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MS 8770 Received 28 September 1998; accepted after revision 7 December 1998.
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ABSTRACT |
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INTRODUCTION |
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K+-Cl- cotransport is a feature of red blood cells from many vertebrate species (see Lauf et al. 1992 for a review). The cotransporter is stimulated by swelling, but other potential physiological modulators include H+ ions, urea and O2 (Lauf et al. 1992; Ellory et al. 1998). Recently, it has become apparent that, in red cells from many species, O2 exerts an overriding control (trout: Borgese et al. 1991; Nielsen et al. 1992; carp: Jensen, 1992; horse: Gibson et al. 1995a; sheep: Campbell & Gibson, 1998a; human: Canessa et al. 1987; Gibson et al. 1998). The K+-Cl- cotransporter requires adequate levels of O2 tension (PO2) for activation, and only following O2 activation can it respond to the modulation by cell volume, H+ ions and moderate (but not high) concentrations of urea (Speake & Gibson, 1997; Speake et al. 1997; Gibson et al. 1998). This 'permissive' action of O2 will be relevant to the red cell in vivo: it occurs over physiological PO2 values (Speake et al. 1997; Gibson et al. 1998).
The mechanism underlying O2 activation remains unclear. As for the other physiological stimuli (volume, H+ ions and urea), cellular protein kinase and phosphatase enzymes are implicated (Cossins et al. 1994; Honess et al. 1996). Dephosphorylation of the transporter per se, or of some unidentified regulatory protein, is associated with increased transport activity; phosphorylation inhibits (see Lauf et al. 1992 for references). Fluctuations in free [Mg2+]i occur in red cells during oxygenation-deoxygenation cycles (Flatman, 1980), and it has been hypothesized that these changes initiate events in the signalling pathway linking changes in PO2 to alteration in cotransport activity (Canessa et al. 1987), presumably via the regulatory phosphorylation cascade. This postulate is widely quoted (Canessa et al. 1987; Joiner, 1993; Joiner et al. 1993, 1998).
We have shown that O2-dependent K+-Cl- cotransport occurs in low potassium-containing (LK) sheep red cells, a species whose Hb lacks the capacity for binding organic phosphates, and Mg2+ clamping in sheep red cells did not abolish the O2-stimulated fluxes (Campbell & Gibson, 1998a). These observations throw doubt on the role of [Mg2+]i in regulation of the cotransporter by O2. Recently, it has been proposed that the cotransporter in Mg2+-clamped human sickle cells shows the opposite response to O2 and that its activity is in fact increased by deoxygenation (Joiner et al. 1998). Because of this, we have assessed the role of O2 and Mg2+ clamping in control of the K+-Cl- cotransporter in equine red cells. Unlike sickle cells, but like those from sheep, we show that O2 stimulates K+-Cl- cotransport even in Mg2+-clamped equine red cells. Results imply that control by O2 is mediated by some other mechanism than changes in [Mg2+]i.
A preliminary account of this work has been presented previously (Gibson et al. 1995b; Campbell & Gibson, 1998b).
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METHODS |
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Materials and salines
The standard saline (MBS) comprised (mM): 145 NaCl, 5 glucose and 10 Mops (pH 7·4 at 37°C; 290 ± 5 mosmol kg-1). For experiments in which Cl- dependence of K+ influx was examined, Cl- was substituted with NO3- or MeSO4-; for those shown in Fig. 4, saline osmolality was reduced by addition of distilled water. K+ was added with the isotope (86Rb+) to give a final [K+] of 7·5 mM. Stocks of A23187 (10 mM) were prepared in DMSO and used at a final concentration of 20 µM, those of ouabain (10 mM) were prepared with water and used at a final concentration of 100 µM.
Sample collection and handling
Blood samples were obtained from horses kept at the Department of Veterinary Clinical Sciences and Animal Husbandry, Leahurst, by jugular venepuncture into heparinized vacutainers and prepared as previously described (Speake et al. 1997).
Tonometry
Before flux measurement, red cell suspensions were incubated at about 50 % haematocrit in glass tonometers flushed with N2 or O2, warmed to 37°C and fully humidified through three humidifiers prior to delivery. In most experiments, samples were deoxygenated for 60 min and then either held in N2 or incubated with O2 for 15 min before dilution into saline at low haematocrit (about 5 %) pre-equilibrated and maintained at the requisite PO2 for influx measurement, alteration of [Mg2+]i or pH determinations, as appropriate (see Campbell & Gibson, 1998a, for details).
K+ influx
K+ influx was measured at 37°C using 86Rb+ (DuPont-NEN, Bad Homburg, Germany) as a tracer for K+ (Dunham & Ellory, 1981). Ouabain (100 µM) was present in all experiments, obviating any K+ influx through the Na+, K+-ATPase. Haematocrit was measured either by the cyanomethaemoglobin method or by microhaematocrit determination. Influxes are expressed as millimoles of K+ per litre of cells per hour (mmol (l cells)-1 h-1).
Measurement of cell volume
Cell water content was determined by the wet weight/dry weight method of Borgese et al. 1991 and expressed as millilitres per gram of dry cell solids.
