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. 1998 Aug 15;511(Pt 1):225–234. doi: 10.1111/j.1469-7793.1998.225bi.x

Differential oxygen sensitivity of the K+-Cl− cotransporter in normal and sickle human red blood cells

J S Gibson *, P F Speake *, J C Ellory *
PMCID: PMC2231113  PMID: 9679176

Abstract

  1. K+ influx and efflux were measured in normal (HbA) and sickle (HbS) red blood cells to investigate the interaction of swelling, H+ ions and urea with O2 (0 to 150 mmHg O2) in the presence of ouabain and bumetanide (both 100 μM).

  2. In HbA cells, K+-Cl− cotransport was O2 dependent. At low oxygen tensions (PO2s) the transporter was inactive and refractory to low pH, swelling or urea.

  3. Cl−-independent K+ influxes in sickle cells were elevated at low PO2s, as previously reported. Cl−-dependent K+ influxes were large at both high and low PO2s, whether stimulated by swelling, H+ ions or urea. In the absence of O2, Cl−-dependent K+ influxes were similar in magnitude to those measured at high PO2s. The minimum for Cl−-dependent K+ influx was observed at PO2s of about 40-70 mmHg.

  4. K+ efflux from HbS cells was stimulated by the addition of urea (500 mm). The rate constants were of similar magnitude whether measured at high PO2 or in the absence of O2, and were predominantly Cl− dependent under both conditions.

  5. In HbS red blood cells, reduction of extracellular Ca2+, addition of 1 mm Mg2+ or nitrendipine (10 μM) to the saline had no effect. Inhibitors of K+-Cl− cotransport, [(dihydroindenyl)oxy] alkanoic acid (DIOA; 100 μM) or calyculin A (0·1 μM), inhibited influxes by a similar magnitude to Cl− substitution.

  6. Results are significant for the pathophysiology of sickle cell disease. Low pH and urea are able to stimulate KCl loss from sickle cells, leading to cellular dehydration, even in regions of low PO2.

The K+-Cl− cotransporter can represent a major route for K+ loss from human red blood cells. The transporter is largely quiescent in normal (HbA) mature cells, but it remains present and can be activated pharmacologically, for example with N-ethylmaleimide (Lauf, 1985; Canessa et al. 1986; Hall & Ellory, 1986), or by subjecting cells to high hydrostatic pressure (Hall & Ellory, 1986). In a fraction of young HbA cells and also in samples of red blood cells from patients with certain Hb variants, in particular HbS (sickle cells) and HbC, the activity of the K+-Cl− cotransporter is unusually high (Brugnara et al. 1986; Canessa et al. 1986; Hall & Ellory, 1986; Brugnara & Tosteson, 1987; Canessa et al. 1987; Olivieri et al. 1992). The main physiological regulators of the cotransporter are thought to be cell volume (Canessa et al. 1986; Hall & Ellory, 1986), H+ ions (Ellory et al. 1989a, 1991) and possibly urea (Kaji & Gasson, 1995). Once activated, the transporter mediates KCl efflux, water will follow, thus reducing the cell volume. The cotransporter may contribute to regulatory volume decrease. More importantly, inappropriate activation will produce shrunken red blood cells with an elevated mean cell haemoglobin concentration, promoting polymerization of HbS, mainly due to a reduced lag time for polymer formation (Eaton & Hofrichter, 1987). K+-Cl− cotransport activity can therefore contribute to the development of sickle cells, whose raised microviscosity and reduced deformability (Ellory et al. 1989b) contribute to vascular occlusion (at least in animal models) and hence to sickle cell disease. The relative importance of K+-Cl− cotransport in these processes, compared with that of the Ca2+-activated K+ channel activity (Gardos channel: Gardos, 1958), also abnormally high in sickle cells, has been the subject of considerable research (for example: Ohnishi et al. 1986; Ellory et al. 1989b; Bookchin et al. 1991; Apovo et al. 1994; Franco et al. 1996).

