Effect of 1-chloro-2,4-dinitrobenzene on K+ transport in normal and sickle human red blood cells

doi:10.1113/jphysiol.2002.036467

Effect of 1-chloro-2,4-dinitrobenzene on K⁺ transport in normal and sickle human red blood cells

M. C. Muzyamba and J. S. Gibson

Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

1-Chloro-2,4-dinitrobenzene (CDNB), which causes oxidative stress through depletion of reduced glutathione (GSH), increases the passive K⁺ permeability of red cells. In this paper, we investigated the effects of CDNB (1 mM) on the activities of the K⁺-Cl^- cotransporter (KCC; measured as Cl^--dependent K⁺ influx) and the Gardos channel (taken as clotrimazole-sensitive K⁺ influx, 5 µM) in human red cells, using ⁸⁶Rb⁺ as a K⁺ congener. ⁴⁵Ca²⁺ was used to study passive Ca²⁺ entry and active Ca²⁺ efflux via the plasma membrane Ca²⁺ pump. Both the Gardos channel and KCC were stimulated in both normal and sickle red cells. In sickle cells, stimulation of KCC was similar in oxygenated and deoxygenated cells; that of the Gardos channel was greater in deoxygenated cells. In normal red cells, stimulation of both pathways was greater in oxygenated cells (by 4 ± 1-fold; all means ± S.E.M., n = 3). The effects on the Gardos channel were dependent on extracellular Ca²⁺ and were associated with inhibition of the plasma membrane Ca²⁺ pump (by 29 ± 3 %, P < 0.01) and increased Ca²⁺ sensitivity of the channel (EC₅₀ for [Ca²⁺]_i reduced from 260 ± 26 to 175 ± 15 nM; P < 0.05). Cell volume, pH_i, ATP levels and passive Ca²⁺ entry were not affected by CDNB. The effects on KCC were inhibited (93 ± 6 %) by prior treatment with the protein phosphatase inhibitor calyculin A (100 nM) and were not additive with stimulation by N-ethylmaleimide (1 mM), regardless of the order of addition. These findings are therefore consistent with inhibition of a regulatory protein kinase, although stimulation of the conjugate protein phosphatase(s) may also occur. KCC stimulation was also Ca²⁺ dependent. These findings are important for understanding how GSH depletion alters membrane permeability and how to protect against red cell dehydration.

(Resubmitted 25 November 2002; accepted after revision 4 January 2003; first published online 7 February 2003)
Corresponding author J. S. Gibson: Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. Email: jsg1001{at}cam.ac.uk

INTRODUCTION

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Oxidants have long been used as in vitro tools with which to study mechanisms of membrane transport in red cells (Stein, 1986). They are also of relevance in vivo because of their role in pathological conditions, including various oxidant toxicities and certain red cell enzymopathies and haemoglobinopathies (e.g. Maridonneau et al. 1983; Edwards & Fuller, 1996). Oxidant damage may play a role in the pathogenesis of sickle cell disease (SCD; Rice-Evans et al. 1986). Thus red cells from sickle cell patients contain haemoglobin (Hb) S (here termed HbS cells), rather than the normal HbA (HbA cells). HbS is more susceptible to auto-oxidation (Hebbel et al. 1982, 1988) thereby producing free Hb chains, hemichromes and Fe²⁺, all of which can cause oxidative damage, a situation that is further exacerbated by the low levels of reduced glutathione (GSH) in HbS cells compared to normal red cells (Lachant et al. 1983).

The transport phenotype of HbS cells is markedly different from that of HbA cells (reviewed by Joiner, 1993; Ellory et al. 1998; Gibson & Ellory, 2002). First, on deoxygenation of HbS cells, a non-specific cation channel(s) (sometimes termed P_sickle, but of unknown molecular identity) is activated (Joiner et al. 1988). Second, partly through Ca²⁺ entry via P_sickle coupled with modest inhibition of the plasma membrane Ca²⁺ pump (Tiffert et al. 1993), activation of the Ca²⁺-activated K⁺ channel (Gardos channel, probably IK1/SK4; Ishii et al. 1997) may also occur on deoxygenation (Rhoda et al. 1990). Third, they also show elevated levels (Brugnara et al. 1985, 1986; Canessa et al. 1986) and altered regulation of the K⁺-Cl^- cotransporter (KCC, probably the KCC1 isoform; Gillen et al. 1996; Pellegrino et al. 1998). The transporter has an abnormal response to O₂, remaining active at low O₂ tensions (P_O2) when, by contrast, in HbA cells it is refractory (J. Gibson et al. 1998). These three passive transport pathways combine to mediate loss of intracellular solutes (mainly KCl) and cell shrinkage (see Gibson & Ellory, 2002). Dehydration further encourages HbS polymerisation (Eaton & Hofrichter, 1987), cell sickling and ensuing deleterious effects, which are features of SCD (Ellory et al. 1989, 1998). Since some of these effects may be due to oxidative damage, a number of therapeutic regimes have been designed to ameliorate SCD through reduction of oxidative stress to HbS cells (Chan et al. 1999), including supplementation with iron chelators, ascorbate or N-acetylcysteine (e.g. Moore et al. 1992; X. Gibson et al. 1998).

1-Chloro-2,4-dinitrobenzene (CDNB) causes oxidative damage to red cells through depletion of GSH (Awasthi et al. 1981). It has been shown to stimulate KCC in red cells from sheep (Lauf et al. 1995) and horse (Muzyamba et al. 2000); in human cells there are reports that its main action is on the Gardos channel (Shartava et al. 2000). There are several outstanding questions, however. First, there is discrepancy as to whether it affects KCC as well as the Gardos channel; second, it is not known how it behaves at different levels of P_O2 (note the greater permeability abnormalities in deoxygenated HbS cells); third, its mechanism of action remains obscure. To address these questions, we investigated the effect of CDNB on K⁺ transport in HbA and HbS cells under both oxygenated and deoxygenated conditions, measuring the activity of KCC and the Gardos channel, together with other cell parameters including volume, pH, GSH, methaemoglobin (metHb), Ca²⁺ transport and Na⁺-K⁺ pump activity.

METHODS

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Chemicals

Bumetanide, CDNB, Mops, N-ethylmaleimide (NEM), ouabain, salts, Tris base and vanadate were purchased from Sigma (Poole, Dorset, UK). Calyculin A, clotrimazole, 1,2-bis(o-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM) and A23187 were purchased from Calbiochem (Nottingham, UK) and ⁸⁶Rb⁺ was from DuPont-NEN (Stevenage, UK). ⁴⁵Ca²⁺ was purchased from Amersham Biosciences (Little Chalfont, UK).

