Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+ exchange

C. H. Joiner1,2, M. Jiang1,2, H. Fathallah3, F. Giraud3, and R. S. Franco1,4

1 Cincinnati Comprehensive Sickle Cell Center and Divisions of 2 Pediatric and 4 Medical Hematology/Oncology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039; and 3 Laboratoire de Biomembranes et Messagers Cellulaires, Centre National de la Recherche Scientifique ERS 571, Université Paris XI, Orsay, France

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

KCl cotransport activated by swelling of sickle red blood cells (SS RBC) is inhibited by deoxygenation. Yet recent studies found a Cl--dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl--dependent component of the deoxygenation-induced net K+ efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl--dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+ exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.

potassium; sodium; erythrocyte; reticulocyte; volume regulation

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

DEHYDRATION OF RED BLOOD CELLS containing sickle hemoglobin (Hb S) (SS RBC) contributes to the pathophysiology of sickling because of the high dependence of polymerization on the concentration of Hb S (19). Although the mechanism of dehydration in vivo is not entirely clear, cation loss from sickle cells in vitro appears to involve both sickling-dependent and sickling-independent mechanisms (35).

Sickling-dependent cation loss results from changes in membrane permeability to Na+, K+, and Ca2+ associated with formation of membrane spicules induced by ordered Hb S polymer. In the presence of physiological external Ca2+ concentrations, sickling-induced K+ loss exceeds Na+ gain, as a result of three different mechanisms: 1) imbalance between Na+ influx and K+ efflux via the sickling-induced pathway per se (4, 37), 2) unbalanced compensation of the Na+/K+ pump (40), and 3) activation of Ca2+-dependent K+ channels by Ca2+ influx via the sickling-induced pathway (4, 6). The relative contribution of these pathways to SS RBC dehydration in vivo is not known and may, in fact, vary among different patients and among SS RBC subpopulations in the same patient.

Sickling-independent cation loss from SS RBC may be mediated by the KCl cotransporter, a volume regulatory pathway exhibiting high activity in reticulocytes and capable of rapid K+ efflux (5, 10). The cotransporter probably contributes to the volume reduction that accompanies normal reticulocyte maturation. KCl cotransport activity is markedly increased in SS RBC (5), perhaps as a consequence of the young age of these cells (10) or possibly secondary to specific interactions of Hb S with the transporter or its regulators (47). The importance of KCl cotransport in dehydration of SS reticulocytes was suggested by our recent finding that reticulocytes that had been dehydrated in vivo had higher apparent KCl cotransport activity than reticulocytes that had remained normally hydrated in vivo (28).

Under isotonic conditions at pH 7.3-7.4, no Cl--dependent component of K+ loss from oxygenated SS RBC can be demonstrated (7, 10, 36, 39). KCl cotransport is known to be activated in swollen or acidified cells by mechanisms involving dephosphorylation of the transporter or its regulator(s) (3, 25, 32, 33). KCl cotransport activation in SS RBC has generally been considered independent of sickling because deoxygenation inhibits swelling-activated KCl cotransport (11). Binding to deoxygenated Hb of the major cellular Mg2+ buffers, 2,3-diphospho-D-glycerate and ATP, results in increased intracellular Mg2+ (24, 48), which inhibits the activated cotransporter (8, 18, 43, 49). In this context, it was not surprising that previous studies found no Cl--dependent K+ efflux in SS RBC subjected to prolonged, continuous deoxygenation (2, 39). These results were difficult to reconcile, however, with later studies showing that the K+ efflux and/or density changes of SS RBC subjected to cyclic deoxygenation under isotonic conditions at normal pH were inhibited by the KCl cotransport inhibitor [(dihydroindenyl)oxy]alkanoic acid (DIOA) (1, 46, 52). Recently we also reported that SS RBC reticulocytes exhibited a significant Cl--dependent change in density when subjected to cyclic deoxygenation in vitro, but not under continuous deoxygenation or oxygenation (29).

All of these findings could be explained by a model in which deoxygenation induces a change in the phosphorylation-dephosphorylation equilibrium of the cell, thereby activating KCl cotransport. Such a deoxygenation-induced change in phosphorylation was recently reported for several red cell membrane proteins and shown to result from activation of okadaic acid-sensitive phosphatase(s) (22). The model predicts that the "activated" cotransporter would, nevertheless, be inhibited by elevated internal Mg2+ during deoxygenation. On reoxygenation, with lowering of cellular Mg2+, the activated KCl cotransporter would be released from inhibition and mediate K+ and Cl- efflux until the slower process of deactivation of the transporter occurred. The present study was undertaken to test the hypothesis that the KCl cotransporter is activated by deoxygenation, by measuring transport under conditions that controlled cellular Mg2+. In addition, we examined whether the Na+/H+ exchanger could contribute to the deoxygenation-induced Na+ influx, since deoxygenation may produce an elevation of cytosolic Ca2+ (21) as well as cell shrinkage, both of which could result in the activation of Na+/H+ exchange (12, 20). A preliminary report of these studies has appeared in abstract form (38).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Incubation media. Unless otherwise noted, chemicals were obtained from Sigma (St. Louis, MO) and were reagent grade or better. HEPES-buffered saline (HBS) contained (in mM) 140 NaCl, 20 HEPES (pH 7.4 at 37°C with NaOH), 0.1 EDTA, and 10 glucose. In experiments in which cellular Mg2+ was manipulated with A-23187, EGTA replaced EDTA, and total external Mg2+ concentration was 0.15 mM, unless indicated otherwise. This concentration of external Mg2+ has been shown to maintain constant internal Mg2+ and cell volume in ionophore-treated RBC (8, 26). HEPES-buffered K+ medium and Rb+ medium were identical to HBS, except that K+ and Rb+ salts, respectively, replaced Na+ salts. In NO-3 media, NO-3 salts replaced Cl- salts. Ouabain was present at 0.1 mM in all solutions. DIOA and okadaic acid were purchased from Research Biochemicals International (Natick, MA).

Blood samples and density separations. After informed consent, blood was obtained by venipuncture into heparinized tubes from volunteers homozygous for Hb S. Unless otherwise stated, all experiments were performed on the least dense 15-25% of the SS RBC population, since these cells have the highest KCl cotransport activity (5, 10). Fresh whole blood was washed in HBS at 4°C without removing the buffy coat and then applied to discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradients. After centrifugation at 3,000 g for 20 min, cells with density (partial ) < 1.085 were isolated as described previously (28, 30), washed, and filtered through glass wool to remove white blood cells. When stored overnight, cells were suspended in HEPES-buffered solution containing 15 mM NaCl and 125 mM KCl. Spun hematocrit values were measured on oxygenated samples in microhematocrit tubes after centrifugation for 5 min at 13,000 g in a hematocrit centrifuge.