More recently, it has become apparent that oxygen also modulates the K+-Cl− cotransporter (Borgese et al. 1991; Nielsen et al. 1992). As it passes through the circulation, the red blood cell will be subjected to different oxygen tensions (PO2s). In particular, capillary beds of active muscle will have a low PO2 concomitant with low pH. Similarly, the renal medulla during antidiuresis represents a region of low PO2 and high urea concentration, with urea being a potential activator of K+-Cl− cotransport. It is therefore important to establish the interaction between O2 and other physiological parameters which act on the cotransporter. In red blood cells from other species (e.g. fish: Borgese et al. 1991; Jensen, 1992; Nielsen et al. 1992; horse: Gibson et al. 1995; Honess et al. 1996; Speake & Gibson, 1997; and sheep: Campbell & Gibson, 1998), the activity of the K+-Cl− cotransporter is increased by O2. In the absence of a sufficiently high PO2, the transporter is inactive and refractory to other stimuli such as H+ ions, cell swelling (Nielsen et al. 1992; Honess et al. 1996; Speake & Gibson, 1997; Speake et al. 1997) and moderate (but not high) concentrations of urea (Speake & Gibson, 1997). From the work of Canessa and others, on HbA and HbS red blood cells, the O2 dependence of the human red blood cell K+-Cl− cotransporter has been reported to behave similarly, at least with respect to swelling and H+-stimulated cotransport (Canessa et al. 1987; Joiner et al. 1993). Research on this aspect, however, remains incomplete. For example, it is unknown what level of PO2 supports activation of the human red blood cell K+-Cl− cotransporter, as most experiments are conducted only in air or pure N2. Recently, we have shown that urea-stimulated K+ fluxes in sickle cells are largely unaffected by PO2 (Culliford et al. 1998), and therefore behave differently from those in equine red blood cells (Speake & Gibson, 1997), the only other species in which this has been studied. These observations, and the lack of data concerning O2 and K+ transport in human red blood cells, prompted us to investigate further the interaction of O2 and other stimuli. In this manuscript, we describe the response of the K+ transport in HbA and HbS cells to swelling, H+ ions and urea over a range of PO2 from 0 to 150 mmHg O2 (encompassing the physiological level of PO2s). Important differences emerge between regulation of K+-Cl− cotransport in sickle and normal human red blood cells, which may be significant for the aetiology of sickling.

METHODS

Chemicals

Bumetanide, [(dihydroindenyl)oxy]alkanoic acid (DIOA), EGTA, Mops, ouabain and salts were all purchased from the Sigma Chemical Co., Calyculin A and nitrendipine were purchased from Calbiochem, and 86Rb from NEN Du Pont.

Blood

Samples of normal (HbA) and sickle (homozygous for HbS) red blood cells were obtained with written ethical permission by venepuncture of consenting volunteers into heparin-containing syringes, and stored at 4°C until required (within 48 h). The erythrocytes were washed × 3 by centrifugation (1500 g; 5 min) with aspiration of the supernatant in saline to remove buffy coat, platelets and plasma. In all experiments, unfractionated cell samples were used.

Solutions

The standard medium was Mops-buffered saline solution (MBS) comprising (mm): NaCl, 145; Mops, 10; glucose, 5; pHo, 7.4, 294 ± 3 mosmol kg−1 (mean ±s.d., n= 8). In some experiments NaCl was replaced by equimolar NaMeSO4 or NaNO3; anion substitution was achieved by washing the cells × 3 in the appropriate medium, incubating for 15 min at 37°C, and then washing once more. To investigate reversibility of the urea effect, and for K+ efflux experiments, a high K+-containing (NaCl 72.5 mm; KCl 72.5 mm) saline was used to obviate net K+ loss and cell shrinkage.

Tonometry

Oxygen tension (PO2) was controlled by incubating cells in Eschweiler tonometers coupled to a Wösthoff gas mixing pump as described previously (Speake & Gibson, 1997). All experiments were carried out at normal atmospheric pressure, with gas saturated with water vapour at 37°C, and PO2 was varied by replacement of O2 with N2. Blood samples were incubated for 10 min at each PO2 before flux measurement (at this same PO2); control experiments showed that this interval was adequate for complete equilibration.

