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(The FASEB Journal. 2001;15:823-832.)
© 2001 FASEB

O2 dependence of K+ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79

J. S. GIBSON1, A. KHAN*, P. F. SPEAKE{dagger} and J. C. ELLORY*

Department of Physiol., St. George’s Hospital Medical School, University of London, Tooting, SW17 0RE;
* University Laboratory of Physiol., Oxford, OX1 3PT; and
{dagger} Academic Unit of Child Health, University of Manchester, Manchester, M13, U.K.

1Correspondence: Department of Physiology, St. George’s Hospital Medical School, University of London, Tooting, SW17 0RE, U.K. E-mail: jsgibson{at}sghms.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular basis of sickle cell disease (SCD) is well known but the pathophysiology is poorly understood. It remains intractable to therapy. Hyperactivity of several membrane transport systems, including the K+-Cl- cotransporter (termed KCC), cause HbS-containing red cells (termed HbS cells) to dehydrate and sickle, leading to the development of sickle cell crises (SCCs). Contrary to normal red cells (HbA cells), KCC in HbS cells is active at low O2 tensions (PO2s), remaining responsive to low pH or urea. Since these stimuli are usually encountered in hypoxic regions, the abnormal O2 dependence increases the contribution of KCC to dehydration, and hence development of SCCs. These differences with HbA cells may be due to the younger population of cells or to polymerization of HbS. We used 86Rb+ as a K+ congener to investigate the activity of KCC at different PO2s, and density gradient separation to investigate different red cell fractions. We found no correlation of O2 dependence with cell fractions. We also used the substituted benzaldehyde 12C79 to increase the O2 affinity of HbS and found that its effect on HbS O2 saturation and cell sickling correlated with that on both Cl--independent and Cl--dependent K+ transport, implying that, at low PO2s, KCC activity correlated with HbS polymerization. The importance of these results to understanding the pathophysiology of SCD, and for the design of chemotherapeutic agents to ameliorate or prevent SCC, is discussed.—Gibson, J. S., Khan, A., Speake, P. F., Ellory, J. C. O2 dependence of K+ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79.


Key Words: oxygen • potassium • HbS • HbA • red cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SICKLE CELL DISEASE (SCD) is an important hematological disorder with a simple molecular basis. A single base mutation results in an amino acid substitution at ß 6, valine replacing glutamic acid, producing HbS (cf HbA in normal individuals) (1 , 2) . The resultant loss of a negative charge allows deoxygenated HbS to polymerize. The rodlike HbS polymers distort the red cell shape into the characteristic sickled appearance, impeding flow through the microvasculature, leading to ischemia, pain and death (3) , features of sickle cell crisis (SCC). The molecular characterization of SCD was achieved some 50 years ago (4) ; it has been called ‘the first molecular disease’, but the details of how it alters red cell and circulatory function remain poorly understood. There is no effective therapy.

The hydration state of HbS-containing red cells (here termed HbS cells) is critical because the lag time to polymerization after deoxygenation is inversely proportional to approximately the 30th power of [HbS] (5) . A small decrease in cell volume and a small increase in [HbS], makes polymerization and SCC much more likely. Unfortunately, the volume of HbS cells is very labile; they are prone to rapid and irreversible shrinkage. Three membrane transporters, quiescent or absent in HbA red cells but active in HbS cells, contribute to dehydration: the K+-Cl- cotransporter (termed KCC and probably the KCC1 isoform) (6 7 8) , the Gardos channel (probably IK1) (9 10 11 12) , and Psickle (13 14 15 16 17) . KCC mediates coupled KCl efflux (18) ; the Gardos channel is a Ca2+-activated K+ channel (9) , mediating conductive K+ efflux, coupled electrically to Cl- efflux through distinct Cl- channels or band 3; Psickle is a cation-selective but fairly nonspecific pathway that allows entry of Ca2+ (as well as passage of other ions) (19) , but whose molecular identity remains obscure. The relative contribution of these pathways to dehydration and sickling remains controversial (20 21 22 23 24) and indeed may be variable. A thorough knowledge of their behavior is required to understand the pathogenesis of SCD.