Solutions

The standard saline (MBS) comprised (mM): 80 KCl, 70 NaCl, 2.5 Ca(NO₃)₂, 0.15 MgCl₂, 10 inosine and 10 Mops (pH 7.4 at 37 °C; 290 ± 5 mosmol (kg H₂O)^-1). Where required, low-K⁺ saline solutions were used and contained (mM): 4 KCl, 145 NaCl, 2.5 Ca(NO₃)₂, 0.15 MgCl₂, 10 inosine and 10 Mops (pH 7.4 at 37 °C; 290 ± 5 mosmol (kg H₂O)^-1). For experiments in which the Cl^- dependence of K⁺ influx was examined, Cl^- was substituted for NO₃^-. Anisotonic cell swelling was achieved by adding distilled water and pH was altered by addition of HNO₃ or NaOH. Stock solutions of bumetanide (1 mM) were prepared daily in 100 mM Tris base and used at a final concentration of 10 µM. Stock solutions of NEM (100 mM) were prepared daily in distilled water and used at 1 mM; a stock solution of ouabain (10 mM) was prepared in distilled water and used at a final concentration of 100 µM. Stock solutions of calyculin A (10^-5 M) and clotrimazole (1 mM) were prepared in DMSO, frozen until required, and used at final concentrations of 100 nM and 5 µM, respectively. Finally, CDNB (500 mM) was also dissolved in DMSO and used at a final concentration of 1 mM; in all cases, oxygenated cells were treated with CDNB (1 mM) at 4 % haematocrit (Hct) before washing (twice with MBS). They were then placed in tonometers (at 40 % Hct) to equilibrate at the requisite P_O2 before measurement of transport activity or other cell parameters. Control experiments established that stimulation by CDNB of the K⁺ pathways, and formation of metHb, increased progressively as the concentration of CDNB ([CDNB]) was elevated from 0.1 to 3 mM. We chose 1 mM because at this concentration GSH levels were totally depleted within 10 min, and for comparison with previously published work. In all experiments, controls and cells treated with reagents were exposed to the same concentrations of solvents.

Sample collection and handling

Blood samples were obtained by venepuncture of healthy donors (HbAA) and sickle cell patients who were typed as homozygous for the sickle gene (HbSS) (with permission under ethical consent and in accordance with the Declaration of Helsinki), and collected into heparinised syringes. The sickle cell patients had not received transfusion for at least 6 months and were not in crisis. Samples were kept at 4 °C until use within 48 h.

Tonometry

Red blood cell suspensions were incubated at about 40 % Hct in glass tonometers (Eschweiler, Kiel, Germany) flushed with gas mixtures with the appropriate P_O2 using a Wösthoff gas mixing pump. The gases were warmed up to 37 °C and fully humidified through three humidifiers.

ATP, GSH, metHb, cell volume and pH_i

For all these assays, cells were first incubated for 60 min ± CDNB (1 mM) at 4 % Hct. ATP was determined using an NADH-based commercial assay from Sigma following the method of Beutler & Duron (1965). GSH was assayed following the procedure of Beutler (1975) based on the reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Determination of metHb content was carried out following the procedure of Hegesh et al. (1970), based on its conversion to cyanmetHb and absorption of light at 632 nm. Cell volume was measured using the method of Borgese et al. (1991) in samples swollen anisotonically by 10 %. pH_i was measured by centrifuging cells through dibutyl phthalate oil, lysing the cells by freeze-thawing and measuring the pH with a micropH probe; these values, coupled with those for pH_o, were used to calculate r values (where r = [H⁺]_i/[H⁺]_o). In the presence of A23187, [Ca²⁺]_i = [Ca²⁺]_o times r² (see Fig. 7).

K⁺ influx

To determine the activity of the K⁺ transport pathways, K⁺ influx was measured at 37 °C using ⁸⁶Rb⁺ as a congener for K⁺ (Dunham & Ellory, 1981). Cells were taken from the tonometers and diluted 10-fold into saline, pre-equilibrated at the appropriate P_O2, at 260 mosmol kg^-1 and pH 7. These conditions were chosen in order to stimulate KCC to a reasonable level in HbA cells so that it could be measured reliably with the high-K⁺ saline solutions used; control experiments showed that similar effects to those presented here were present under isotonic conditions at pH 7.4. The high [K⁺] (80 mM) in the saline solutions was chosen to prevent cell dehydration following transporter activation. ⁸⁶Rb⁺ was added in distilled water. Except for experiments on the Na⁺-K⁺ pump, ouabain (100 µM) and bumetanide (10 µM) were present in all experiments to obviate any K⁺ transport through the Na⁺-K⁺-ATPase and the Na⁺-K⁺-2Cl^- cotransporter, respectively. Either microhaematocrit determination or the cyanohaemoglobin method was used to measure Hct. KCC activity was assayed as Cl^--dependent K⁺ influx; Gardos channel activity as the clotrimazole-sensitive (5 µM) K⁺ influx; Na⁺-K⁺ pump activity as the ouabain-sensitive K⁺ influx.

Ca²⁺ efflux and influx

In these experiments, transport determinations were carried out at 37 °C and ⁴⁵Ca²⁺ was used as a tracer for Ca²⁺. Plasma membrane Ca²⁺ pump activity (plasma membrane Ca²⁺-ATPase, PMCA) was assayed following the method of Tiffert et al. (1993). Briefly, cells were washed twice with MBS lacking Ca²⁺ and containing 100 µM EGTA (MBS-0Ca-E; to remove contaminant Ca²⁺), then twice further with MBS-0Ca (without EGTA). They were then suspended at 10 % Hct and loaded with ⁴⁵Ca²⁺ using the ionophore A23187 (10 µM) at [Ca²⁺]_o of 120 µM. The ionophore was then blocked using Co²⁺ (0.4 mM), after which Ca²⁺ is progressively pumped out of the cells by the PMCA. Serial aliquots (50 µl) of cells taken during these procedures were washed twice with ice-cold MBS-0Ca-E plus 0.2 mM Co²⁺ (1.3 ml). The maximum negative slope of the curve (Ca²⁺ content vs. time, see Fig. 6) after addition of Co²⁺ was used as a measure of PMCA V_max (Tiffert et al. 1993). For influx experiments, the saline (MBS-0Ca-P) comprised (mM): 80 KCl, 65 NaCl, 5 sodium pyruvate, 0.15 MgCl₂, 10 inosine and 10 Mops (pH 7.4 at 37 °C; 290 ± 5 mosmol (kg H₂O)^-1). Pyruvate was added to protect cells from ATP depletion by formaldehyde released following ester hydrolysis (Tiffert et al. 1984; Garcia-Sancho, 1985). Cells were preloaded with MAPTAM, a Ca²⁺ chelator, for 90 min (0.25 mM MAPTAM at 20 % Hct) before being incubated with CDNB (60 min, 1 mM, 4 % Hct; see above). They were taken to 4 % Hct and treated with vanadate (1 mM) to inhibit PMCA, prior to addition of ⁴⁵Ca²⁺ (final [Ca²⁺]_o of 1 mM). Serial aliquots (100 µl) were taken and added to 10 ml ice-cold MBS-0Ca-P, pelletted and washed once more (see Tiffert et al. 1993).