Flux measurements

In most experiments, potassium fluxes were determined at 37°C using 86Rb as a congener, added in KNO3 to give a final K+ concentration of 7.5 mm (see Dunham & Ellory, 1981). Ouabain and bumetanide (0.1 mm) were present in all experiments to inhibit the Na+-K+ pump and Na+-K+-2Cl− cotransporter. Packed cell volume was determined by microhaematocrit centrifugation. K+ influxes are expressed in standard units of mmol K+ (l cells)−1 h−1 and given as means ±s.d. or s.e.m. of n measurements. For efflux measurements, cells were preloaded with 86Rb and then suspended at about 4 % haematocrit in high K+-containing solutions (as for urea reversibility). Aliquots were taken at the appropriate time intervals, centrifuged rapidly to remove red blood cells, and 86Rb in the supernatant was then counted. Results were expressed as ln (fraction of 86Rb remaining in cells) and plotted against time to estimate the rate constant for K+ efflux.

Statistics

Results are presented as the mean ±s.d. for single experiments, representative of at least two others, or as the means ±s.e.m. for n experiments. Statistical comparisons were carried out using Student's paired t test (SigmaStat, Jandel Scientific Ltd).

RESULTS

HbA red blood cells

Interaction between oxygen, H+ ions and cell volume

In the first series of experiments, normal human red blood cell samples at high haematocrit (approximately 30 %) were equilibrated in tonometers at various PO2s between 0 and 150 mmHg O2 (1 atm, N2 replacement), pHo 7.4. Red blood cells were then diluted into the appropriate saline, also pre-equilibrated at the same PO2, and subjected to anisosmotic swelling (10 %) at pHo 7.0, before addition of isotope to measure K+ influx (Fig. 1). Experiments were carried out both in the presence and absence of Cl− (substituted with NO3−). Influxes were measured under the combined stimuli of swelling and H+ ions because a low capacity for K+-Cl− cotransport was expected for normal, mature HbA red blood cells. In equine red blood cells, the O2 dependence of the transporter is the same for either stimulus (Speake & Gibson, 1997), and this was indeed confirmed in preliminary experiments with HbA red blood cells in which influxes were measured in swollen cells at pH 7.4 and in cells in isotonic saline at pH 7.0 (data not shown). The Cl−-dependent K+ influx, which is usually taken as representing the activity of the K+-Cl− cotransporter, increased with PO2 (Fig. 1). In N2, only a minimal Cl−-dependent K+ flux was observed and the cotransporter was refractory to the combined stimuli of swelling and H+ ions, and hence presumably to either stimulus on its own. In most cases, maximal K+ influx was obtained with PO2s of > 80-100 mmHg, with a PO2 for half-maximal activation of K+ influx (P50) of 36 ± 3 mmHg (mean ±s.e.m., n= 6) and thus the cotransporter was sensitive to PO2s within the physiological range. By contrast, in the absence of Cl−, K+ influxes were low and independent of PO2.

Figure 1. The effect of oxygen tension on swelling/H+-stimulated K+ influx in normal human red blood cells.

Figure 1

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K+ influx (mmol (l cells)−1 h−1) was measured in the presence and absence of Cl− (substituted with NO3−), 260 mosmol kg−1, pH 7.0. Cl−-dependent K+ influx was calculated as the difference in influx ± Cl−. Points represent the means ±s.e.m. for 6 samples from different individuals.

Interaction between oxygen and urea

In a similar set of experiments, HbA red blood cells were equilibrated with different PO2s and then diluted into saline, again pre-equilibrated at the same PO2, containing urea at a final concentration of 500 mm (Fig. 2). Urea-stimulated Cl−-dependent K+ influxes were minimal in N2 and increased with PO2. Peak Cl−-dependent K+ influxes were measured at PO2s > 70 mmHg with a P50 of 30 ± 3 mmHg (mean ±s.e.m.n= 5). Again K+ influxes in NO3−-containing saline were low and independent of PO2. The O2 dependence of the urea-stimulated K+ influx was therefore similar to that observed for swelling- and H+-stimulated fluxes but in all cases the O2 dependence was shifted towards lower values. P50 when measured simultaneously in cells stimulated with swelling/H+ ions or urea declined by 6 ± 2 mmHg (mean ±s.e.m., n= 5; P < 0.05, Student's paired t test).

Figure 2. The effect of oxygen tension on urea-stimulated K+ influx in normal human red blood cells.