KCC activity in red cells is regulated by a number of physiological factors (25) . Well-established stimuli include cell swelling (18 , 26) , H+ ions (27 , 28) , and urea (29 30 31 32) . In common with many other red cell membrane transport systems (33 , 34) , it is also regulated by physiological O2 tensions (PO2s). In normal human red cells (termed HbA cells) and red cells from many other vertebrate species (35 36 37 38 39) , KCC is fully O2 dependent. It is activated at high PO2s, whereas at lower levels it is inactive and refractory to other stimuli. We have shown that this requirement for O2 is lost in HbS cells (39) : the transporter remains active and stimulated by swelling, H+ ions, and urea even in the complete absence of O2.

The mechanism responsible for the different O2 dependence of KCC in HbA and HbS cells is not understood. HbS cells are markedly heterogeneous and have a reduced life span (40 , 41) . As suggested for other properties of sickle cells (see, for example, refs 42 , 43 ), the abnormal O2 response may reside in a subpopulation of HbS cells. It may result from their younger age. In addition, polymerization of HbS at low PO2s is an obvious difference. In the experiments described in this paper, HbS and HbA cell samples were fractionated by density gradient centrifugation and the substituted benzaldehyde 12C79 (44) was used to increase the O2 affinity of HbS, thereby altering the relationship between HbS saturation/polymerization and PO2. For both series of experiments, the effects on the O2 dependence of KCC was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Arabinogalactan (Stractan), bumetanide, 3-[N-morpholino] propane sulfonic acid (MOPS), N-ethylmaleimide, ouabain, and salts were all purchased from Sigma Chemical Co. (Poole, Dorset, U.K.), 86Rb was from NEN Du Pont (Stevenage, U.K.), and calyculin A from Calbiochem (Nottingham, U.K.). 12C79 was a gift from Glaxo Wellcome (Stevenage, U.K.).

Solutions
The standard medium was MOPS-buffered saline (MBS) comprising (in mM); NaCl (145), MOPS (10), glucose (5), pH 7.4, at 37°C. MBS was nominally free of Ca2+. To investigate the effects of Cl- substitution, NaCl was replaced by equimolar NaNO3. In some experiments pH was adjusted with HNO3, giving pH 7 after addition of cells. These salines had an osmolality of 290 mOsm.kg-1. In experiments at pH 7 with HbA cells, osmolality was reduced to 260 mOsm.kg-1 by addition of distilled water. The two stimuli were combined because HbA cells have a low KCC activity. To investigate the effect of urea, stock solutions of 5 M urea were made daily in the appropriate saline and diluted 10-fold to give a final urea concentration of 500 mM.

Blood
Samples of normal (HbAA) and sickle (HbSS) red blood cells were obtained with ethical permission by venepuncture of consenting volunteers. Blood was collected in heparin-containing syringes and stored at 4°C until needed (within 48 h). White cells were removed by filtration through glass wool and red cells were then washed 3 times by centrifugation (2000 g; 5 min at 4°C) in MBS to remove platelets and plasma. Discontinuous arabinogalactan gradients were prepared as described by Ortiz et al. (45) , with densities 1.090 g/ml-1 and 1.100 g/ml-1 for HbS cells, and 1.087 g/ml-1, 1.089 g/ml-1 and 1.095 g/ml-1 for HbA cells, resting on a dense cushion of 1.160 g/ml-1. Red cell samples were layered on top of the gradients and centrifuged for 45 min at 52,000 g, 4°C. Cells were harvested from the top and bottom of the gradients using a Pasteur pipette to yield low- and high-density red cells, respectively. Contaminant arabinogalactan was removed by washing 3 times in MBS (2000 g, 5 min at 4°C). The red cells were then washed an additional 3 times in MBS ± Cl- to achieve Cl- substitution. Unfractionated controls were centrifuged and washed for the same number of times, but were not exposed to the arabinogalactan gradients.