Statistics

Results are presented as single observations representative of at least three others, or as means ± S.E.M. of n observations. Where appropriate, comparisons were made using Student's paired t tests.

RESULTS

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Methods
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Cellular effects of CDNB

The effects of CDNB (1 mM) treatment on a number of cell parameters, relevant to K⁺ transport, are shown in Table 1. GSH was fully depleted, and metHb accumulation was observed. Total intracellular ATP was unaffected, as was ouabain-sensitive K⁺ influx. The latter is a measure of the activity of the plasma membrane Na⁺-K⁺ pump, indicative of the membrane pool of ATP accessible to these pumps. pH_i and cell volume were unaffected.

Effects of CDNB on KCC and Gardos channel

The activities of KCC and the Gardos channel in oxygenated and deoxygenated HbA cells are shown in Fig. 1A. In control cells, KCC activity was modest in oxygenated cells and inactivated upon deoxygenation (91 ± 3 % inhibition, mean ± S.E.M., n = 3), whilst Gardos channel activity was minimal (< 0.1 mmol (l cells)^-1 h^-1 irrespective of P_O2. In cells treated with CDNB, both KCC and Gardos channel activities were increased. In oxygenated cells, the fold increase in KCC following application of CDNB was 7 ± 1 (mean ± S.E.M., n = 3). The absolute magnitudes of both K⁺ transport pathways following CDNB treatment were greater in oxygenated cells, the activities of both the Gardos channel and KCC being 4 ± 1-fold higher in air (means ± S.E.M., n = 3; P < 0.05). Figure 1B shows that the effects of CDNB on cells in low-K⁺ (4 mM) saline were qualitatively similar.

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Figure 1. Effects of CDNB treatment on normal human red blood cells
A, cells were treated with CDNB (1 mM) for 60 min prior to measurement of the activity of the K⁺-Cl^- cotransporter (KCC; measured as Cl^--dependent K⁺ influx) and the Gardos channel (measured as clotrimazole-sensitive K⁺ influx, 5 µM) in fully oxygenated (Air) or deoxygenated (N₂) cells. B, identical experiment to that in A but cells were incubated under isotonic conditions, in low-K⁺-containing saline and at pH 7.4 during the flux measurements. Ouabain (100 µM) and bumetanide (10 µM) were present to prevent transport via the Na⁺-K⁺ pump and Na⁺-K⁺-2Cl^- cotransporter. Transport is expressed as mmol K⁺ (l cells)^-1 h^-1, means ± S.E.M., n = 3 different experiments.

A similar experiment was carried out on HbS cells (Fig. 2). In this case, KCC activity was markedly increased compared to HbA cells, and control fluxes were similar in oxygenated and deoxygenated cells. Gardos channel activity was minimal in air, but was stimulated by deoxygenation, presumably following P_sickle activation and increased Ca²⁺ influx. As for HbA cells, in air, CDNB stimulated both KCC and the Gardos channel. By contrast, however, transport rates were not diminished by deoxygenation, in fact that for the Gardos channel was further elevated.

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Figure 2. Effects of CDNB treatment on red blood cells from sickle cell patients
Details, methodology and statistics are as given in the legend to Fig. 1A.

These findings show that CDNB stimulates both KCC and Gardos channels, and that transporters are reduced by deoxygenation in HbA, but not in HbS, cells.

Mechanism of action of CDNB

We went on to study further the action of CDNB on HbA cells. In the first series of experiments, the effects of Ca²⁺ removal were examined. As expected, CDNB was unable to stimulate Gardos channel activity in HbA cells suspended in Ca²⁺-free saline (Fig. 3), implying that Ca²⁺ entry is important for the activation of this channel. An unexpected finding, however, was that CDNB-induced stimulation of KCC was also sensitive to Ca²⁺ removal. The effects of the protein phosphatase inhibitor calyculin A (100 nM) are also shown in Fig. 3. Calyculin A did not have a significant effect on CDNB-induced stimulation of the Gardos channel. In the case of KCC, however, prior treatment of cells with calyculin A abolished the stimulatory effect of CDNB (93 ± 6 % inhibition, mean ± S.E.M., n = 3). The KCC activity in cells to which calyculin A was applied after CDNB was similar to that in cells treated with CDNB alone, showing that calyculin A was without effect (3 ± 1 % inhibition, mean ± S.E.M., n = 3).

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Figure 3. Effects of inhibitors on CDNB-induced K⁺ transport in normal human red blood cells
Apart from control cells (i), all aliquots were incubated for 60 min with CDNB before measurement of transporter activity, as described in the legend to Fig. 1. Cells were also treated with calyculin A (Cal, 100 nM) for 10 min, either prior to 60 min with CDNB (iii) or for 10 min after 60 min with CDNB (CDNB/Cal; (iv)). [Ca²⁺]_o was 2.5 mM, except for the 0 Ca²⁺ condition (v) where saline was nominally Ca²⁺ free with an addition of 50 µM EGTA. Histograms represent means ± S.E.M., n = 3 different experiments.

These findings show that extracellular Ca²⁺ is required for stimulation of the Gardos channel, implying that Ca²⁺ entry is required. Results with calyculin A imply that CDNB-induced stimulation of KCC requires protein phosphatase activity. The finding that calyculin A was without effect after CDNB application is consistent with CDNB acting on an inhibitory protein kinase, although additional stimulation of the conjugate protein phosphatase(s) cannot be excluded.

The interaction of CDNB with NEM was also studied (Fig. 4). NEM, like calyculin A (Fig. 3), had no effect on the Gardos channel, either in the absence or presence of CDNB. NEM alone and CDNB alone both stimulated KCC, with CDNB being more powerful. When applied together, stimulation was no greater than that for CDNB alone, and no additive or synergistic effect was observed. In isotonic conditions at pH 7.4, KCC activity in cells treated with NEM or CDNB alone was 4.53 ± 0.16 and 6.55 ± 0.41 mmol (l cells)^-1 h^-1, respectively, compared with 6.56 ± 1.12 mmol (l cells)^-1 h^-1 in cells treated with CDNB followed by NEM, and 6.32 ± 0.63 mmol (l cells)^-1 h^-1 when the order was reversed. These findings show that the order of NEM/CDNB addition, isotonicity or hypotonicity, or pH change from 7.4 to 7.0, were without effect on the pattern of KCC activity. Again, results are consistent with CDNB-induced inhibition of the regulatory protein kinase for KCC.