Cellular cation measurements. Cellular cations were measured by methods described in detail previously (37, 39). Briefly, samples containing 5-10 µl of cells were taken into iced tubes containing 110 mM MgCl2 layered over dibutyl phthalate (Fisher, Pittsburgh, PA). Cells exposed to ionophore were not washed before exposure to MgCl2, but there was no indication of cell swelling or hemolysis before centrifugation through phthalate oil. There was also no difference in Na+ or K+ fluxes in NO-3 media between cells incubated with or without ionophore, indicating that the short exposure of cells to the combination of isotonic MgCl2 and ionophore at 0°C did not affect the monovalent cation content of the cells. After the tubes had been washed, the oil was removed and the cells were lysed for analysis of cations by flame emission spectroscopy (Perkin-Elmer model 370 atomic absorption spectrophotometer, Norwalk, CT). Hemoglobin was measured at 540 nm using a Beckman DU spectrophotometer (Beckman Instruments). Cation content was calculated as millimoles per kilogram hemoglobin; all cation measurements were made in triplicate, unless otherwise noted.

Net cation fluxes. As in previous studies (37-40), SS RBC were suspended at 2% hematocrit in appropriate media, and ionophore or drugs were added as needed in stock solutions in DMSO. DMSO was present in a final concentration of 0.4-1 vol% and was added to controls. Cells were warmed to 37°C for 10 min, and an initial cation sample was taken. Oxygenated samples were capped, whereas deoxygenated samples were flushed with humidified N2 for 1 h, after which the flasks remained sealed. This deoxygenation protocol did not change the osmolality of media, indicating that N2 humidification was adequate. A second cation sample was taken after 2 h of incubation, and the net flux was calculated from the change in cation content.

Na+/H+ exchange measurements. Blood was centrifuged on Percoll gradients, and a density fraction (1.076 < partial  < 1.106) was isolated. Cells were washed in solution A [in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 NaH2PO4-Na2HPO4 (20:80), 10 HEPES-Tris (pH 7.4 at 37°C), and 10 glucose] and stored at 4°C for 12-36 h. Just before the experiments, cells were washed once in solution A and incubated at 15% hematocrit at 37°C for 45 min in the same solution containing 2 mM adenine and 10 mM inosine. Cells were then washed four times in solution B (solution A without PO3-4 and supplemented with 1 mM CaCl2, 0.1 mM ouabain, and 0.01 mM bumetanide) and resuspended in the same solution at 10% hematocrit. Cells were incubated at 37°C for 5 min with 40 µM dimethyl amiloride (DMA) and then for 10 min with 0.4 µM phorbol 12-myristate 13-acetate (PMA). DMSO from the stock solutions of DMA and PMA was present at a final concentration of 0.4-0.8 vol% and was added to control suspensions. Cells were then either kept oxygenated or flushed with humidified N2, as described previously (22, 23). After 90 min, cells were washed twice in a solution containing 110 mM MgCl2 and 10 mM HEPES-Tris (pH 7.4 at 4°C). Pellets were lysed, and lysate was analyzed for hemoglobin and Na+.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cl- dependence of K+ efflux in deoxygenated SS RBC. Figure 1 depicts a series of experiments in which ouabain-treated SS RBC were incubated under oxygenated or deoxygenated conditions in either Cl- or NO-3 media and net fluxes of Na+ and K+ were measured. Figure 1A depicts cells under control conditions (without ionophore). In oxygenated cells there was no Cl--dependent transport of either Na+ or K+; fluxes in NO-3 exceed those in Cl-, as reported by others (7, 39). On deoxygenation, both Na+ and K+ fluxes increased, as expected from previous studies (2, 4, 37, 39), and Na+ influx was independent of Cl-. K+ efflux, however, was 12.5% higher in Cl- compared with NO-3 media (P < 0.04), suggesting KCl cotransport activity stimulated by deoxygenation. This result contrasts with previous findings from our laboratory (36, 39) and by others (2) that there was no Cl--dependent component to the K+ efflux in deoxygenated SS RBC. This difference may arise from the fact that the current studies utilized a low-density fraction of SS RBC (least dense 25-50% of cells) containing a higher number of young cells with high KCl cotransport activity (10), whereas previous studies used unfractionated cells. In addition, the deoxygenation-induced K+ efflux (difference between fluxes in deoxygenated and oxygenated cells) in this fraction exceeded deoxygenation-induced Na+ influx, which is also in contrast to previous results obtained in unfractionated SS RBC showing balanced sickling-induced Na+ and K+ fluxes (2, 36, 39, 40). The activation of KCl cotransport in this low-density fraction appears to account for this difference.

Figure 1B depicts parallel incubations in the presence of the ionophore A-23187, which causes Mg2+ to distribute across the membrane according to the membrane potential, so that free internal Mg2+ is approximately twice the external concentration (26). In the experiments in Fig. 1, external Mg2+ was 0.15 mM, which in the presence of ionophore "clamps" internal Mg2+ at normal levels in oxygenated cells (8, 26) and prevents the increase in cellular free Mg2+ associated with deoxygenation (11, 24, 26, 48). There was no effect of ionophore on Na+ and K+ fluxes in oxygenated cells, as expected, and in deoxygenated cells, no Cl- dependence of Na+ influx was noted. However, K+ efflux from deoxygenated SS RBC under these conditions was 34% higher in Cl- media compared with NO-3 media (P < 0.0001), whereas K+ efflux from deoxygenated SS RBC in NO-3 media was the same without and with ionophore (means ± SD, 33.9 ± 4.9 vs. 34.5 ± 6.5 mmol · kg Hb-1 · h-1). Thus the Cl--dependent component of the K+ efflux in deoxygenated SS RBC was increased in the presence of ionophore. In this series of experiments, the Cl--dependent K+ efflux in deoxygenated SS RBC without ionophore (Fig. 1A) averaged 5.4 ± 3.8 mmol · kg Hb-1 · h-1, which is rather small compared with the total K+ efflux (38.3 ± 4.9 mmol · kg Hb-1 · h-1). However, when internal Mg2+ was clamped in the presence of A-23187 (Fig. 1B), Cl--dependent K+ efflux was stimulated to 12.4 ± 5.2 mmol · kg Hb-1 · h-1 (P < 0.005 vs. ionophore-free control). To establish that this Cl--dependent component of the K+ efflux in deoxygenated SS RBC was mediated by KCl cotransport, the effects of pharmacological inhibitors of this transport system were examined.