Tonometry
Oxygen tension (PO2) was controlled by incubating cells in Eschweiler tonometers coupled to a Wösthoff gas mixing pump as described previously (31) . All experiments were carried out at 1 ATA. PO2 was varied by replacement of O2 with N2, and all gas mixtures were fully saturated with water vapor at 37°C prior to delivery to the tonometers. Blood samples in Cl--free MBS were incubated at ~40% hematocrit for 15 min at each PO2 before influx measurement; control experiments showed that this interval was adequate for complete equilibration. Samples were then removed and diluted 10-fold into MBS (± Cl-) containing urea (500 mM; pH 7.4), pH 7 (for HbS cells) or MBS 260 mOsm.kg-1 at pH 7 (for HbA cells), pre-equilibrated to the same PO2, for measurement of K+ influx. HbA cells were exposed to both swelling and reduction in pH because these cells have a relatively modest KCC activity. Radioisotope was added after 10 min to allow sufficient time for pH stabilization or urea stimulation. To measure O2 saturation, samples from the tonometers were processed following the method of Tucker (46) . To assess sickling, other cell aliquots were taken directly from tonometers and fixed in MBS with 1% glutaraldehyde equilibrated at the same PO2 as the tonometers. Percentage of sickled cells were then counted using an hemocytometer.

12C79
Stock solutions of 12C79 (282 mM) were made daily in Tris base (500 mM) and diluted in the appropriate saline to give a final concentration of 5 mM. Cell samples at 40% hematocrit were incubated with 12C79 (5 mM) in air for 15 min before placing them in tonometers to adjust PO2. Measurement of both O2 saturation and K+ influx was made in the presence of 12C79 (5 mM).

Flux measurements
K+ influxes were determined at 37°C over a 10 min period (during which uptake was linear). Although K+ influx was measured, because of the outwardly facing chemical gradient for K+ there would in fact be a net loss of ions through the various K+ pathways; nevertheless, influx measurements indicate their activity (18) . 86Rb was used as a congener added in KNO3 to give a final K+ concentration of 7.5 mM (see ref 18 ). Ouabain and bumetanide (100 µM) were present in all experiments to inhibit the Na/K pump and NaK2Cl cotransporter, respectively. The Cl--dependence of K+ influx was taken as a measure of KCC activity (Cl- substituted with NO3-): in some cases, influxes in both Cl- and NO3- media are presented; in others, for clarity, only the Cl--dependent flux is given (calculated as the difference in influx ± Cl-). Hematocrit was determined by optical density using Drabkins solution (following the method of Beutler; see refs 18 , 47 ). K+ influxes are expressed in standard units of mmol K+.(l cells.h)-1.

Statistics
Results are presented as the mean ± SD for single experiments, representative of at least 2 others, or as mean ± SE for n experiments. Comparisons were made using Student’s t test. For Fig. 4b and Fig. 5b, c , the lines of best fit were made by linear regression using Sigmaplot (Jandel Scientific, Corte Madera, Calif.).



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  		Figure 4. Effect of 12C79 on Cl--independent K+ influx in HbS cells. Data for control cells are represented by open circles, those from cells treated with 12C79 (5 mM) by filled circles, protocol similar to that described in Fig. 3 . a) Cl--independent K+ influxes, expressed as a percentage of influx measured at 0 mmHg (2.82 and 1.85 mmol/l cells.h for control and 12C79-treated HbS cells, respectively) against O2 tension. b) Relationship between Cl--independent influxes and sickling (from Fig. 3b ). Cl--independent K+ influxes and percentage sickling at O2 tensions of <= 40 mmHg are plotted against each other. The plot is drawn by linear regression (r2=0.92) and shows a significant correlation (P<0.01) between the two components of K+ influx. Symbols represent mean percentage influxes ± SE (n=5–8 different patients) or n=3 for sickling.