Figure 2

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K+ influx (mmol (l cells)−1 h−1) was measured in the presence and absence of Cl− (substituted with NO3−), pH 7.4, with 500 mm urea. Cl−-dependent K+ influx was calculated as the difference in influx ± Cl−. Points represent the means ±s.e.m. for 5 samples from different individuals.

HbS red blood cells

Interaction between oxygen, H+ and volume

Experiments similar to those described above for HbA red blood cells were carried out with HbS cells, except that swelling and low pH were tested separately. The O2 dependence of swelling-stimulated K+ influxes are shown in Fig. 3 and that for H+-stimulated K+ influx in Fig. 4. At high PO2s, the influxes were large and predominantly Cl− dependent, as for HbA cells. As PO2 was reduced, these Cl−-dependent influxes began to decline, usually reaching a nadir between 40 and 70 mmHg. At PO2s lower than about 40 mmHg, however, the Cl−-dependent influxes increased again so that, in N2, they were of similar magnitude to those observed in high PO2. Combining the results for all methods of stimulation (swelling, H+ ions and urea), the ratio of Cl−-dependent K+ influxes when measured in pure N2 compared with air was 0.93 ± 0.09 (n= 15). The Cl−-independent K+ influxes were smaller in magnitude, unaffected by PO2s of 40 mmHg or above, but at lower PO2s also increased (Figs 3 and 4). The stimulation of Cl−-independent K+ influxes at very low PO2s, < 20 mmHg, was variable between samples, but was sometimes very marked and, in all cases, Cl−-independent influx was maximal in pure N2.

Figure 3. The effect of oxygen tension on swelling-stimulated K+ influx in sickle human red blood cells.

Figure 3

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K+ influx (mmol (l cells)−1 h−1) was measured in the presence and absence of Cl− (substituted with NO3−) in samples swollen anisosmotically by 10 %, pH 7.4. Cl−-dependent K+ influx was calculated as the difference in influx ± Cl−. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

Figure 4. The effect of oxygen tension on H+-stimulated K+ influx in human sickle red blood cells.

Figure 4

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K+ influx (mmol (l cells)−1 h−1) was measured in the presence and absence of Cl− (substituted with NO3−), at pH 7.0. Cl−-dependent K+ influx was calculated as the difference in influx ± Cl−. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

It was noticeable that, at the lower PO2s, HbS red blood cells became markedly ‘stickier’ and more difficult to wash. Because of this, we were concerned that the high K+ influxes at these PO2s might be artefactual, although it is difficult to understand why they should be Cl− dependent if this was the case. We therefore tested the effect of various inhibitors on H+-stimulated K+ influx at high and low PO2s (150 mmHg and pure N2, respectively). Results are shown in Tables 1 and 2

Table 1.

Effect of Cl− substitution or reduction in extracellular Ca2+ on H+- and urea-stimulated K+ influx in sickle cells

pH 7 Urea
High PO2 Low PO2 High PO2 Low PO2
Control 3.55 ± 0.13 4.72 ± 0.06 4.19 ± 0.16 3.48 ± 0.08
Cl− free 0.38 ± 0.09 1.94 ± 0.03 0.33 ± 0.02 0.53 ± 0.03
EGTA 3.74 ± 0.21 4.91 ± 0.33 4.42 ± 0.03 4.16 ± 0.11
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Influxes (mmol (l cells)−1 h−1) were measured in air (high PO2) or N2(low PO2) in normal Cl−-containing saline (control influx), in the absence of Cl− (substituted with NO3−), or in normal saline with the addition of EGTA (100 μM). Extracellular pH was 7.0 (H+ stimulation) or 7.4 (urea-stimulated influxes, with a final urea concentration of 500 mm). Data are given as means ± S.D. for triplicate determinations on a single sample.

Table 2.