View larger version (26K):
[in this window]
[in a new window]
 


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of deoxygenation and cellular Mg2+ clamping on net Na+ and K+ fluxes in sickle cells. Cells were incubated at 2% hematocrit under oxygenating (Oxy) or deoxygenating (Deoxy) conditions in media containing 0.1 mM ouabain, and net cation movements were calculated over 2 h. External Mg2+ was 0.15 mM and 0.1 mM EGTA was present. A: cells incubated without ionophore. B: cells incubated with A-23187 (20 µmol/l cells). Paired samples in Cl- [HEPES-buffered saline (HBS)] or NO-3 [HEPES-buffered NaNO3 (HBN)] media were subjected to all conditions. Data are means ± SD of 9 separate experiments. Statistical analysis of K+ efflux in deoxygenated cells was by paired t-test: without A-23187 (A), Cl- vs. NO-3 media, P < 0.04; with A-23187 (B), Cl- vs. NO-3 media, P < 0.0001.

Cl--dependent K+ efflux stimulated by deoxygenation is blocked by okadaic acid and DIOA. In a separate series of experiments of similar design, we tested the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor that blocks the swelling-induced activation of KCl cotransport (33), and DIOA, a compound that blocks the cotransport pathway (46, 52). Figure 2 shows net K+ efflux in deoxygenated SS RBC. The small Cl--dependent component of K+ efflux in deoxygenated SS RBC was apparent in the control (no drug) cells without ionophore; this was not present in oxygenated cells (not shown) and was therefore stimulated by deoxygenation. This Cl--dependent K+ efflux was augmented by clamping Mg2+, as before, and was completely blocked by both okadaic acid and DIOA, supporting its mediation by the KCl cotransporter.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of KCl cotransport inhibitors on K+ efflux in deoxygenated sickle cell red blood cells (SS RBC). Cells were incubated with or without ionophore A-23187, as described for Fig. 1, in either Cl- (HBS) or NO-3 (HBN) media. When present, [(dihydroindenyl)oxy]alkanoic acid (DIOA) concentration was at 50 µM and okadaic acid (OA) at 10 µM, and net fluxes were measured; total net K+ efflux in deoxygenated cells is shown as means ± SD of 6 experiments. Inhibitors had no effect on Na+ or K+ fluxes in oxygenated cells or on Na+ influx in deoxygenated cells (not shown). Statistical analysis was by paired t-test without correction for multiple testing. Without A-23187, Cl- vs. NO-3 media, P < 0.006; with A-23187, Cl- vs. NO-3 media, P < 0.006. Drug effects without ionophore (Cl- media): DIOA vs. Control, P < 0.001; OA vs. Control, P < 0.002. Drug effects with ionophore (Cl- media): DIOA vs. Control, P < 0.0001; OA vs. Control, P < 0.0001.

In Fig. 3, the data have been recalculated, by subtracting the flux in oxygenated cells from that in deoxygenated cells, to yield the deoxygenation-induced K+ efflux (39). In control samples of these low-density SS RBC fractions, a portion of the deoxygenation-induced K+ flux was Cl- dependent without ionophore, and this Cl--dependent component was augmented by clamping cellular Mg2+ at oxygenated levels with ionophore. The Cl--dependent component of these deoxygenation-induced fluxes was abolished by DIOA and okadaic acid both with and without ionophore.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of KCl cotransport inhibitors on deoxygenation-induced K+ efflux. Data from Fig. 2 were recalculated to yield deoxygenation-induced flux by subtracting flux in oxygenated cells from that in deoxygenated cells for each condition. Effects of DIOA and OA are shown in cells without and with A-23187.

Although not shown in Fig. 3, the deoxygenation-induced K+ efflux in NO-3 media was equivalent to deoxygenation-induced Na+ influx under all conditions, as previously described (2, 37), and is presumably mediated by the passive, diffusional pathway activated by sickling and inhibited by stilbenes (2, 34, 39, 40). Although the term "deoxygenation-induced" has been used to describe this pathway, the current data demonstrate that, in low-density SS RBC at least, the deoxygenation-induced K+ efflux consists of two components: one mediated by KCl cotransport stimulated by deoxygenation and another mediated by the sickling-induced leak pathway.

Stimulation of KCl cotransport by deoxygenation is not a consequence of the Bohr effect. The Bohr effect of hemoglobin (9) produces two changes in RBC on deoxygenation that might have secondary effects on KCl cotransport. Cellular pH is increased by 0.05-0.10 pH units (24), but this should inhibit, rather than stimulate, the system (7, 43, 49). The reduced negative charge on deoxygenated Hb leads to Cl- uptake and slight cell swelling on deoxygenation. To assess the effect of deoxygenation on cell volume, hematocrit was measured on paired suspensions of normal (AA) RBC subjected to both oxygenated and deoxygenated conditions, taking care to ensure that cells remained deoxygenated during measurement. The cell volume of deoxygenated cells relative to oxygenated cells, calculated as the ratio of hematocrit values, was 1.008 ± 0.0004 (mean ± SD; n = 3). Such a change in volume of <1% produces minimal activation of KCl cotransport (41) and is insufficient to account for the magnitude of the stimulatory effect of deoxygenation.

Cl--dependent K+ efflux stimulated by deoxygenation is a function of Mg2+. We predicted that, if the effect of A-23187 to augment deoxygenation stimulation of KCl cotransport activity in SS RBC is due to clamping of cellular Mg2+ at the normal oxygenated level, raising external Mg2+ in A-23187-treated cells would blunt the deoxygenation stimulation. To test this prediction, SS RBC were deoxygenated as before in the presence of A-23187 at total external Mg2+ concentrations of 0.15 and 1.5 mM. Figure 4 illustrates that at 0.15 mM Mg2+ there was a marked stimulation of Cl--dependent K+ efflux in deoxygenated cells, as was apparent in previous experiments (Fig. 1). At high Mg2+ (1.5 mM), however, the stimulation of the Cl--dependent component of the K+ efflux in deoxygenated SS RBC was markedly reduced compared with stimulation at 0.15 mM Mg2+. These data are consistent with the notion that increased cellular Mg2+ in deoxygenated SS RBC (in the absence of ionophore) counteracts the stimulation of KCl cotransport activity by partially inhibiting the transporter under continuous deoxygenation.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of Mg2+ on K+ efflux in SS RBC treated with ionophore A-23187. Cells were incubated with ionophore under oxygenated and deoxygenated conditions as described in Fig. 1. Total external Mg2+ concentrations are indicated; 0.1 mM EGTA was present to chelate external Ca2+. There was no effect of Mg2+ on Na+ influx under these conditions (not shown).