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  		Figure 5. Effect of 12C79 on Cl--dependent K+ influx in HbS cells. Protocol as described in legend to Fig. 4 . Data for control cells are represented by open circles, those from cells treated with 12C79 (5 mM) by filled circles. a) Cl--dependent K+ influxes, expressed as a percentage of influx measured at 150 mmHg at different PO2s (2.98 and 2.03 mmol/l cells.h for control and 12C79-treated cells, respectively). b) Relationship between Cl--dependent and sickling (from Fig. 3b ). Cl--dependent K+ influxes and percentage sickling at O2 tensions of <= 40 mmHg are plotted against each other; r2=0.97, P<0.002. c) Relationship between Cl--independent (raw data from Fig. 4 ) and Cl--dependent K+ influxes. Cl--dependent K+ influxes and Cl--independent K+ influxes (given as mmol/l cells h) at O2 tensions of <= 40 mmHg are plotted against each other; r2 = 0.77, P<0.01. Symbols represent mean influxes (n=5–8 different patients) or mean percentages of sickled cells (n=3).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
O2 dependence of KCC activity in HbA and HbS cells
Figure 1 shows a direct comparison of Cl--dependent K+ influx (a measure of KCC activity) in HbA and HbS cells at different O2 tensions. HbA cells showed the typical pattern for KCC activity: maximal activity at high PO2s, inhibition as PO2 was reduced, and minimal activity at low PO2s. By contrast, in HbS cells, KCC activity had a biphasic relationship with PO2. At higher PO2s, it followed the relationship observed in HbA cells, decreasing with PO2 from 100 to 40 mmHg. At lower PO2s, from 40 to 0 mmHg, KCC in HbS cells was progressively stimulated so that at the lowest PO2s, activity was not dissimilar in magnitude to that observed in fully oxygenated cells. We have shown previously that this Cl--dependent K+ influx in HbS cells held at low PO2s was inhibited by [(dihydroindenyl)oxy]alkanoic acid (DIOA, a direct inhibitor of KCC148,49) and by calyculin A (a specific protein phosphatase inhibitor that interferes with the dephosphorylation that activates KCC in red cells from many species (50) . By contrast, it was unaffected by Ca2+ chelation or clotrimazole (39) (which will inhibit the Gardos channel). This pharmacological profile is consistent with mediation by KCC.



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  		Figure 1. Comparison of the O2 dependence of Cl--dependent K+ influx in HbA and HbS cells. HbS cells were incubated in tonometers for 15 min at the indicated O2 tensions before 10-fold dilution into hypotonic (260 mOsm.kg-1, pH 7.4) MBS ± Cl- (substituted with NO3-), pre-equilibrated at the same O2 tension. HbA cells were treated similarly except that influxes were measured at pH 7. Cl--dependent K+ influxes were calculated as the difference in K+ influx measured in the presence or absence of Cl-. Ouabain and bumetanide (both at 100 µM) were present in all cases. Values are expressed as a percentage of the influx at high PO2 (150 mmHg: 1.31 and 2.98 mmol/l cells.h for HbA and HbS cells, respectively). Symbols represent means ± SE, n=5 (HbS) or 7 (HbA).

KCC activity in subpopulations of red cells
The pattern of K+ influxes was investigated in subfractions of HbA and HbS cells after density gradient separation. In all cases, K+ influx was greatest in the light fraction and smallest in the dense fraction, with the Cl--dependent component of K+ influx responsible for most of this difference. For HbS cells, combining results for stimulation by both urea and H+ ions, Cl--dependent K+ influx in oxygenated low-density cells was 1.8 ± 0.2-fold greater than that in unfractionated cells, whereas influx in the high-density fraction was reduced to 19 ± 4% that in unfractionated cells (both means ± SE, n=10). For HbA cells, Cl--dependent K+ influxes in oxygenated low-density fractions were 2.7 ± 0.6-fold (mean ± SE, n=6) larger than those in unfractionated blood.

The O2 dependence of KCC in fractionated red cells is shown in Fig. 2a for HbS cells and in Fig. 2b for HbA cells. As in Fig. 1 , Cl--dependent K+ influxes in unfractionated HbS cell samples were similar in magnitude when measured in air or N2 (Fig. 2a ). K+ influx in the low-density fraction, although larger in magnitude, was also largely independent of PO2. Figure 2a presents activity of KCC in HbS cells after stimulation by urea, but similar results were observed after stimulation by H+ ions (data not shown). Sensitivity of KCC to calyculin A was also investigated. For low-density HbS cells and when combining results for stimulation by H+ ions or urea, mean inhibition of KCC by calyculin A (100 nM) was 80 ± 5% in air and 83 ± 10% in N2 (means ± SE, n=4).