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Figure 4. Effects of NEM on CDNB-induced K⁺ transport in normal human red blood cells
Apart from control cells, all aliquots were exposed to NEM for 10 min and/or CDNB for 60 min, before measurement of transporter activity, as described in the legend to Fig. 1: (i) control cells; (ii) cells treated with NEM (1 mM) only for 10 min; (iii) cells treated with CDNB for 60 min; (iv) cells treated with NEM for 10 min followed by CDNB for 60 min. Ouabain (100 µM) and bumetanide (10 µM) were present to prevent transport via the Na⁺-K⁺ pump and Na⁺-K⁺-2Cl^- cotransporter. Transport is expressed as mmol K⁺ (l cells)^-1 h^-1, means ± S.E.M., n = 3 different experiments.

Effects of CDNB on Ca²⁺-activated K⁺ transport

Stimulation of Gardos channels by CDNB could be mediated by three possible mechanisms, singly or in combination: increased passive Ca²⁺ influx, decreased active Ca²⁺ exit or an increased sensitivity of the Gardos channel to [Ca²⁺]_i. These possibilities were examined.

Passive Ca²⁺ influx in HbA cells was not significantly different in control cells and in those treated with CDNB (e.g. Fig. 5). Mean influx was lower in CDNB-treated cells than in controls (44 ± 18 vs. 59 ± 14 µmol (l cells)^-1 h^-1 in control cells, means ± S.E.M., n = 4), but not significantly so (P > 0.1). There was no evidence of CDNB-induced stimulation of Ca²⁺ entry.

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Figure 5. Effects of CDNB treatment on Ca²⁺ influx into normal human red blood cells
Cells were preincubated for 90 min at 37 °C with MAPTAM (see Methods), before being incubated with or without CDNB for a further 60 min. Aliquots were then treated with vanadate (1 mM) before addition of ⁴⁵Ca²⁺ ([Ca²⁺]_o = 1 mM) to measure Ca²⁺ influx, which is expressed as µmol (l cells)^-1. Symbols represent single determinations and are representative of four additional experiments.

The activity of the plasma membrane Ca²⁺ pump was then investigated (Fig. 6) following the method developed by Tiffert et al. (1993). Results show that Ca²⁺ loading with ⁴⁵Ca²⁺ in the presence of the ionophore A23187 was greater in CDNB-treated cells than in control cells (481 ± 38 vs. 538 ± 42 µmol (l cells)^-1 h^-1, means ± S.E.M., n = 3), but this effect was not significant. After addition of Co²⁺ to prevent further activity of A23187, ⁴⁵Ca²⁺ was actively pumped out of the cells. The maximal slope of the resulting plots estimates the pumping capacity of the cells. The mean activity of PMCA was 6.6 ± 1.2 mmol (l cells)^-1 h^-1 in control cells and 4.8 ± 1.1 mmol (l cells)^-1 h^-1 in cells treated with CDNB, giving a mean inhibition of 29 ± 3 % (all means ± S.E.M., n = 3; P < 0.01).

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Figure 6. Effects of CDNB treatment on Ca²⁺ efflux from normal human red blood cells
Plasma membrane Ca²⁺ pump activity (PMCA) was measured following the method of Tiffert et al. (1993). Cells were loaded with ⁴⁵Ca²⁺ using the ionophore A23187 (10 µM) before addition of Co²⁺ (0.4 mM) to inhibit ionophore permeability. Symbols represent single determinations and are representative of four further experiments.

Finally, we examined the Ca²⁺ sensitivity of the Gardos channel (Fig. 7). Maximal activities (at [Ca²⁺]_i > 1 mM) were 185 ± 46 and 121 ± 35 mmol (l cells)^-1 h^-1 for control and CDNB-treated cells, respectively (P < 0.05). The mean EC₅₀ ([Ca²⁺]_i required for half-maximal activation) was 260 ± 26 and 175 ± 15 nM (means ± S.E.M., n = 3; P < 0.05) for control and CDNB-treated cells, respectively. There was therefore a 1.5 ± 0.2-fold increase in the sensitivity of the channel. Thus, for CDNB-treated cells, a [Ca²⁺]_i of ~80 nM would give a 20 % activation of the channel, corresponding to a K⁺ influx of about 24 mmol (l cells)^-1 h^-1.

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Figure 7. Effects of CDNB treatment on Ca²⁺ sensitivity of the Gardos channel in normal human red blood cells
Cells were incubated for 60 min in the presence or absence (Control) of CDNB (1 mM). They were then divided into six aliquots and treated with A23187 and combinations of EGTA and different [Ca²⁺]_o to provide the [Ca²⁺]_i indicated; r² = 1.6. Gardos channel activity was determined as the clotrimazole-sensitive (5 µM) K⁺ influx. Symbols represent triplicate determinations on a single sample, and are representative of three further experiments.

DISCUSSION

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Methods
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References

In this study, we have examined the effects of CDNB on passive K⁺ transport in human red cells, and attempted to define its mechanism of action. We show that CDNB stimulates both major passive K⁺ transport pathways (the Gardos channel and KCC) in normal and sickle human red cells. Stimulation of the Gardos channel was dependent on the presence of extracellular Ca²⁺ but independent of effects on passive Ca²⁺ transport. Increased activity was associated with inhibition of the plasma membrane Ca²⁺ pump and increased sensitivity of the channel to Ca²⁺. Stimulation of KCC by CDNB was inhibited by prior treatment with the protein phosphatase inhibitor calyculin A, but not on application after CDNB. The effect of CDNB was not additive with that of NEM. These findings suggest that inhibition of the regulatory protein kinase that normally inhibits KCC is a major site of action of CDNB.