The data of Fig. 4 are also consistent with previously reported effects of Mg2+ and other divalent cations on swelling-induced KCl cotransport (8, 11, 18, 43, 49). This effect is illustrated in Fig. 5A. Cells were incubated in hypotonic solutions (220 mosM), and net K+ efflux was measured over 1 h in Cl- or NO-3 media. Cellular Mg2+ was clamped at various levels, dictated by the external Mg2+ concentrations shown in Fig. 5; Flatman and Lew (26) have shown that internal Mg2+ equilibrates at approximately twice the concentration of external Mg2+ under these conditions. The lower curve of Fig. 5A shows the flux from cells incubated in hypotonic NaCl (94 mM Na+, 5.6 mM K+), whereas the upper curve of Fig. 5A and the curve in Fig. 5B represent cells incubated in hypotonic RbCl (94 mM Rb+, 5.6 mM K+). In hypotonic NaCl, KCl cotransport was highest at nominal concentrations of zero Mg2+, with progressive inhibition even at external Mg2+ <0.15 mM, which approximates normal equilibrium values in oxygenated cells (26, 48); these findings are similar to those reported by Brugnara and Tosteson (8) in human RBC and by Lauf and colleagues (18, 43) in low-K+ sheep RBC.


View larger version (13K):
[in this window]
[in a new window]
 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of Mg2+ on swelling-stimulated K+ efflux in SS RBC treated with ionophore A-23187. SS RBC were washed in isotonic HEPES-buffered KCl (HBK; or in corresponding KNO3 buffer) and resuspended at 55% hematocrit. To begin flux, an aliquot of suspension was added to warmed hypotonic medium containing A-23187 (20 µmol/l cells) and the concentrations of external Mg2+ shown. Final osmolarity was 220 mosM. After addition of cells, hypotonic HBS (hypo NaCl) contained 94 mM Na+ and 5.6 mM K+ (+ 20 mM HEPES, 0.1 mM EGTA, 10 mM glucose, and 0.1 mM ouabain); hypo RbCl was identical except that Rb+ replaced Na+. In NO-3 media, NO-3 salts replaced Cl- salts of Na+ and Rb+. K+ efflux and Rb+ influx were calculated from cellular cation measurements made before addition of cells to hypotonic buffers, and after 1 h of incubation. A: K+ efflux in cells in hypotonic Na+ and Rb+ buffers. B: Rb+ influx measured simultaneously in cells in hypotonic Rb+ buffers. Fluxes in NO-3 media (hypotonic NaNO3 or hypotonic RbNO3) were measured at 0 Mg2+. Data are means of triplicate measurements in 1 experiment representative of 3 others; SD, where not shown, were smaller than symbols.

Results from SS RBC in hypotonic high-Rb+ media are depicted in Fig. 5, A and B. Unlike Na+, Rb+ substitutes for K+ as a transported ion on the KCl cotransporter and has been shown to stimulate K+ efflux via the transporter in human RBC (41). This trans-stimulation is apparent in hypotonic RbCl media, in which cells show higher K+ efflux at all Mg2+ concentrations than in hypotonic NaCl (Fig. 5A). A very different response to Mg2+ is also noted in high-Rb+ media: the swelling-induced K+ efflux is less sensitive to increasing Mg2+ concentrations, including the range of external Mg2+ concentrations (0.1-0.2 mM) that equilibrate with physiological internal Mg2+ levels in oxygenated cells (26). On deoxygenation, RBC Mg2+ levels increase ~50% (24, 48). Because 0.15 mM external Mg2+ reflects the equilibrium concentration in oxygenated cells, deoxygenation would be expected to produce a cellular Mg2+ concentration corresponding to an external concentration of 0.2-0.3 mM. As shown in Fig. 5A, this range of concentrations (0.15-0.3 mM) has a profound effect on swelling-activated K+ efflux in low-K+ medium but a much smaller effect in high-Rb+ medium. A similar Mg2+ dependence is noted for Rb+ influx measured simultaneously in the same cells, as shown in Fig. 5B. Thus swelling-activated K+ efflux and Rb+ influx mediated by the KCl cotransporter are less susceptible to Mg2+ inhibition in high-Rb+(K+) buffers. Therefore, if activation of the KCl cotransporter in SS RBC by continuous deoxygenation is partially masked by elevated Mg2+ under physiological conditions, then such stimulation should be apparent without Mg2+ clamping in cells incubated in high-Rb+(K+) buffers. This prediction was borne out in experiments presented below.

KCl cotransport activity in isotonic high-Rb+(K+) buffers is stimulated by deoxygenation of SS RBC without manipulating cellular Mg2+. Figure 6 depicts an experiment in which Rb+ influx was measured in SS RBC incubated under oxygenated and deoxygenated conditions (without addition of ionophore) in buffers containing high external Rb+. Cells were deoxygenated in isotonic HBS or HEPES-buffered NaNO3 medium containing 0.1 mM ouabain and 0.1 mM EDTA, and at time 0 isotonic Rb+ buffer (either Cl- or NO-3) was added to give 70 mM Rb+ (+ 70 mM Na+). Cellular Rb+ uptake was measured at various time points, as depicted in Fig. 6; the slopes of the curves represent flux rates. Under oxygenated conditions, Rb+ influx was greater in Cl- medium than in NO-3 medium, indicating a Cl--dependent component in this experiment; however, this was not a consistent finding, and in the pooled data of Fig. 7 this is not apparent. As illustrated in Fig. 6, deoxygenation increased Rb+ influx approximately twofold in NO-3 medium, reflecting the sickling-induced increase in passive membrane permeability (39). In Cl- media, however, deoxygenation increased Rb+ influx even more strikingly. Rb+ influx measured in saline media with external Rb+ = 5.6 mM showed no significant Cl-dependent component in either oxygenated or deoxygenated cells (not shown), indicating that the deoxygenation stimulation of Cl--dependent Rb+ influx was unique to the condition of high external Rb+. These data demonstrate that under conditions of high external Rb+, a substantial component of the Rb+ influx stimulated by deoxygenation of SS RBC was Cl- dependent.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Rb+ uptake in SS RBC incubated in high-Rb+ buffer. Cells were preincubated under oxygenated or deoxygenated conditions for 1 h in isotonic HBS or HBN containing 0.1 mM ouabain and 0.1 mM EDTA. Flux was initiated by addition of isotonic HEPES-buffered Rb+ medium (Cl- or NO-3) to give 70 mM Rb+ and 70 mM Na+. Cellular Rb+ was measured at indicated times; slopes of these uptake curves represent flux rates. This experiment was representative of 4 others.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of KCl cotransport inhibitors on K+ efflux and Rb+ influx in deoxygenated SS RBC incubated in high-K+(Rb+) buffer. SS RBC were deoxygenated for 1 h in HBK (Cl-) or KNO3 media containing 0.1 mM ouabain and 0.1 mM EDTA; no ionophore was present. Flux was initiated by addition of a mixture of Na+ and Rb+ media (Cl- or NO-3) to give final external concentrations of (in mM) 35 Na+, 65 K+, and 40 Rb+. DIOA was added as indicated to give 50 µM; OA was present as indicated at 1 µM. K+ efflux and Rb+ influx were measured over subsequent 2-h period. In parallel oxygenated incubations, there was no Cl--dependent K+ efflux or Rb+ influx, and neither DIOA nor OA affected flux rates (not shown). Data are means ± SD of 4 experiments.