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  		Figure 2. O2 dependence of urea-stimulated K+ influx in different fractions of HbS or HbA cells. Cells were separated into low- or high-density fractions or left unfractionated. They were then incubated at 40% hematocrit for 15 min at the PO2 indicated before 10-fold dilution into MBS ± Cl- (substituted with NO3-), pre-equilibrated at the same O2 tension and containing urea (500 mM, pH 7.4). K+ influx (mmol(l cells.h)-1) was measured and the Cl--dependent component calculated as the difference in K+ influx measured in the presence or absence of Cl-. Ouabain and bumetanide (both 100 µM) were present in all cases. a) Cl--dependent K+ influx in different fractions of HbS cells. Histograms represent means ± SE for 3 different patients. b) K+ influx in low-density HbA red cells. Symbols represent means ± SD of triplicate determinations in cells from a single individual.

The effect of PO2 on K+ influxes in low-density HbA cells stimulated by urea is shown in Fig. 2b . As for unfractionated cells (Fig. 1) , the relationship between PO2 and Cl--dependent K+ influx was sigmoidal, with flux increasing with PO2. Peak influxes were observed by ~80 mmHg whereas PO2 required for half-maximal activation was 29 ± 2 (urea) and 37 ± 3 (H+ ions/swelling) mmHg (means ± SE, n=3). In N2, Cl--dependent K+ influxes were low: 14 ± 2% or 15 ± 5% (means ± SE, n=5) measured at PO2 of 100 mmHg for urea or H+ ion/swelling stimulation.

Effect of 12C79 on O2 saturation and sickling
The next series of experiments was designed to test the hypothesis that HbS polymerization is involved in the abnormal O2 response of KCC in HbS cells. We used the substituted benzaldehyde 12C79 (44) to stabilize HbS in the oxy conformation and thereby reduce HbS polymerization. The effect of 12C79 on the O2 saturation curve of HbS cells is shown in Fig. 3a . In its presence, the relationship between O2 saturation and PO2 was shifted toward lower PO2s. The PO2 required for half-maximal O2 saturation decreased considerably (Table 1 ), indicative of a marked increase in O2 affinity. Since polymers of deoxygenated HbS are responsible for distorting the red cell shape into the sickled appearance, sickling represents a direct measure of polymerization. We therefore investigated the effect of 12C79 on the O2 dependence of sickling. In the presence of 12C79, sickling was also shifted toward significantly lower PO2s (Fig. 3b , Table 1 ).



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  		Figure 3. Effect of 12C79 on O2 saturation and sickling of HbS cells. Oxygenated HbS cell samples were incubated for 15 min in the presence or absence of 12C79 (5 mM). They were then placed in tonometers at the indicated O2 tensions for a further 15 min before determination of a) O2 saturation or b) sickling. O2 saturation was determined following the method of Tucker (46) and given as a percentage of the maximal value in fully oxygenated cells. For determination of sickling, cell aliquots were taken from the tonometers and immediately fixed in MBS containing 1% glutaraldehyde for cell counting in an hemocytometer. The number of sickled cells is expressed as a percentage of those present at 0 mmHg (76% and 65% for control and 12C79-treated HbS cells, respectively). Symbols represent means ± SD of triplicate determinations in cells from a single patient.


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  		Table 1. The effect of 12C79 on O2 saturation, cell sickling and K+ transport in HbS cellsa

Effect of 12C79 on Psickle
The nonselective cation channel (Psickle) activated stochastically upon deoxygenation (43) is also associated with HbS polymerization. We used Cl--independent K+ influx as a measure of its activity. This component of K+ influx was increased by deoxygenation (Fig. 4a ). Upon addition of 12C79, activation of Cl--independent K+ influxes by deoxygenation was shifted to lower PO2s. In both untreated HbS cells and in those treated with 12C79, the PO2s required for half-maximal activation of Cl--independent K+ influxes were similar to those required for half-maximal saturation of HbS with O2 (Table 1) . Figure 4b shows the correlation between sickling and activity of Cl--independent K+ influxes, which was significant (r2=0.92, P<0.003).