Effect of Cl− substitution or transport inhibitors on H+- and urea-stimulated K+ influx in sickle cells

pH 7 Urea
High PO2 Low PO2 High PO2 Low PO2
Control 2.27 ± 0.08 3.03 ± 0.05 4.69 ± 0.11 4.50 ± 0.07
Cl− free 0.48 ± 0.07 1.41 ± 0.01 0.45 ± 0.02 0.77 ± 0.06
DIOA 0.97 ± 0.03 1.79 ± 0.03 0.83 ± 0.03 1.02 ± 0.09
Calyculin A 0.30 ± 0.01 0.72 ± 0.04 0.15 ± 0.01 0.36 ± 0.01
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Influxes (mmol (l cells)−1 h−1) were measured in air (high PO2) or N2(low PO2) in normal Cl−-containing saline (control influx), in the absence of Cl− (substituted with NO3−), or in normal saline following pretreatment of cells for 5 min with [(dihydroindenyl)oxy]alkanoic acids (DIOA, 100 μM) or calyculin A (0.1 μM). Extracellular pH was 7.0 (H+ stimulation) or 7.4 (urea-stimulated influxes, with a final urea concentration of 500 mm). Data are given as means ± S.D. for triplicate determinations on a single sample.

At both gas tensions, the K+ influx was predominantly Cl− dependent, consistent with the results shown in Fig. 4. Influx was unaffected by addition of EGTA (100 μM; Table 1) but was markedly inhibited by DIOA (100 μM; Table 2) or the protein phosphatase inhibitor calyculin A (0.1 μM; Table 2), consistent with the presence of a large component of K+-Cl− cotransport at both low and high PO2s. A further possible artefact, that of Mg2+ efflux via the deoxygenation-induced non-specific cation channel, acting via the protein kinase/phosphatase enzymes which regulate the K+-Cl− cotransporter, was also addressed: however, influxes in air or N2, in the presence or absence of Cl−, were unaffected by addition of 1 mm Mg2+ to the saline (Table 3).

Table 3.

Effect of extracellular Mg2+ on H+- and urea-stimulated K+ influx in sickle cells

H+-stimulated K+ influxes
High PO2 Low PO2
-Mg2+ +Mg2+ -Mg2+ +Mg2+
Control 2.27 ± 0.08 2.06 ± 0.13 3.03 ± 0.05 2.99 ± 0.05
Cl− free 0.48 ± 0.07 0.36 ± 0.05 1.41 ± 0.01 1.12 ± 0.03
Urea-stimulated K+ influxes
High PO2 Low PO2
-Mg2+ +Mg2+ -Mg2+ +Mg2+
Control 4.69 ± 0.11 4.61 ± 0.12 4.50 ± 0.07 4.50 ± 0.17
Cl− free 0.45 ± 0.02 0.78 ± 0.08 0.77 ± 0.06 1.57 ± 0.08
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Influxes (mmol (l cells)−1 h−1) were measured in air (high PO2) or N2(low PO2) in normal Cl−-containing saline (control influx) or in the absence of Cl− (substituted with NO3−) in paired samples with or without the addition of Mg2+ (1 mm). Extracellular pH was 7.0 (H+ stimulation) or 7.4 (urea-stimulated influxes, with a final urea concentration of 500 mm). Data are given as means ± S.D. for triplicate determinations on a single sample.

Interaction between oxygen and urea

Recently we have described the effects of varying PO2 on the urea-stimulated K+ influx in sickle cells (Culliford et al. 1998). A typical response for K+ transport in HbS red blood cells stimulated with 500 mm urea is shown in Fig. 5. Again, peak Cl−-dependent K+ influxes were attained at the lowest and highest PO2s; influxes measured in NO3−-containing saline were insensitive to high PO2s but showed stimulation at the lowest PO2s (0-20 mmHg). By washing away urea, and measuring K+ influx in normal saline, we show that stimulation of K+ influx by urea was fully reversible (Fig. 6).

Figure 5. The effect of oxygen tension on urea-stimulated K+ influx in human sickle red blood cells.

Figure 5

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K+ influx (mmol (l cells)−1 h−1) was measured in the presence and absence of Cl− (substituted with NO3−), at pH 7.4 with 500 mm urea. Cl−-dependent K+ influx was calculated as the difference in influx ± Cl−. Points represent the means ±s.d. for triplicate determinations on a single sample, representative of 3 other experiments.

Figure 6. Reversibility of the effect of urea on K+ influx in human sickle cells.