This Cl- dependence is illustrated in the series of experiments in Fig. 7, in which both Rb+ influx and K+ efflux were measured in deoxygenated SS RBC incubated in isotonic high-K+(Rb+) media, conditions of trans-stimulation of the cotransporter. Final external cation concentrations were 35 mM Na+, 65 mM K+, 40 mM Rb+. These conditions permit measurement of KCl cotransport-mediated K+ efflux, as well as Rb+ influx. In oxygenated SS RBC, there was no Cl--dependent flux and no effect of DIOA or okadaic acid (not shown). However, in deoxygenated SS RBC (Fig. 7), 40% of both K+ efflux and Rb+ influx was inhibited in NO-3 medium, without cell swelling, acidification, or manipulation of Mg2+. This deoxygenation-stimulated, Cl--dependent component was substantially inhibited by DIOA and abolished by okadaic acid, confirming its mediation by the KCl cotransporter. Thus, in high-Rb+(K+) buffers, KCl cotransport is activated by continuous deoxygenation. Under these conditions of trans-stimulation, the increase in cellular Mg2+ produced by deoxygenation has a minimal inhibitory effect on cotransporter activity (Fig. 5).

Na+/H+ exchange is not activated by deoxygenation. To investigate whether a component of the deoxygenation-induced Na+ influx (Fig. 1) was mediated by the Na+/H+ exchanger, we examined the effect of DMA, an inhibitor of the antiporter (41). These experiments utilized a large density fraction of SS RBC (1.076 < partial  < 1.106); Na+/H+ exchange has been found to be independent of density over this range and decreased only in cells of higher density (12). Net Na+ influx was measured under oxygenated and deoxygenated conditions in isotonic media at pH 7.4 as described in MATERIALS AND METHODS, with and without DMA. DMA had no effect on Na+ influx in oxygenated SS RBC or in oxygenated or deoxygenated AA RBC (not shown), indicating that the Na+/H+ exchanger was quiescent under these conditions. The deoxygenation-induced Na+ flux was calculated as the difference between the values in paired deoxygenated and oxygenated samples, and was normalized to the value in control cells (no added drugs). These normalized values are presented in Fig. 8 as averages of six experiments. Deoxygenation-induced Na+ influx was not inhibited by DMA, indicating that the Na+/H+ exchanger was not activated by deoxygenation. In contrast, deoxygenation-induced Na+ influx was increased by 45% after a pretreatment of SS RBC with PMA, as reported previously (22), and this PMA-stimulated component was completely inhibited by DMA. These data demonstrate that the Na+/H+ exchanger is stimulated by PMA in deoxygenated SS red blood cells, as previously observed in acid-loaded erythrocytes (14) and nucleated cells (45). Interestingly, PMA had no effect on Na+ influx under oxygenated conditions in SS RBC or under oxygenated or deoxygenated conditions in normal RBC (not shown). That activation of Na+/H+ exchange in isotonic media at normal pH occurs only in SS RBC and requires both deoxygenation and PMA implies a complex mechanism of activation. In any case, the data indicate that the Na+/H+ exchanger is present in SS RBC and can be stimulated in deoxygenated SS RBC by PMA but that the exchanger is not activated by deoxygenation alone and thus is not likely to contribute to cation homeostasis under physiological conditions.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of dimethyl amiloride (DMA) and phorbol 12-myristate 13-acetate (PMA) on deoxygenation-induced Na+ influx in SS RBC. SS RBC were incubated with or without DMA (40 µM) for 5 min and with or without PMA (0.4 µM) for an additional 10 min and then deoxygenated (with parallel oxygenated samples) for 90 min. Deoxygenation-induced Na+ flux was calculated as difference in net fluxes in deoxygenated and oxygenated cells and was normalized to value in control cells without DMA. Data are means ± SD of 6 experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Early studies of cation fluxes in unfractionated SS RBC under continuous deoxygenation in isotonic media at normal pH found no Cl--dependent component to the K+ efflux (2, 36, 39). This was consistent with previous findings that the KCl cotransporter was inactive under isotonic conditions at normal pH under oxygenated conditions (7, 11), and, when activated by cell swelling or acid pH, KCl cotransport was inhibited by deoxygenation (11). Canessa et al. (11) showed that this inhibitory effect was a consequence of two well-described phenomena: increased cellular Mg2+ due to binding of organic anions to deoxygenated Hb (24, 26, 48) and inhibition of the cotransporter by internal Mg2+ (8, 11, 18). Deoxygenation also was shown to reduce KCl cotransport in trout and horse RBC incubated in isotonic media at pH 7.0 (15, 31), although the authors of these reports suggested that factors other than cellular Mg2+ were involved.

Later studies, however, suggested that the K+ loss from deoxygenated SS RBC was mediated, at least in part, by the KCl cotransporter. Beuzard and colleagues (46, 52) reported that a portion of the K+ efflux from deoxygenated cells was inhibited by DIOA. Apovo et al. (1) found that cyclic deoxygenation of SS RBC resulted in dense cell formation that was independent of the presence of Ca2+ and inhibited by DIOA. Franco et al. (29) found that Cl--dependent dense cell formation in SS reticulocytes occurred with cyclic, but not continuous, deoxygenation under isotonic conditions at normal pH. We proposed an explanation for these findings that takes into account the increase in Mg2+ associated with deoxygenation (24, 48) and its effect on KCl cotransport (11, 18, 49) and the alteration in protein phosphorylation equilibrium induced by deoxygenation (22). Fathallah et al. (22) found that deoxygenation of SS RBC, as well as AA RBC, resulted in nonspecific dephosphorylation of RBC membrane proteins. We proposed (29) that the KCl cotransporter or its regulators might be dephosphorylated on deoxygenation of SS RBC but its activity attenuated by the accompanying increase in cellular Mg2+ (24, 26). On reoxygenation, Mg2+ levels fall rapidly, whereas dephosphorylation may persist for some period of time, during which KCl cotransport could take place.

In the present study, we have demonstrated a small Cl--dependent K+ efflux in continuously deoxygenated, low-density SS RBC (Fig. 1A). The Cl--dependent component of the deoxygenation-induced K+ efflux was augmented more than twofold in cells treated with ionophore A-23187 to minimize the increase in cellular Mg2+ associated with deoxygenation (Fig. 1B). This supports the proposed mechanism whereby the activated KCl cotransporter is partially inhibited by elevated cellular Mg2+ in continuously deoxygenated SS RBC. Inhibition of the Cl--dependent component of the deoxygenation-induced flux by DIOA (Fig. 2) identifies it as a manifestation of KCl cotransport activity. Blockade by the phosphatase inhibitor okadaic acid further supports the idea that the stimulation is due to dephosphorylation of the cotransporter (or its regulators) by deoxygenation.