Effect of 12C79 on KCC activity
We then examined the effects of 12C79 on the activity of KCC, measured as Cl--dependent K+ influx. We concentrated on the PO2 range over which the O2 dependence of KCC differs markedly when comparing HbA and HbS cells: at low PO2s, decreasing from 40 to 0 mmHg, KCC in HbA red cells is progressively inhibited; that in HbS cells is progressively stimulated (Fig. 1) . In untreated HbS cells, stimulation of KCC at low PO2s was half-maximal at a PO2, similar to that required for half-maximal O2 saturation of HbS (Table 1) . In the presence of 12C79, several changes are apparent (Fig. 5a ; Table 1 ). First, from PO2s of 40 to 100 mmHg, inactivation of KCC was shifted slightly toward lower PO2s. Second, inhibition of KCC activity at 40 mmHg was less marked. Third, the low PO2 stimulation (between 0 and 40 mmHg) of KCC was shifted significantly toward lower PO2s (Table 1) . As for Psickle, the PO2 required for half-maximal stimulation of this component of KCC was also similar to that required for half-maximal O2 saturation and cell sickling (Table 1) . The relationship between activity of KCC at low PO2s and sickling correlated significantly (Fig. 5b ; r2=0.87, P<0.001). Finally, we examined the relationship between Cl--dependent K+ influxes (via KCC) and Cl--independent ones at PO2s of <= 40 mmHg (Fig. 5c ). This also showed a significant correlation (r2=0.77, P<0.01) between K+ influx through the two pathways. Thus, all four parameters measured in Table 1 showed a similar O2 dependence under control conditions or after treatment with 12C79.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results described in this paper demonstrate that, in HbS cells at low PO2s, both Cl--independent K+ influxes and Cl--dependent K+ influxes (indicative of the activities of Psickle and KCC, respectively) correlate with deoxygenation of HbS and cell sickling. Results also confirm that KCC activity stimulated by swelling or H+ ions was highest in the least dense fractions of HbS and HbA cells and show that the same applied to urea-stimulated KCC. The opposite responses of KCC to low PO2 in HbA and HbS cells (i.e., inactivation of KCC at in HbA cells but not in HbS cells) occurred in all density separated fractions. These findings are important because they imply that the high activity of KCC in HbS cells at low PO2 is not due to the presence of a younger population of cells per se; they also emphasize the stimulatory effect of HbS deoxygenation, consistent with a role for HbS polymerization.

A thorough characterization of cation transport in HbS cells is directly relevant to understanding the pathophysiology of SCD because of its contribution to dehydration and sickling. Three pathways—KCC, the Gardos channel, and Psickle—are involved (reviewed in refs 51 , 52 ). Two of these transport pathways are O2 sensitive. First, Psickle opens stochastically upon deoxygenation, probably subsequent to HbS polymerization (43 , 52 53 54) . Second, KCC activity in HbA red cells (and those from a number of other vertebrate species (reviewed by Gibson et al., ref 34 ), is O2 dependent [e.g., trout (35 , 36) , horse (55) , sheep (38) , human HbA (39) ]. KCC in HbS cells, however, has an abnormal O2 response, remaining active at low PO2s(39) . These findings are important because red cells will encounter H+ ions predominantly in active muscle beds or ischemic tissues and urea in the renal medulla, all relatively hypoxic areas of the circulation. The abnormal O2 dependence of HbS cell KCC enables it to respond to these stimuli (32 , 39) despite the hypoxia and thereby mediate inappropriate and deleterious cell dehydration. In addition, it will enable stimulation of KCC by intracellular acidification due to K+ efflux after activation of the Gardos channel (56) , an event that occurs only in deoxygenated HbS cells. In this paper, we have investigated the O2 dependence of K+ transport in red cells in an attempt to uncover the mechanism responsible for the abnormal O2 dependence of KCC in HbS cells.