Figure 6

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The cell sample was divided into three; two aliquots were pre-incubated for 60 min at 37 °C in the presence of urea (500 mm), one in its absence (control). The three aliquots were then washed, leaving urea present in only one of the urea-treated aliquots. K+ influx (mmol (l cells)−1 h−1) was then measured simultaneously in all aliquots. Histograms represent means ±s.d. for triplicate determinations on a single sample, representative of 2 others.

Unlike the situation for cells equilibrated with low PO2s and stimulated by swelling or H+ ions, those in urea were not obviously ‘sticky’, making a possible exaggeration of the true influx through incomplete washing unlikely. Nevertheless, we were concerned that the high Cl−-dependent K+ influx at low PO2s might be inaccurate and therefore examined the effect of PO2 on urea-stimulated K+ efflux. Cell samples loaded with 86Rb were equilibrated with 150 mmHg O2 or 100 % N2. K+ efflux was then measured in the presence and absence of 500 mm urea. Rate constants for K+ efflux in the absence of urea were low, slightly higher in N2 than O2, and largely insensitive to Cl− (data not shown). The addition of 500 mm urea stimulated K+ efflux and this stimulation was markedly Cl− dependent at both PO2s: efflux rate constants for Cl−-dependent K+ efflux were 0.21 and 0.24 h−1 in pure N2 and 150 mmHg O2, respectively. Thus, as for influx measurements, Cl−-dependent K+ effluxes were considerable in the absence of O2 and similar in magnitude to those measured in air.

The effect of various inhibitors was also tested on urea-stimulated K+ influxes, in the presence and absence of O2. As for H+-stimulated influxes, at both 0 and 150 mmHg O2, K+ influxes were predominantly Cl− dependent, largely unaffected by addition of EGTA (100 μM; Table 1) but markedly inhibited by pretreatment of cells for 5 min with DIOA (100 μM; Table 2) or calyculin A (0.1 μM; Table 2) before addition of the urea (500 mm). Nitrendipine (10 μM) had no effect on the influxes (data not shown) and neither did the addition of Mg2+ (1 mm; Table 3). This pharmacological profile is consistent with the presence of a large component of urea-stimulated K+-Cl− cotransport at both low and high PO2s.

DISCUSSION

The results described in this paper represent the first description of the oxygen sensitivity of K+ transport in human red blood cells from 0 to 150 mmHg O2, encompassing the physiological range. We show that Cl−-dependent K+ transport (commonly taken as representing transport through the K+-Cl− cotransporter) in normal (HbA-containing) red blood cells is markedly O2 dependent, and refractory to physiological stimuli such as swelling, H+ ions and urea (500 mm) in the absence of an adequate PO2. By contrast, the K+-Cl− cotransporter in sickle (HbS-containing) cells has similar transport rates in high PO2 and in the absence of O2. These results are directly relevant to understanding the physiology of both normal and sickle red blood cells. They are also contrary to the prevailing view concerning the role of O2 in regulation of K+-Cl− cotransport in sickle cells (Canessa et al. 1987; Joiner et al. 1993; Apovo et al. 1994).

Although it is widely believed that the human red blood cell K+-Cl− cotransporter, in both normal and sickle cells, is O2 dependent, in fact data in support of this view are scant (Canessa et al. 1987; Joiner et al. 1993). Our results confirm this assertion only for normal (HbA) red blood cells. We show that these normal human red blood cells behave like those of fish (trout: Nielsen et al. 1992; carp: Jensen, 1992), horse (Gibson et al. 1995; Honess et al. 1996; Speake & Gibson, 1997) and sheep (Campbell & Gibson, 1998). The relationship between the activity of the cotransporter and PO2 is approximately sigmoidal (present data and Speake et al. 1997). In horse red blood cells, the P50 required for half-maximal activation of the swelling- or H+-stimulated Cl−-dependent K+ influx were 30-40 mmHg O2 (Speake & Gibson, 1997; Speake et al. 1997); that for urea-stimulated K+-Cl− cotransport was smaller (10-15 mmHg at 500 mm urea, Speake & Gibson, 1997). Exact P50s were more difficult to ascertain for HbA red blood cells, since the smaller magnitude of the fluxes makes the estimate less accurate, but a P50 of 36 ± 3 mmHg (mean ±s.e.m., n= 5) was observed for swelling/H+-stimulated cells, similar to that in horse red blood cells. Although 500 mm urea reduced the P50, the decrease (to 30 ± 3 mmHg; mean ±s.e.m., n= 5) was not as great as that observed previously in cells from horses (Speake & Gibson, 1997): the P50, however, is markedly dependent on the urea concentration and higher concentrations of urea applied to human cells might have reduced it further. The O2 sensitivity of K+-Cl− cotransport (or its regulatory pathways) in normal human red blood cells is such that arterial PO2s would support maximal activation by O2, the low PO2s in certain capillary beds would inactivate the transporter, whilst PO2s in mixed venous blood would support a level of activation intermediate between these extremes. Our experiments do not address the rates of activation/inactivation although these will obviously be relevant for determining the mean level of activation as the red blood cell circulates from high to low PO2s.