Thus stimulation of KCl cotransport does indeed occur on deoxygenation of low-density SS RBC. The activation of KCl cotransport in a population of cells may be influenced by a variety of factors, including the age and density distribution of the cell population studied, cellular Mg2+ content and buffering capacity, pH, conditions of deoxygenation, and flux measurement techniques. Differences in these variables may explain why a Cl--dependent component of the deoxygenation-induced flux was not apparent in some previous studies (2, 11, 39).

Stimulation of KCl cotransport by deoxygenation could also be demonstrated by flux measurements in high-Rb+(K+) medium. Under these conditions of trans-stimulation, it was shown that the transporter was more resistant to inhibition by cellular Mg2+, presumably due to the kinetic alterations induced by the trans-stimulation of the transporter in high-K+ medium (41). In terms of a kinetic model for KCl cotransport activity, this phenomenon could be explained if the return of the empty carrier (outside to inside) were rate limiting to KCl cotransport activity at low external K+ and were also inhibited by internal Mg2+. In high external K+(Rb+), if return of the filled carrier were no longer rate limiting (hence, the trans-stimulation), transport inhibition by Mg2+ might be reduced. The data in Fig. 5 are consistent with this hypothesis, but further experiments will be required to provide rigorous kinetic support. Nevertheless, the stimulation of Cl--dependent K+ and Rb+ fluxes by deoxygenation of SS RBC is clear in high-Rb+(K+) medium (Figs. 6 and 7). As with the K+ flux in Mg2+-clamped cells in low-K+ medium, these fluxes were inhibited by okadaic acid and DIOA, indicating respectively their activation by a dephosphorylation mechanism and mediation by KCl cotransport.

Stimulation of KCl cotransport in these experiments occurred in the absence of Ca2+, indicating that sickling-induced Ca2+ influx, with K+ channel activation, cellular dehydration, and acidification as proposed by Bookchin et al. (4), was not the trigger for cotransport activation by deoxygenation under these conditions. The simplest explanation of our data is the direct activation of KCl cotransport by deoxygenation of SS RBC, presumably by a dephosphorylation event. Blockade of this activation by okadaic acid implicates a serine/threonine phosphatase in the process, probably protein phosphatase 1 (PP1), at least in the case of swelling-activated KCl cotransport (33). Involvement of tyrosine kinases and/or phosphatases in the regulation of KCl cotransport has recently been suggested. However, depending on species, tyrosine kinase inhibitors were reported either to inhibit (25, 50, 53) or to stimulate (3, 17, 25) cotransport. In fact, Flatman et al. (25) reported that the stimulation of KCl cotransport by one kinase inhibitor, staurosporine, was blocked by another inhibitor, genistein, suggesting two separate tyrosine phosphorylation sites. Inhibition of PP1 activity and direct activation of the cotransporter have been proposed to account for these effects, although the precise targets of tyrosine phosphorylation are not known and may vary among species. The involvement of both tyrosine kinase and PP1 in the activation of KCl cotransport implies the existence of two additional regulatory proteins: a tyrosine phosphatase and a serine/threonine kinase. Each of these enzymes is a potential target for modulators of KCl cotransport activation such as cell swelling, pH, Mg2+, oxidation, and phosphorylation-dephosphorylation. Furthermore, interactions among these modulators are likely to be quite complex. It is known that cell swelling, pH, and Mg2+ levels alter the effect of each other on KCl cotransport activity (8, 18, 43, 49). In addition, there is evidence for dual effects of Mg2+ on the cotransporter, both altering phosphorylation (18) and inhibiting the transporter directly (49). The interactions of multiple phosphorylation-dephosphorylation events (25, 50) in the activation of KCl cotransport and how they are affected by deoxygenation remain to be determined.

Our data suggest a new stimulus for activation of the KCl cotransporter, deoxygenation, which does not require the abnormal conditions of cell swelling or acidification and may be important both physiologically in normal RBC and pathologically in SS RBC. The magnitude of the K+ efflux via the KCl cotransporter stimulated by cyclic deoxygenation of RBC in vivo would be a complex function of circulatory transit times, dynamics of dephosphorylation-rephosphorylation of target proteins in relation to changing cellular Mg2+ levels, and the number of transporter molecules in individual cells. In vitro studies of the activation and inactivation of KCl cotransport in SS RBC by swelling and shrinking revealed delay times for activation of 1.7 ± 0.3 min and for inactivation of 3.6 ± 0.4 min (13). These delay times are somewhat longer than overall circulatory transit time (total blood volume divided by cardiac output), estimated at ~1 min in the healthy adult (44). However, transit times vary considerably among different vascular beds, bone and bone marrow having transit times 10-fold longer than kidney, brain, and lung (44). In addition, the adherence of SS reticulocytes to the endothelium of postcapillary venules (42) may further prolong circulatory times. Thus it is quite possible that individual cells might experience, at least intermittently, oxygenation-deoxygenation cycles in vivo that could activate the KCl cotransporter, with important effects in these cells on volume regulation.

Recently we reported that KCl cotransport activity was higher in SS reticulocytes that had become moderately dense in vivo relative to cells that retained normal density as reticulocytes (28), suggesting that KCl cotransport plays a role in reticulocyte dehydration in vivo. The fact that moderately dehydrated reticulocytes have the same levels of fetal hemoglobin (Hb F) as normally hydrated reticulocytes (27) implies that KCl cotransport, which is independent of Hb F (30), may be more important in the process of moderate reticulocyte dehydration than sickling-dependent mechanisms, which are inhibited by Hb F (19). Sickling-dependent processes, facilitated by moderate dehydration of reticulocytes, may contribute to more severe dehydration of certain reticulocytes (particularly those without Hb F), and to dehydration of more mature cells.