The activity of KCC is highest in young mammalian red cells, decreasing as the cells mature (25) . In unfractionated HbA cells, the activity of KCC is very low and stimulation by physiological factors such as H+ ions and swelling is small (26) . The transporter is present, however, and its activity can be markedly stimulated pharmacologically, e.g., by treatment with N-ethylmaleimide or staurosporine or by application of high hydrostatic pressure (8 , 57 , 58) . It is probable that part of the necessary signaling cascade is lost as cells mature rather than the transporter per se. When HbA cells are density fractionated, the low-density fraction of HbA cells, representing the youngest subpopulation, is enriched for KCC activity and exhibits greater responses to swelling and other stimuli than unfractionated cells (26 , 58 , 59) . Because of their decreased life span, HbS cells represent a younger average population than HbA red cells. As for HbA cells, most KCC activity is found in cells from the lowest density fraction (21 , 27 , 42 , 58 59 60 61) (although not all of the youngest cells are found in this fraction because some will have undergone rapid ‘fast-track’ dehydration (21 , 42) . Young red cells could have different O2 responses from mature red cells, thus accounting for the unusual behavior of KCC in HbS cells. We found, however, that low-density HbA red cells exhibited a cotransporter activity that was completely inhibited at low PO2s, like that in unfractionated HbA red cells. Stimulation by urea or H+ ions and swelling was inhibited at low PO2s. KCC activity in low-density HbS cells also retained the features observed in unfractionated samples, remaining highly active at low PO2s. These findings imply that the abnormal O2 response of KCC in HbS cells is not confined to a particular subfraction separable by density.

The ability of HbS to form polymers represents another obvious difference between HbS and HbA cells (2 , 51) . HbS polymerization has several consequences: membrane distortion and damage, sequestration of a large proportion of cell water, significant metabolic effects and changes in membrane permeability, including increased cation permeability via Psickle (2 , 52 , 53) . We used the substituted benzaldehyde 12C79 to increase O2 affinity and thereby inhibit polymer formation (44) . Cl--independent K+ influx, which in HbA cells is unaffected by 12C79 (63) , was taken as a measure of Psickle. With sufficient Ca2+ entry via Psickle, Cl--independent K+ transport would also occur via the Gardos channel; however, the present experiments were carried out in the nominal absence of extracellular Ca2+. Under the conditions of the present experiments (15 min deoxygenation), addition of Ca2+ (2.5 mM) or EGTA (100 µM) to the nominally Ca2+-free MBS or addition of clotrimazole (10 µM) did not affect results (data not shown), suggesting that the Gardos channel remained quiescent. While this may appear to suggest a lack of deoxygenation-induced Ca2+ permeability incompatible with the properties of Psickle, it is not a unique finding. Joiner and colleagues (22 , 62) have shown that measurable transport via the Gardos channel is not always observed in unfractionated HbS cells upon deoxygenation. They suggest that most activity resides in a small subpopulation of HbS cells (perhaps those with least Ca2+ pump activity) or that experimental conditions (for example, duration of deoxygenation, which in some cases may be several hours) are critical to allow sufficient Ca2+ entry via Psickle. In an additional set of experiments in which cells were deoxygenated in the presence of 2.5 mM Ca2+ for 60 min, we did observe activation of a Ca2+-dependent, clotrimazole-sensitive component.

We confirm that Cl--independent K+ influxes were stimulated as PO2 was reduced, as expected for mediation via Psickle. In control HbS cells, O2-dependence was similar to that in a previous report that involved a different method (passive K+ and Na+ fluxes) to monitor Psickle (22) . Upon addition of 12C79, the stimulation of Cl--independent K+ influxes occurred at lower PO2s, so that in both control cells and those treated with 12C79, the PO2s required for half-maximal activation of Cl--independent K+ influx were similar to those required for half-maximal saturation of HbS with O2 and half-maximal sickling (Table 1) . Percentage sickling was significantly correlated with activity of the Cl--independent K+ pathway, again consistent with mediation via Psickle. These findings are fully consistent with activation of Psickle by HbS polymerization.