K+-Cl− cotransport activity in sickle red blood cells has a markedly different response to O2 compared with normal cells. At lower PO2s, the K+-Cl− cotransporter in sickle cells remains activated and capable of responding to H+ ions, swelling and urea. Indeed, the transport rate in pure N2 was not dissimilar to that observed at high ‘arterial’PO2s.

We were concerned that our data with sickle cells may be artefactual, particularly because of the marked ‘stickiness’ of HbS red blood cells at low PO2s, which may have prevented efficient removal of extracellular isotope during washing, thus artificially elevating K+‘influx’. This explanation, however, would not easily account for the Cl− dependence of the influx and a number of other observations also support the validity of our supposition. First, urea-treated cells were not ‘sticky’ at low PO2 but high Cl−-dependent K+ influxes were observed. Second, K+ efflux experiments gave the same pattern as those measuring K+ influx. Third, inhibitors of K+-Cl− cotransport (DIOA; Garay et al. 1989; Vitoux et al. 1989) and calyculin A (a specific protein phosphatase I and IIA inhibitor which also inhibits K+-Cl− cotransport, Starke & Jennings, 1993) reduced K+ influx to a similar degree at both high and low PO2s, and to that observed by substitution of Cl−; conversely, inhibitors of Ca2+-activated K+ channels (EGTA and nitrendipine) were without effect. Finally, we excluded the possibility that a decreased [Mg2+]i, following Mg2+ efflux via the deoxygenation-stimulated non-specific cation pathway (see later), may have modified the activity of the protein kinase/phosphatase enzymes regulating K+-Cl− cotransport (e.g. Lauf et al. 1992). The activation of this cation transport pathway at low PO2 was indicated in our experiments by the increase in the Cl−-independent K+ influx at very low PO2s. The addition of 1 mm external Mg2+, however, had no effect on K+-Cl− cotransport. (By a similar argument, at low PO2, EGTA could deplete cells of Mg2+, again via this non-specific cation pathway, and hence stimulate K+-Cl− cotransport. This was unlikely in our experiments, which were short < 10 min, and increased influx with EGTA (Table 1) was not significant.)

All our data with sickle cells are consistent with an active K+-Cl− cotransporter at low PO2s, in contradiction to claims in the literature. In fact, however, an examination of the relevant published work shows that substantial Cl−-dependent K+ fluxes were found in sickle cells at low PO2s (Canessa et al. 1987; Joiner et al. 1993), at variance with the authors’ interpretation that low PO2 inactivated K+-Cl− cotransporter in sickle cells, as in HbA-containing red blood cells: we believe that their findings should be re-evaluated.

It has been established previously that either the Ca2+-activated K+ channels or the K+-Cl− cotransporter can mediate dehydration of sickle cells in vitro, but their relative importance in vivo remains the subject of considerable debate. Sickle cells display a non-selective cation permeability, particularly at low PO2s when it may be stimulated by HbS polymerization. This pathway transports K+, Na+, Mg2+ and Ca2+ amongst other ions (for example: Tosteson, 1955; Berkowitz & Orringer, 1985; Joiner et al. 1988; Ortiz et al. 1990). The increased entry of Ca2+ mediated via this ‘channel’ (also known as Psickle, Lew et al. 1997) is thought to be important in elevating intracellular [Ca2+]i (Etzion et al. 1993) with consequent activation of the Ca2+-dependent K+ channel (or Gardos channel: Gardos, 1958). Dehydration of sickle cells, dependent on the presence of extracellular Ca2+, has indeed been observed under oxygenated or deoxygenated conditions (Ohnishi et al. 1986; Bookchin et al. 1991; Apovo et al. 1994; Franco et al. 1996; Lew et al. 1997), indicative of a role for Ca2+-activated K+ channels in solute loss.