Effect of CDNB on K⁺ transport

CDNB forms 2,4-dinitrophenyl-s-glutathione with GSH through a reaction that is catalysed by GSH-s-transferase (Awasthi et al. 1981; Chiu et al. 1993). Since GSH represents a major part of the antioxidant defence of the red cell (Board & Agar, 1983; Kurata et al. 1993; Edwards & Fuller, 1996; Sies, 1997), its depletion applies oxidative stress to the cells, with accumulation of metHb (Awasthi et al. 1981). A number of oxidants stimulate K⁺ transport in human red cells, including the activity of KCC (Olivieri et al. 1993). CDNB also perturbs the membrane permeability of red cells, notably stimulating passive K⁺ transport (Chiu et al. 1993; Zou et al. 2002). There is some discrepancy, however, as to whether these effects are mediated via stimulation of KCC, or the Gardos channel, or both (Lauf et al. 1995; Shartava et al. 2000). Recent reports on the activity of CDNB on HbS cells suggest that the cotransporter is unaffected and only the Gardos channel is stimulated (Shartava et al. 2000). In other species, however, CDNB has been observed to stimulate KCC (sheep - Lauf et al. 1995; horse - Muzyamba et al. 2000); the red blood cells of both of these species lack a Ca²⁺-activated K⁺ pathway. The cotransporter has a complicated regulatory apparatus involving protein (de)phosphorylation, which may vary between species, thereby accounting for differences in activity (Dunham et al. 1993; Flatman et al. 1996). Conversely, it may be that HbS cells differ from HbA cells. Since CDNB treatment has been used to assess the protection of cell dehydration by N-acetylcysteine (NAC; Shartava et al. 1999), it is important to investigate these possibilities. In addition, there are no reports on the interaction between CDNB and P_O2, which represents an important influence on KCC activity.

We show here that CDNB stimulates both the Gardos channel and KCC in HbA and HbS cells. Stimulation was present at all levels of P_O2 tested. In HbA cells, as in the horse red cell KCC (Muzyamba et al. 2000), the effect of CDNB is greater at high values of P_O2 and may correlate with development of metHb. In HbS cells, stimulation of KCC was as great in N₂ as in air, similar to that observed for reduction in pH, swelling and low concentrations of urea (J. Gibson et al. 1998). Previous reports suggesting a lack of action on KCC in HbS cells may be due to treatment at low temperature (4 °C), which would markedly disturb the enzymatic control of this transporter. Any protective effect of NAC in red cells from SCD patients must therefore be assessed on both transporters and at a range of P_O2 values.

Mechanism of action of CDNB

The Gardos channel is regulated mainly by [Ca²⁺]_i (Gardos, 1958), which is dependent upon the level of passive Ca²⁺ entry and the activity of the PMCA. Its Ca²⁺ sensitivity may also be variable (Lew & Ferreira, 1976) and can increase in response to protein phosphorylation or exposure to cytokines (e.g. Pellegrino & Pellegrini, 1998; Rivera et al. 2002). We show here that increased Gardos channel activity following CDNB treatment was not associated with increased Ca²⁺ influx; rather the PMCA was inhibited (by about 30 %) and the Ca²⁺ sensitivity of the channel increased (by about 1.5-fold). The measured EC₅₀ (of ~300 nM) is similar to that reported previously (Ishii et al. 1997; Rivera et al. 2002) and similar increases in Ca²⁺ sensitivity do occur (Rivera et al. 2002). In intact red cells, unlike in isolated red cell membranes, the PMCA is fairly resistant to inhibition (e.g. Tiffert et al. 2000; Raftos et al. 2001), but levels of inhibition similar to those reported here have been observed. Possible secondary effects of CDNB that might cause inhibition of the pump (intracellular acidification, cell shrinkage or ATP depletion: Gardos, 1958; Tiffert et al. 1993) were not present. The lack of inhibition of Na⁺-K⁺ pump activity also suggests that any privileged membrane pool (Hoffman, 1997) of ATP is also unaffected by CDNB. The insensitivity of Gardos channel activity to calyculin A and NEM also suggests that CDNB does not affect Ca²⁺ homeostasis through the regulatory protein (de)phosphorylation steps that control the KCC. The relatively small inhibitory effect of CDNB on PMCA, together with the increased Ca²⁺ sensitivity, may be sufficient to take [Ca²⁺]_i into the range required for the level of Gardos channel activation observed ([Ca²⁺]_i < 80 nM would give > 20 mmol K⁺ (l cells)^-1 h^-1). Conversely, it may be that only a subpopulation of cells is activated.

KCC activity is regulated by protein phosphorylation (Jennings & Al-Rohil, 1990; Dunham et al. 1993; Flatman et al. 1996). Our results are consistent with CDNB acting via this pathway to stimulate the cotransporter. Thus, CDNB stimulation was abolished by protein phosphatase inhibition, and application of NEM (which can act as a protein kinase inhibitor) did not increase CDNB-induced KCC activity. In the absence of changes in ATP levels, these findings imply that CDNB acts by protein kinase inhibition, as suggested previously in sheep red cells (Lauf et al. 1995). It may also stimulate conjugate protein phosphatase(s), as observed for NEM (Bize et al. 2000), but this cannot be the sole action, otherwise the KCC would remain sensitive to calyculin A after CDNB treatment.

Comparison of HbA and HbS cells

An additional aim of the present work was to compare the effects of CDNB on HbA and HbS cells, and the response of CDNB-treated HbA cells with normal HbS cells. We found that in air, the effect of CDNB was similar in both HbA and HbS cells, with stimulation of both Gardos channels and KCC. In HbS cells, the activity of KCC following CDNB treatment was similar in air and N₂. Gardos channel activity, which is already observed in deoxygenated control HbS cells, presumably following activation of the deoxygenation-induced cation pathway (P_sickle), was greater following treatment with CDNB. These findings represent important differences with HbA cells. GSH depletion, therefore (at least over the time course of the present experiments), cannot account for the difference in transport phenotype between normal and sickle red cells, since CDNB-induced KCC and Gardos channel activities were inhibited by deoxygenation in HbA, but not HbS, cells. In HbS cells, the additive stimulatory effects on the Gardos channel by deoxygenation and CDNB treatment may follow if CDNB treatment also inhibits PMCA and increases the Ca²⁺ sensitivity of the channel (as in HbA cells), coupled with increased Ca²⁺ entry via P_sickle.