The O2 dependence of Cl--dependent K+ transport pathway (usually taken as via KCC) was also altered by 12C79. As for the Cl--independent flux, the PO2 required for half-maximal stimulation (P50) of Cl--dependent K+ transport at low PO2s (0–40 mmHg) was also similar to that required for half-maximal O2 saturation and half-maximal sickling (Table 1) . Cl--dependent K+ influxes correlated significantly with sickling and with Cl--independent influxes (Fig. 5b , c ). These correlations may indicate a common underlying mechanism involved in the activation of the two transport pathways. Although the existence of parallel transduction pathways is a possibility, a role for the O2 saturation of Hb is suggested by the similarities in P50s and also argues against a role for other heme-containing proteins. Taken together, our findings suggest that the stimulation of KCC in HbS cells at low PO2s, like sickling and activation of Psickle, is also caused by HbS deoxygenation. They may indicate a role for HbS polymerization. Psickle may open subsequent to membrane distortion by HbS polymers, and the same mechanism could account for stimulation of KCC. In many cells, KCC mediates volume regulatory decrease (RVD); in these responses, it is stimulated by cell swelling (64) . It is conceivable that distortion of the red cell membrane by HbS polymers may activate KCC via this pathway. An intriguing alternative possibility is that loss of soluble HbS into polymers reduces macromolecular crowding (65) , thereby reducing the set point volume of the cell and activating RVD mechanisms (C. H. Joiner, personal communication). The transporter is also controlled by protein phosphorylation involving a cascade of, as yet, unidentified regulatory kinase and phosphatase enzymes (66 , 67) . The function of these may be subverted by polymerization of HbS, although it is important to note that HbS cell KCC retains its sensitivity to calyculin A at low PO2s. Finally, the interaction of Hb with other membrane-bound proteins, notably band 3, has been implicated in the response of membrane transporters to O2 (reviewed in ref 68 ). The abnormal O2 dependence of HbS cell KCC may follow from the altered behavior of HbS at this site.

Entrainment of KCC activity with HbS deoxygenation and polymerization represents a novel hypothesis and would provide a further mechanism to add to the other detrimental features of HbS cell pathophysiology (see Fig. 6 for a model). Once cells start to sickle, stimulation of KCC would mediate further solute loss, raising [HbS] and thus promoting polymerization. These events not only represent positive feedback contributing to the marked instability of HbS cells, but also indicate future chemotherapeutic strategies (as presented schematically in Fig. 6 ). For example, although it is difficult to achieve sufficiently high levels of transport inhibitors to abrogate completely the individual transport pathways, combination therapy with low doses of ‘left shift’ reagents, coupled with KCC and/or Gardos channel inhibitors, may be tolerated clinically and act synergistically to stabilize sickle volume and prevent the development of sickle cell crises.



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  		Figure 6. Model of the transport pathways involved in sickle cell dehydration. a) High O2 tension (PO2): Psickle is inactive because oxyHb does not polymerize; insufficient Ca2+ enters the cell to activate the Gardos channel (ascribed IKCa); the K+-Cl- cotransporter (KCC) is active, but is not stimulated unless pH is low or urea levels are high. b) Low O2 tension (PO2): deoxyHb forms polymers that open Psickle stochastically; Ca2+ entry via Psickle activates IKCa; KCC is stimulated by sickling, by intracellular acidification on K+ loss via IKCa and in areas of low pH and high urea. Solute loss via IKCa and KCC increase [HbS] further promoting sickling. There is considerable positive feedback, cell volume is unstable, and the cell rapidly progresses to an irreversibly sickled state. c) Irreversible sickling may arise from state (b) via positive feedback or, more rarely (hence dashed arrow), from state (a) if KCC stimulation by low pH or urea is adequate to cause dehydration. d) Stabilization: inhibition of the transporters IKCa (e.g., by clotrimazole) and KCC (e.g., by DIOA), together with increased O2 affinity of HbS induced by 12C79, act synergistically to stabilize cell volume. KCC, K+-Cl- cotransporter; IKCa, Ca2+-activated K+ channel (Gardos channel); Psickle, deoxygenation-induced cation-selective channel; oxyHb, oxygenated Hb; deoxyHb, dexoygenated Hb; Hb, either oxy or deoxyHb; ISC, irreversibly sickled cells; DIOA, [(dihydroindenyl)oxy] alkanoic acid; +++, stimulation; --, inhibition.


   ACKNOWLEDGMENTS
 
This work is supported by Action Research and the Wellcome Trust. We thank Mrs. S. Stevens for acquisition of the sickle cell samples and Glaxo Wellcome for the gift of 12C79.

Received for publication April 5, 2000. Revision received August 11, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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