Ascribing a role for K+-Cl− cotransport is more problematical. Certainly in oxygenated sickle cells it is activated by low pH or swelling, and in these cells can mediate solute loss and dehydration (Brugnara et al. 1986, 1989; Ellory et al. 1989b; Fabry et al. 1991). The model of red blood cell transport systems developed by Lew and colleagues (Lew et al. 1991) shows that transient activation of the Ca2+-activated K+ channel, for example via deoxygenation-stimulated Ca2+ influx, results in intracellular acidification. Extracellular acidification (for example in exercise-induced acidosis) and high concentrations of urea in the renal medulla would also provide stimulatory conditions to the K+-Cl− cotransporter. Such stimuli, however, would be encountered by predominantly deoxygenated sickle cells and their effect on the activity of the K+-Cl− cotransporter would depend on the O2 sensitivity of the system. Our results suggest that the K+-Cl− cotransporter would be able to respond to the stimulatory effects of low pH or high concentrations of urea irrespective of the PO2 encountered.

Consistent with our data are observations of Cl−-dependent dehydration of sickle cells during cyclical deoxygenation/oxygenation (Apovo et al. 1994; Franco et al. 1996). These cycles per se would cause transient intracellular acidifications (Dr V. L. Lew, Physiological Laboratory, University of Cambridge, personal communication), through effects on the isoelectric point (pI) of Hb, and thus lead to secondary stimulation of the K+-Cl− cotransporter. On the other hand, the abrogation of Cl−-dependent shrinkage during continuous deoxygenation (Ohnishi et al. 1986; Apovo et al. 1994; Franco et al. 1996) may suggest an O2 dependence of the K+-Cl− cotransporter, contrary to our findings, although prolonged deoxygenation in these experiments may have resulted in inactivation of the cotransporter.

An obvious question to address is what mechanism accounts for the different O2 dependence of the K+-Cl− cotransporter in normal and sickle red blood cells? There are a number of possible explanations. First, the high reticulocytosis may result in the presence in the circulation of younger red blood cells with unusual properties (as observed for LK sheep red blood cells, Lauf & Valet, 1983). It is certainly true that there is a marked cell heterogeneity in HbS blood, and the presence of ‘fast track’ dehydrated sickle cells is thought to be important (Bookchin et al. 1991; Fabry et al. 1991; Lew et al. 1991). Second, the amino acid mutation in the Hb β chain may be directly responsible - HbA and HbS have different O2 affinities and isoelectric points, and possibly different interactions with cytoskeletal or membrane moieties (as suggested by Brugnara et al. 1989). Polymers of HbS sequester a significant proportion of intracellular water, making it inaccessible to cytoplasmic constituents over a certain molecular weight (Lew et al. 1995). This may affect the activity of protein kinase/phosphatase enzymes, which regulate cotransport activity including its response to O2 (Cossins et al. 1994; Honess et al. 1996; Speake & Gibson, 1997), and thus distort the normal response to changes in PO2. Third, secondary changes in cell metabolism or other intracellular constituents, following repeated stimulation of the deoxygenation-activated Psickle, may occur. We are currently addressing these possibilities.

The present findings are of potential clinical importance. The notion that low PO2 inactivates the K+-Cl− cotransporter in sickle cells appears to be invalid. The transporter can thus respond to stimuli such as low pH or high urea concentrations even in regions of low PO2, resulting in loss of cellular solutes and dehydration, precipitating cell sickling. The development of therapeutic agents to inhibit specifically the K+-Cl− cotransporter of sickle cells remains a valuable goal (Garay et al. 1989; Ellory et al. 1990).

Acknowledgments

This work was supported by the Wellcome Trust. We thank Mrs S. Stevens for provision of the sickle cell samples.

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