Conclusion

This manuscript presents findings on the action of CDNB on both the KCC and Gardos channels in normal and sickle red cells. The site of action of CDNB that accounts for the effects on Gardos channels or the KCC cannot be deduced from the present results. We suggest, however, that it follows the oxidation of protein thiol groups, subsequent to GSH depletion. Future work will be aimed at the identification of possible sites of action.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

Awasthi YC, Gary HS, Dao DD & Partridge CA (1981). Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4-dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on haemoglobin. Blood 58, 733-738 [Abstract]

Beutler E, (1975). Red Cell Metabolism. A Manual of Biochemical Methods. Grune & Stratton, New York

Beutler E , & Duron O (1965). Effect of pH on preservation of red cell ATP. Transfusion 5, 17-22

Bize I, Güvenç B, Buchbinder G & Brugnara C (2000). Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg²⁺. J Membr Biol 177, 159-168 [Medline]

Board PG , & Agar NS (1983). Glutathione metabolism in erythrocytes. In Red Blood Cells of Domestic Mammals, ed. Agar NS & Board PG, pp. 253-270. Elsevier Science, Amsterdam

Borgese F, Motais R & Garcia-Romeu F (1991). Regulation of Cl dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells. Biochim Biophys Acta 1066, 252-256 [Medline]

Brugnara C, Bunn HF & Tosteson DC (1986). Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232, 388-390

Brugnara C, Kopin AS, Bunn HF & Tosteson DC (1985). Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease. J Clin Invest 75, 1608-1617 [Medline]

Canessa M, Spalvins A & Nagel RL (1986). Volume-dependent and NEM-stimulated K⁺, Cl^- transport is elevated in oxygenated SS, SC and CC human red cells. FEBS Lett 200, 197-202 [Medline]

Chan AC, Chow CK & Chiu D (1999). Interaction of antioxidants and their implication in genetic anaemia. Proc Soc Exp Biol Med 222, 274-282 [Abstract/Full Text]

Chiu DTY, Lai K-M, Xu C-M, Liour SSS, Lee J & Liu TZ (1993). Direct alteration of erythrocyte membrane properties by 1-chloro-2,4-dinitrobenzene without oxidative challenge. Exp Haematol 21, 114-118

Dunham PB , & Ellory JC (1981). Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride. J Physiol 318, 511-530 [Abstract]

Dunham PB, Klimczak J & Logue PJ (1993). Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process. J Gen Physiol 101, 733-766 [Abstract]

Eaton WA , & Hofrichter J (1987). Hemoglobin S gelation and sickle cell disease. Blood 70, 1245-1266 [Medline]

Edwards CJ , & Fuller J (1996). Oxidative stress in erythrocytes. Comp Haematol Int 6, 24-31

Ellory JC, Gibson JS & Stewart GW (1998). Pathophysiology of abnormal cell volume in human red cells. Contrib Nephrol 123, 220-239 [Medline]

Ellory JC, Player M, Chalder SM & Stuart J (1989). Rheological effect of activation of the KCl-cotransport pathway in normal and sickle erythrocytes. Clin Hemorheol 9, 1009-1016

Flatman PW, Adragna NC & Lauf PK (1996). Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport. Am J Physiol 271, C255-263 [Medline]

Garcia-Sancho J, (1985). Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell. Biochim Biophys Acta 813, 148-150 [Medline]

Gardos G, (1958). The function of calcium and potassium permeability of human erythrocytes. Biochim Biophys Acta 30, 653-654

Gibson JS , & Ellory JC (2002). Membrane transport in sickle cell disease. Blood Cells Mol Dis 28, 303-314 [Medline]

Gibson JS, Speake PF & Ellory JC (1998). Differential oxygen sensitivity of the K⁺-Cl^- cotransporter in normal and sickle human red blood cells. J Physiol 511, 225-234 [Abstract/Full Text]

Gibson XA, Shartava A, McIntyre J, Monteiro CA, Zhang Y, Shah A, Campbell NF & Goodman SR (1998). The efficacy of reducing agents or antioxidants in blocking the formation of dense cells and irreversibly sickled cells in vitro. Blood 91, 4373-4378 [Abstract/Full Text]

Gillen CM, Brill S, Payne JA & Forbusch IB (1996). Molecular cloning and functional expression of the KCl cotransporter from rabbit, rat and human. J Biol Chem 217, 16237-16244

Hebbel RP, Eaton JW, Balasingam M & Steinberg MH (1982). Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest 70, 1253-1259 [Medline]

Hebbel RP, Morgan WT, Eaton JW & Hedlund BE (1988). Accelerated autoxidation and heme loss due to instability of sickle hemoglobin. Proc Natl Acad Sci U S A 85, 237-241 [Medline]

Hegesh E, Gruener N, Cohen S, Bochkovsky R & Shuval HI (1970). A sensitive micromethod for the determination of methemoglobin in blood. Clin Chim Acta 30, 679-682 [Medline]

Hoffman JF, (1997). ATP compartmentalization in human erythrocytes. Curr Opin Hematol 4, 112-115 [Medline]

Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP & Maylie J (1997). A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A 94, 11651-11656 [Abstract/Full Text]

Jennings ML , & Al-Rohil N (1990). Kinetics of activation and inactivation of swelling-stimulated K/Cl transport: The volume-sensitive parameter is the rate constant for inactivation. J Gen Physiol 95, 1021-1040 [Abstract]

Joiner CH, (1993). Cation transport and volume regulation in sickle red blood cells. Am J Physiol 264, C251-270 [Medline]

Joiner CH, Dew A & Ge DL (1988). Deoxygenation-induced fluxes in sickle cells. I. Relationship between net potassium efflux and net sodium influx. Blood Cells 13, 339-348 [Medline]

Kurata M, Suzuki M & Agar NS (1993). Antioxidant systems and erythrocyte life-span in mammals. Comp Biochem Physiol B 106, 477-487 [Medline]

Lachant NA, Davidson WD & Tanaka KR (1983). Impaired pentose phosphate shunt function in sickle cell disease: a potential mechanism for increased Heinz body formation and membrane lipid peroxidation. Am J Hematol 15, 1-13 [Medline]

Lauf PK, Adragna NC & Agar NS (1995). Glutathione removal reveals kinases as common targets for K-Cl cotransport stimulation in sheep erythrocytes. Am J Physiol 269, C234-241 [Medline]

Lew VL , & Ferreira HG (1976). Variable Ca sensitivity of a K-selective channel in intact red-cell membranes. Nature 263, 336-338 [Medline]

Maridonneau I, Braquet P & Garay RP (1983). Na⁺ and K⁺ transport damage induced by oxygen free radicals in human red cell membranes. J Biol Chem 258, 3107-3113 [Medline]

Moore RB, Hulgan TM, Green JW & Jenkins LD (1992). Increased susceptibility of the sickle cell membrane Ca²⁺ + Mg²⁺-ATPase to t-butylhydroperoxide: protective effects of ascorbate and desferal. Blood 79, 1334-1341 [Abstract]

Muzyamba MC, Speake PF & Gibson JS (2000). Oxidants and regulation of KCl cotransport in equine red blood cells. Am J Physiol Cell Physiol 279, C981-989 [Abstract/Full Text]

Olivieri O, Bonollo M, Friso S, Girelli D, Corrocher R & Vettore L (1993). Activation of K⁺/Cl^- cotransport in human erythrocytes exposed to oxidative agents. Biochim Biophys Acta 1176, 37-42 [Medline]