The abnormalities of volume regulation in SS RBC could theoretically arise from perturbations of Na+ transport pathways. The Na+/H+ exchanger is an important Na+ uptake pathway in human red blood cells (20), and pharmacological and kinetic data suggest that red blood cells express at least the NHE1 isoform (14, 20). SS RBC exhibit elevated Na+/H+ exchange activity (12), and we hypothesized that deoxygenation might stimulate this pathway. The Na+/H+ exchanger in RBC is activated by cell shrinkage (12) or, in the presence of a pH gradient, by incubation with PMA or with Ca2+ and ionophore (20), both treatments resulting in protein kinase C alpha  (PKCalpha ) translocation to the membrane (23). Deoxygenation of SS RBC in the presence of external Ca2+ leads to transient elevation of cytosolic Ca2+ and subsequent cell volume reduction (via activation of Ca2+-dependent K+ channels) (4, 6). The present data, however, show that Na+/H+ exchange activity was not stimulated by deoxygenation alone. The transient rise in Ca2+ associated with deoxygenation is not sufficient to induce PKCalpha translocation (23), which may explain the failure of deoxygenation to activate the transporter. Likewise, the addition of PMA to oxygenated SS RBC was insufficient to activate the transporter, although under these conditions PMA induces translocation of PKCalpha to the membrane (23). The combined requirement for deoxygenation of SS RBC and the presence of PMA implies that both elevated Ca2+ and PKCalpha activation are required for stimulation of Na+/H+ exchange, in agreement with the proposed mechanism of NHE1 activation in other cells (45). Nevertheless, it does not appear that the Na+/H+ exchanger mediates a component of deoxygenation-induced Na+ influx under physiological conditions.

    ACKNOWLEDGEMENTS

The technical assistance of Mary Palascak and graphic skills of William Claussen are gratefully acknowledged. We thank Dr. Donald Rucknagel for many useful discussions during the course of this work and for obtaining blood samples from the University of Cincinnati sickle cell clinic. We are also grateful to Drs. D. Bachir and F. Galacteros for providing blood samples from the Centre de la Drépanocytose et des Thalassémies (Hopital Henri Mondor, Créteil, France).

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-37515 and HL-57614 (to C. H. Joiner) and HL-51174 (to R. S. Franco) and by Fondation pour la Recherche Médicale Grant 40000 187S.05 to F. Giraud.

Address for reprint requests: C. H. Joiner, Comprehensive Sickle Cell Center, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 3 November 1997; accepted in final form 12 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Apovo, M., Y. Beuzard, F. Galacteros, D. Bachir, and F. Giraud. The involvement of the Ca-dependent K channel and of KCl cotransport in sickle cell dehydration during cyclic deoxygenation. Biochim. Biophys. Acta 1225: 255-258, 1994[Medline].

2.   Berkowitz, L. R., and E. P. Orringer. Passive sodium and potassium movements in sickle erythrocytes. Am. J. Physiol. 249 (Cell Physiol. 18): C208-C214, 1985[Abstract].

3.   Bize, I., and P. B. Dunham. Staurosporine, a protein kinase inhibitor, activates K-Cl cotransport in LK sheep erythrocytes. Am. J. Physiol. 266 (Cell Physiol. 35): C759-C770, 1994[Abstract/Free Full Text].

4.   Bookchin, R. M., O. E. Ortiz, and V. L. Lew. Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia. J. Clin. Invest. 87: 113-124, 1991[Medline].

5.   Brugnara, C., H. F. Bunn, and D. C. Tosteson. Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232: 388-390, 1986[Medline].

6.   Brugnara, C., L. De Franceschi, and S. L. Alper. Inhibition of Ca++-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J. Clin. Invest. 92: 520-526, 1993[Medline].

7.   Brugnara, C., T. V. Ha, and D. C. Tosteson. Acid pH induces formation of dense cells in sickle erythrocytes. Blood 74: 487-495, 1989[Abstract].

8.   Brugnara, C., and D. C. Tosteson. Inhibition of K transport by divalent cation in sickle erythrocytes. Blood 70: 1810-1815, 1987[Abstract].

9.   Bunn, H. F., and B. G. Forget. Hemoglobin: Molecular, Genetic, and Clinical Aspects. Philadelphia: Saunders, 1986.

10.   Canessa, M., M. E. Fabry, N. Blumenfeld, and R. L. Nagel. Volume-stimulated, Cl--dependent K+ efflux is highly expressed in young human red cells containing normal hemoglobin or HbS. J. Membr. Biol. 97: 97-105, 1987[Medline].

11.   Canessa, M., M. E. Fabry, and R. L. Nagel. Deoxygenation inhibits the volume-stimulated, Cl--dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation. Blood 70: 1861-1866, 1987[Abstract].

12.   Canessa, M., M. E. Fabry, S. M. Suzuka, K. Morgan, and R. L. Nagel. Na+/H+ exchange is increased in sickle cell anemia and young normal red cells. J. Membr. Biol. 116: 107-115, 1990[Medline].

13.   Canessa, M., J. R. Romero, C. Lawrence, R. L. Nagel, and M. E. Fabry. Rate of activation and deactivation of K:Cl cotransport by changes in cell volume in hemoglobin SS, CC, and AA red cells. J. Membr. Biol. 142: 349-362, 1994[Medline].

14.   Ceolotto, G., P. Conlin, G. Clari, A. Semplicini, and M. Canessa. Protein kinase C and insulin regulation of red blood cell Na+/H+ exchange. Am. J. Physiol. 272 (Cell Physiol. 41): C818-C826, 1997[Abstract/Free Full Text].

15.   Cossins, A. R., Y. R. Weaver, G. Lykkeboe, and O. B. Nielsen. Role of protein phosphorylation in control of K flux pathways in trout red blood cells. Am. J. Physiol. 267 (Cell Physiol. 36): C1641-C1650, 1994[Abstract/Free Full Text].

17.   De Francheschi, L., L. Fumagalli, O. Olivieri, R. Corrocher, C. A. Lowell, and G. Berton. Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport. J. Clin. Invest. 99: 220-227, 1997[Abstract/Free Full Text].

18.   Delpire, E., and P. K. Lauf. Magnesium and ATP dependence of K-Cl cotransport in low K sheep red blood cells. J. Physiol. (Lond.) 441: 219-231, 1991[Abstract].

19.   Eaton, W. A., and J. Hofrichter. Hemoglobin S gelation and sickle cell disease. Blood 70: 1245-1266, 1987[Medline].

20.   Escobales, N., and M. Canessa. Ca++-activated Na+ fluxes in human red cells: amiloride sensitivity. J. Biol. Chem. 260: 11914-11923, 1985[Abstract/Free Full Text].

21.   Etzion, Z., T. Tiffert, R. M. Bookchin, and V. L. Lew. Effects of deoxygenation on active and passive Ca++ transport and on the cytoplasmic Ca++ levels of sickle cell anemia red cells. J. Clin. Invest. 92: 2489-2498, 1993[Medline].

22.   Fathallah, H., E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells. Blood 86: 1999-2007, 1995[Abstract/Free Full Text].

23.   Fathallah, H., M. Sauvage, J. R. Romero, M. Canessa, and F. Giraud. Effects of protein kinase C alpha  activation on Ca pump and KCa channel in deoxygenated sickle cells. Am. J. Physiol. 273 (Cell Physiol. 42): C1206-C1214, 1997[Abstract/Free Full Text].

