCs and demonstrated its abnormal response to deoxygenation compared with normal cells.…”

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confidence: 99%

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Joiner

Rettig

Jiang

et al. 2006

Blood

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KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and IntroductionThe KCl cotransporter (KCC) mediates electroneutral coupled transport of K ϩ and Cl Ϫ in a variety of cell types. 1,2 KCC activation results in net efflux of KCl from cells with high potassium content, with accompanying water loss and volume reduction. KCC is active in reticulocytes and diminishes with red cell maturation. 3,4 It is thought to function physiologically to reduce red blood cell (RBC) volume and establish the high cellular hemoglobin concentration (CHC) characteristic of mature cells. KCC is activated in vitro by cell swelling, acid pH, sulfhydryl oxidation/alkylation, and exposure to urea, but the relative importance of these stimuli in vivo is unknown.In RBCs containing sickle hemoglobin (Hb S), KCC activity is high, in part because of the high percentage of reticulocytes. 4 However, Hb S or Hb C in trait RBCs (AS or AC) with normal mean cell age, or after incorporation into normal RBC ghosts, appears to increase KCC activity. 5,6 Expression of Hb S or C in transgenic mice also increases red cell KCC activity. 7 Recently, we demonstrated an abnormal response to acid pH in SS RBCs that was partially corrected by incubation with the sulfhydryl reducing agent, dithiothreitol. 8 These findings suggested that regulation of KCC is abnormal in SS RBCs.As a volume regulator, KCC activity is responsive to CHC. 9,10 KCC is activated on cell swelling, and, as KCl is lost, cell volume falls, cellular hemoglobin concentration increases, and transport activity diminishes. The resultant CHC thus reflects the physiologic "volume set point" of the system. The inactivation of KCC as the cell shrinks toward its new steady state is most likely a key factor in defining the volume regulatory function of the transporter, which in turn is crucial for establishing the steady state CHC for RBCs in vivo.Volume reduction mediated by KCC has been demonstrated with RBC density measurements. 7,11,12 However, these studies, like flux studies, are complicated by varying numbers of reticulocytes (or young cells) in unfractionated SS blood, which preclude comparisons of KCC-mediated volume reduction in SS and AA cells. We recently compared the RVD mediated by KCC and stimulated by cell swelling and acid pH in SS and AA reticulocytes by tracking the changes in reticulocyte CHC by means of density gradient analysis. 8 Acid-stimulated volume reduction was exaggerated in SS reticulocytes and was diminished by sulfhydryl reduction, parallel to the behavior of KCC flux activity. However, despite the normal response of KCC flux activation to cell swelling in SS RBCs, swelling-stimulated volume reduction was markedly abnormal in SS reticulocytes and resulted in higher final CHC than in AA reticulocytes. Thus, volume reduction and KCC fluxes appear to reflect different functional aspects of the KCC system in RBCs and may be subject to different pathologic influences in SS RBCs...

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Membrane Transport in Sickle Cell Disease

Gibson

Ellory

2002

Blood Cells, Molecules, and Diseases

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Urea-stimulated K-Cl cotransport in equine red blood cells

Cited by 25 publications

References 23 publications

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

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

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Membrane Transport in Sickle Cell Disease

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