Pellegrino CM, Rybicki AC, Musto S, Nagel RL & Schwartz RS (1998). Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells. Blood Cells Mol Dis 24, 31-40 [Medline]

Pellegrino M , & Pellegrini M (1998). Modulation of Ca²⁺-activated K⁺ channels of human erythrocytes by endogenous cAMP-dependent protein kinase. Pflugers Arch 436, 749-756 [Medline]

Raftos JE, Edgley A, Bookchin RM, Etzion Z, Lew VL & Tiffert T (2001). Normal Ca²⁺ extrusion by the Ca²⁺ pump of intact red blood cells exposed to high glucose concentrations. Am J Physiol Cell Physiol 280, C1449-1454 [Abstract/Full Text]

Rhoda MD, Apovo M, Beuzard Y & Giraud F (1990). Ca²⁺ permeability in deoxygenated sickle cells. Blood 75, 2453-2458 [Abstract]

Rice-Evans C, Omorphos SC & Baysal E (1986). Sickle cell membranes and oxidative damage. Biochem J 237, 265-269 [Medline]

Rivera A, Jarolim P & Brugnara C (2002). Modulation of Gardos channel activity by cytokines in sickle erythrocytes. Blood 99, 357-603 [Abstract/Full Text]

Shartava A, McIntyre J, Shah AK & Goodman SR (2000). The Gardos channel is responsible for CDNB-induced dense sickle cell formation. Am J Hematol 64, 184-189 [Medline]

Shartava A, Shah AK & Goodman SR (1999). N-acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1-chloro-2,4-dinitrobenzene. Am J Hematol 62, 19-24 [Medline]

Sies H, (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82, 291-295 [Medline]

Stein WD, (1986). Transport and Diffusion Across Cell Membranes. Academic Press, Orlando

Tiffert T, Etzion Z, Bookchin RM & Lew VL (1993). Effects of deoxygenation on active and passive Ca²⁺ transport and cytoplasmic Ca²⁺ buffering in normal human red cells. J Physiol 464, 529-544 [Abstract]

Tiffert T, Garcia-Sancho J & Lew VL (1984). Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta 773, 143-156 [Medline]

Tiffert T, Lew VL, Perdomo D & Ginsburg H (2000). Effect of ferriprotoporphyrin IX and non-heme iron on the Ca²⁺ pump activity of intact human red cells. J Membr Biol 175, 107-113 [Medline]

Zou CG, Agar NS & Jones GL (2002). Chlorodinitrobenzene-mediated damage in the human erythrocyte membrane leads to haemolysis. Life Sci 71, 735-746 [Medline]

Acknowledgements

This work was supported by Action Research and The Wellcome Trust.