24.   Flatman, P. W. The effect of buffer composition and deoxygenation on the concentration of ionized magnesium inside human red blood cells. J. Physiol. (Lond.) 300: 19-30, 1980[Abstract].

25.   Flatman, P. W., N. C. Adragna, and P. K. Lauf. Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport. Am. J. Physiol. 271 (Cell Physiol. 40): C255-C261, 1996[Abstract/Free Full Text].

26.   Flatman, P. W., and V. L. Lew. Magnesium buffering in intact human red blood cells measured using the ionophore A23187. J. Physiol. (Lond.) 305: 13-30, 1980[Abstract].

27.   Franco, R. S., R. Barker-Gear, M. A. Miller, S. M. Williams, C. H. Joiner, and D. L. Rucknagel. Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes. Blood 84: 2013-2020, 1994[Abstract/Free Full Text].

28.   Franco, R. S., M. Palascak, H. Thompson, and C. H. Joiner. KCl cotransport activity in light and dense transferrin receptor-positive sickle reticulocytes. J. Clin. Invest. 95: 2573-2580, 1995[Medline].

29.   Franco, R. S., M. Palascak, H. Thompson, D. L. Rucknagel, and C. H. Joiner. Dehydration of TfR+ sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium. Blood 88: 4359-4365, 1996[Abstract/Free Full Text].

30.   Franco, R. S., H. Thompson, M. Palascak, and C. H. Joiner. The formation of transferrin receptor-positive sickle reticulocytes with intermediate density is not determined by HbF content. Blood 90: 3195-3203, 1997[Abstract/Free Full Text].

31.   Gibson, J. S., H. Godart, J. C. Ellory, H. Staines, N. A. Honess, and A. R. Cossins. Modulation of K+-Cl- cotransport in equine red blood cells. Exp. Physiol. 79: 997-1009, 1995.

32.   Jennings, M. L., and N. Al-Rohil. 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, 1990[Abstract].

33.   Jennings, M. L., and R. K. Schulz. Okadaic acid inhibition of KCl cotransport: evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethyl-maleimide. J. Gen. Physiol. 97: 799-818, 1991[Abstract].

34.   Joiner, C. H. Deoxygenation-induced fluxes in sickle cells: II. Inhibition by stilbene disulfonates. Blood 76: 212-220, 1990[Abstract].

35.   Joiner, C. H. Cation transport and volume regulation in sickle red blood cells. Am. J. Physiol. 264 (Cell Physiol. 33): C251-C270, 1993[Abstract/Free Full Text].

36.   Joiner, C. H., A. Dew, and D. L. Ge. Deoxygenation-induced fluxes in sickle cells: I. Relationship between net potassium efflux and net sodium influx. Blood Cells 13: 339-348, 1988[Medline].

37.   Joiner, C. H., M. Jiang, and R. S. Franco. Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium. Am. J. Physiol. 269 (Cell Physiol. 38): C403-C409, 1995[Abstract/Free Full Text].

38.   Joiner, C. H., M. Jiang, and R. S. Franco. KCl cotransport is activated by deoxygenation of sickle red blood cells (Abstract). Blood 88: 649a, 1996.

39.   Joiner, C. H., C. L. Morris, and E. S. Cooper. Deoxygenation-induced cation fluxes in sickle cells: III. Cation selectivity and response to pH and membrane potential. Am. J. Physiol. 264 (Cell Physiol. 33): C734-C744, 1993[Abstract/Free Full Text].

40.   Joiner, C. H., O. S. Platt, and S. E. Lux. Cation depletion by the sodium pump in red cells with pathological cation leaks: sickle cells and xerocytes. J. Clin. Invest. 78: 1487-1496, 1986[Medline].

41.   Kaji, D. M. Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry. Am. J. Physiol. 256 (Cell Physiol. 25): C1214-C1223, 1989[Abstract/Free Full Text].

42.   Kaul, D. K., M. E. Fabry, and R. L. Nagel. Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications. Proc. Natl. Acad. Sci. USA 86: 3356-3362, 1989[Abstract].

43.   Lauf, P. K., A. Erdmann, and N. C. Adragna. K-Cl cotransport, pH, and role of Mg in volume-clamped low-K sheep erythrocytes: three equilibrium states. Am. J. Physiol. 266 (Cell Physiol. 35): C95-C103, 1994[Abstract/Free Full Text].

44.   Leggett, R. W., and L. R. Williams. A proposed blood circulation model for Reference Man. Health Phys. 69: 187-201, 1995[Medline].

45.   Noël, J., and J. Pouysségur. Hormonal regulation, pharmacology, and membrane sorting of the vertebrate Na/H exchanger isoforms. Am. J. Physiol. 268 (Cell Physiol. 37): C283-C296, 1995[Abstract/Free Full Text].

46.   Olivieri, O., D. Vitoux, D. Bachir, and Y. Beuzard. K+ efflux in deoxygenated sickle cells in the presence or absence of DIOA, a specific inhibitor of the [K-Cl] cotransport system. Br. J. Haematol. 77: 117-120, 1991[Medline].

47.   Olivieri, O., D. Vitoux, F. Galacteros, D. Bachir, Y. Blouquit, Y. Beuzard, and C. Brugnara. Hemoglobin variants and activity of the (K+Cl-) cotransport system in human erythrocytes. Blood 79: 793-797, 1992[Abstract].

48.   Ortiz, O. E., V. L. Lew, and R. M. Bookchin. Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J. Physiol. (Lond.) 427: 211-226, 1990[Abstract].

49.   Ortiz-Carranza, O., N. C. Adragna, and P. K. Lauf. Modulation of K-Cl cotransport in volume-clamped low-K sheep erythrocytes by pH, magnesium, and ATP. Am. J. Physiol. 271 (Cell Physiol. 40): C1049-C1058, 1996[Abstract/Free Full Text].

50.   Sachs, J. R., and D. W. Martin. The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway. J. Gen. Physiol. 102: 551-560, 1993[Abstract].

51.   Sardet, C., I. Counillon, A. Franchi, and J. Pouysségur. Growth factors induce phosphorylation of the Na/H antiporter, a glycoprotein of 110 kD. Science 247: 123-126, 1990.

52.   Vitoux, D., O. Olivieri, R. P. Garay, E. J. Cragoe, Jr., F. Galacteros, and Y. Beuzard. Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K+Cl- cotransport system. Proc. Natl. Acad. Sci. USA 86: 4273-4276, 1989[Abstract].

53.   Weaver, Y. R., and A. R. Cossins. Protein tyrosine phosphorylation and the regulation of KCl cotransport in trout erythrocytes. Pflügers Arch. 432: 727-734, 1996[Medline].


Am J Physiol Cell Physiol 274(6):C1466-C1475
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society