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Awasthi YC, Gary HS, Dao DD & Partridge CA (1981). Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4-dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on haemoglobin. Blood 58, 733-738	[Abstract]
Beutler E, (1975). Red Cell Metabolism. A Manual of Biochemical Methods. Grune & Stratton, New York
Beutler E , & Duron O (1965). Effect of pH on preservation of red cell ATP. Transfusion 5, 17-22
Bize I, Güvenç B, Buchbinder G & Brugnara C (2000). Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg²⁺. J Membr Biol 177, 159-168	[Medline]
Board PG , & Agar NS (1983). Glutathione metabolism in erythrocytes. In Red Blood Cells of Domestic Mammals, ed. Agar NS & Board PG, pp. 253-270. Elsevier Science, Amsterdam
Borgese F, Motais R & Garcia-Romeu F (1991). Regulation of Cl dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells. Biochim Biophys Acta 1066, 252-256	[Medline]
Brugnara C, Bunn HF & Tosteson DC (1986). Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232, 388-390
Brugnara C, Kopin AS, Bunn HF & Tosteson DC (1985). Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease. J Clin Invest 75, 1608-1617	[Medline]
Canessa M, Spalvins A & Nagel RL (1986). Volume-dependent and NEM-stimulated K⁺, Cl^- transport is elevated in oxygenated SS, SC and CC human red cells. FEBS Lett 200, 197-202	[Medline]
Chan AC, Chow CK & Chiu D (1999). Interaction of antioxidants and their implication in genetic anaemia. Proc Soc Exp Biol Med 222, 274-282	[Abstract/Full Text]
Chiu DTY, Lai K-M, Xu C-M, Liour SSS, Lee J & Liu TZ (1993). Direct alteration of erythrocyte membrane properties by 1-chloro-2,4-dinitrobenzene without oxidative challenge. Exp Haematol 21, 114-118
Dunham PB , & Ellory JC (1981). Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride. J Physiol 318, 511-530	[Abstract]
Dunham PB, Klimczak J & Logue PJ (1993). Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process. J Gen Physiol 101, 733-766	[Abstract]
Eaton WA , & Hofrichter J (1987). Hemoglobin S gelation and sickle cell disease. Blood 70, 1245-1266	[Medline]
Edwards CJ , & Fuller J (1996). Oxidative stress in erythrocytes. Comp Haematol Int 6, 24-31
Ellory JC, Gibson JS & Stewart GW (1998). Pathophysiology of abnormal cell volume in human red cells. Contrib Nephrol 123, 220-239	[Medline]
Ellory JC, Player M, Chalder SM & Stuart J (1989). Rheological effect of activation of the KCl-cotransport pathway in normal and sickle erythrocytes. Clin Hemorheol 9, 1009-1016
Flatman PW, Adragna NC & Lauf PK (1996). Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport. Am J Physiol 271, C255-263	[Medline]
Garcia-Sancho J, (1985). Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell. Biochim Biophys Acta 813, 148-150	[Medline]
Gardos G, (1958). The function of calcium and potassium permeability of human erythrocytes. Biochim Biophys Acta 30, 653-654
Gibson JS , & Ellory JC (2002). Membrane transport in sickle cell disease. Blood Cells Mol Dis 28, 303-314	[Medline]
Gibson JS, Speake PF & Ellory JC (1998). Differential oxygen sensitivity of the K⁺-Cl^- cotransporter in normal and sickle human red blood cells. J Physiol 511, 225-234	[Abstract/Full Text]
Gibson XA, Shartava A, McIntyre J, Monteiro CA, Zhang Y, Shah A, Campbell NF & Goodman SR (1998). The efficacy of reducing agents or antioxidants in blocking the formation of dense cells and irreversibly sickled cells in vitro. Blood 91, 4373-4378	[Abstract/Full Text]
Gillen CM, Brill S, Payne JA & Forbusch IB (1996). Molecular cloning and functional expression of the KCl cotransporter from rabbit, rat and human. J Biol Chem 217, 16237-16244
Hebbel RP, Eaton JW, Balasingam M & Steinberg MH (1982). Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest 70, 1253-1259	[Medline]
Hebbel RP, Morgan WT, Eaton JW & Hedlund BE (1988). Accelerated autoxidation and heme loss due to instability of sickle hemoglobin. Proc Natl Acad Sci U S A 85, 237-241	[Medline]
Hegesh E, Gruener N, Cohen S, Bochkovsky R & Shuval HI (1970). A sensitive micromethod for the determination of methemoglobin in blood. Clin Chim Acta 30, 679-682	[Medline]
Hoffman JF, (1997). ATP compartmentalization in human erythrocytes. Curr Opin Hematol 4, 112-115	[Medline]
Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP & Maylie J (1997). A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A 94, 11651-11656	[Abstract/Full Text]
Jennings ML , & Al-Rohil N (1990). Kinetics of activation and inactivation of swelling-stimulated K/Cl transport: The volume-sensitive parameter is the rate constant for inactivation. J Gen Physiol 95, 1021-1040	[Abstract]
Joiner CH, (1993). Cation transport and volume regulation in sickle red blood cells. Am J Physiol 264, C251-270	[Medline]
Joiner CH, Dew A & Ge DL (1988). Deoxygenation-induced fluxes in sickle cells. I. Relationship between net potassium efflux and net sodium influx. Blood Cells 13, 339-348	[Medline]
Kurata M, Suzuki M & Agar NS (1993). Antioxidant systems and erythrocyte life-span in mammals. Comp Biochem Physiol B 106, 477-487	[Medline]
Lachant NA, Davidson WD & Tanaka KR (1983). Impaired pentose phosphate shunt function in sickle cell disease: a potential mechanism for increased Heinz body formation and membrane lipid peroxidation. Am J Hematol 15, 1-13	[Medline]
Lauf PK, Adragna NC & Agar NS (1995). Glutathione removal reveals kinases as common targets for K-Cl cotransport stimulation in sheep erythrocytes. Am J Physiol 269, C234-241	[Medline]
Lew VL , & Ferreira HG (1976). Variable Ca sensitivity of a K-selective channel in intact red-cell membranes. Nature 263, 336-338	[Medline]
Maridonneau I, Braquet P & Garay RP (1983). Na⁺ and K⁺ transport damage induced by oxygen free radicals in human red cell membranes. J Biol Chem 258, 3107-3113	[Medline]
Moore RB, Hulgan TM, Green JW & Jenkins LD (1992). Increased susceptibility of the sickle cell membrane Ca²⁺ + Mg²⁺-ATPase to t-butylhydroperoxide: protective effects of ascorbate and desferal. Blood 79, 1334-1341	[Abstract]
Muzyamba MC, Speake PF & Gibson JS (2000). Oxidants and regulation of KCl cotransport in equine red blood cells. Am J Physiol Cell Physiol 279, C981-989	[Abstract/Full Text]
Olivieri O, Bonollo M, Friso S, Girelli D, Corrocher R & Vettore L (1993). Activation of K⁺/Cl^- cotransport in human erythrocytes exposed to oxidative agents. Biochim Biophys Acta 1176, 37-42	[Medline]
Pellegrino CM, Rybicki AC, Musto S, Nagel RL & Schwartz RS (1998). Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells. Blood Cells Mol Dis 24, 31-40	[Medline]
Pellegrino M , & Pellegrini M (1998). Modulation of Ca²⁺-activated K⁺ channels of human erythrocytes by endogenous cAMP-dependent protein kinase. Pflugers Arch 436, 749-756	[Medline]
Raftos JE, Edgley A, Bookchin RM, Etzion Z, Lew VL & Tiffert T (2001). Normal Ca²⁺ extrusion by the Ca²⁺ pump of intact red blood cells exposed to high glucose concentrations. Am J Physiol Cell Physiol 280, C1449-1454	[Abstract/Full Text]
Rhoda MD, Apovo M, Beuzard Y & Giraud F (1990). Ca²⁺ permeability in deoxygenated sickle cells. Blood 75, 2453-2458	[Abstract]
Rice-Evans C, Omorphos SC & Baysal E (1986). Sickle cell membranes and oxidative damage. Biochem J 237, 265-269	[Medline]
Rivera A, Jarolim P & Brugnara C (2002). Modulation of Gardos channel activity by cytokines in sickle erythrocytes. Blood 99, 357-603	[Abstract/Full Text]
Shartava A, McIntyre J, Shah AK & Goodman SR (2000). The Gardos channel is responsible for CDNB-induced dense sickle cell formation. Am J Hematol 64, 184-189	[Medline]
Shartava A, Shah AK & Goodman SR (1999). N-acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1-chloro-2,4-dinitrobenzene. Am J Hematol 62, 19-24	[Medline]
Sies H, (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82, 291-295	[Medline]
Stein WD, (1986). Transport and Diffusion Across Cell Membranes. Academic Press, Orlando
Tiffert T, Etzion Z, Bookchin RM & Lew VL (1993). Effects of deoxygenation on active and passive Ca²⁺ transport and cytoplasmic Ca²⁺ buffering in normal human red cells. J Physiol 464, 529-544	[Abstract]
Tiffert T, Garcia-Sancho J & Lew VL (1984). Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta 773, 143-156	[Medline]
Tiffert T, Lew VL, Perdomo D & Ginsburg H (2000). Effect of ferriprotoporphyrin IX and non-heme iron on the Ca²⁺ pump activity of intact human red cells. J Membr Biol 175, 107-113	[Medline]
Zou CG, Agar NS & Jones GL (2002). Chlorodinitrobenzene-mediated damage in the human erythrocyte membrane leads to haemolysis. Life Sci 71, 735-746	[Medline]

				I. Petrushanko, N. Bogdanov, E. Bulygina, B. Grenacher, T. Leinsoo, A. Boldyrev, M. Gassmann, and A. Bogdanova Na-K-ATPase in rat cerebellar granule cells is redox sensitive Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R916 - R925. [Abstract] [Full Text] [PDF]

				A. Bogdanova, B. Grenacher, M. Nikinmaa, and M. Gassmann Hypoxic responses of Na+/K+ ATPase in trout hepatocytes J. Exp. Biol., May 15, 2005; 208(10): 1793 - 1801. [Abstract] [Full Text] [PDF]

				C. H. Joiner, R. K. Rettig, M. Jiang, and R. S. Franco KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes Blood, November 1, 2004; 104(9): 2954 - 2960. [Abstract] [Full Text] [PDF]