Measurement of extracellular and intracellular pH
Extracellular pH (pHo) was measured directly in the presence of cells under the same experimental conditions as for Mg2+ measurement (see below). For measurement of intracellular pH (pHi), 1 ml of the same cell suspension used for Mg2+ determination was centrifuged through dibutylphthalate oil to separate the red cells from the incubation medium. The cell pellet was lysed (by freezing and thawing) and pHi measured directly (see Campbell & Gibson, 1998a, for details). pH values were measured in both oxygenated and deoxygenated samples. Control experiments showed that the oil was not significantly permeable to O2 over the time course of the pHi measurements.
Measurement of Mg2+ content
Total intracellular Mg2+ concentration ([Mg2+]i) was determined by atomic absorption spectroscopy following the method of Flatman & Lew (1980). Briefly, cell suspensions of about 5 % haematocrit were incubated for 20 min at 37°C in MBS containing 20 µM A23187, the required extracellular Mg2+ concentration ([Mg2+]o) and 50 µM EGTA. Cell aliquots were then diluted into ice-cold MBS containing 2 mM EDTA layered on dibutylphthalate oil and centrifuged. The resultant cell pellet was lysed and deproteinated for determination of the Mg2+ absorbance. Controls showed that trapped extracellular fluid had no effect on the measurements.
Statistics
Unless stated otherwise, all values are the mean ±
Effect of A23187 on equine red cells
In control experiments, total [Mg2+]i was 2·55 ± 0·07 mM (n = 6). In the presence of A23187, cells rapidly became loaded with or depleted of Mg2+ depending on [Mg2+]o, with stable values reached within 5 min (data not shown). In subsequent experiments, cells were exposed to A23187 for 20 min to ensure complete equilibration. Figure 1 shows the change in total [Mg2+]i for both oxygenated and deoxygenated cells in the presence of A23187 (continuous lines) and different free [Mg2+]i values (with ionophore, free [Mg2+]i = [Mg2+]o r 2, where r 2 = ([H+]i/[H+]o)2 and was 1·52 ± 0·15 and 1·40 ± 0·19, respectively, in oxygenated and deoxygenated cells; n = 3). Total [Mg2+]i is also shown for control cells in the absence of A23187 (dashed line). With A23187, total [Mg2+]i values were greater in O2, implying a larger Mg2+ buffering capacity in these cells, as expected. The plot was also used to estimate physiological free [Mg2+]i at both PO2s, given by the free [Mg2+]is at which the cell Mg2+ content did not change. Values of 0·45 ± 0·04 and 0·68 ± 0·03 mM were estimated for cells in O2 and N2, respectively (n = 4). Cell water content was unaffected by A23187 and [Mg2+]o from 0 to 1 mM (data not shown).
Total [Mg2+]i was measured in red cell aliquots which had been fully oxygenated or deoxygenated and treated with A23187 (20 µM) at different [Mg2+]o values. The abscissa is plotted as [Mg2+]i, which in the presence of ionophore is given by [Mg2+]o r 2, where r 2 was 1·5 for oxygenated and 1·4 for deoxygenated cells, respectively. The dashed horizontal line gives total [Mg2+] in control cells, untreated with ionophore. Arrows indicate the estimated physiological free [Mg2+]i in O2 and N2, i.e. the free [Mg2+]i when cells neither gain nor lose Mg2+. Data are given as means ±
Effect of Mg2+ depletion on O2-dependent Cl--dependent K+ influx in equine red cells
Cl--dependent K+ fluxes, usually taken as evidence for K+-Cl- cotransport, are O2 dependent in red cells from a number of animals, including horse (Gibson et al. 1995a). Control experiments showed that K+ influx was substantial in O2 and < 0·1 mmol (l cells)-1 h-1 in N2 (data not shown), as expected. Figure 2 demonstrates the effect of Mg2+ depletion on K+ influxes in deoxygenated cells. Again, control cells had a minimal K+ influx. A high K+ influx was observed when cells were incubated with A23187 and 0·1 mM [Mg2+]o, and this K+ influx was abolished by Cl- removal (substituted with NO3- or MeSO4-) or by subsequent treatment of cells with calyculin A (100 nM), a specific phosphatase inhibitor which has been shown to abrogate K+-Cl- cotransport in red cells. In all experiments, K+ influxes in the absence of Cl- were low (< 0·2 mmol (l cells)-1 h-1), unaffected by treatment with A23187, by Mg2+ depletion or loading, or by changes in PO2. Changes in total K+ influx therefore represent modulation of Cl--dependent K+ transport, i.e. a measure of the activity of the K+-Cl- cotransporter. Control experiments also showed that K+ influxes in the presence of A23187 were unaffected by the addition of 200 µM EGTA, excluding the possibility that they were mediated via a Ca2+-activated K+ channel.
K+ influx (mmol (l cells)-1 h-1) was measured in deoxygenated cells, either in control cells without ionophore, or in cells treated with A23187 (20 µM) and [Mg2+]o of 0·1 mM to activate a K+ influx in N2. This influx was inhibited by removal of Cl- (substituted with MeSO4-) or by subsequent treatment with calyculin A (Cal A, 100 nM). Data are given as means ±
Can physiological changes in free [Mg2+]i account for O2 dependence of K+-Cl- cotransport in equine red cells?
The above shows that on oxygenation of red cells (i) free [Mg2+]i decreases by about 230 µM, (ii) Cl--dependent K+ influx is activated, and (iii) depleting deoxygenated cells of Mg2+ activated the flux. In this section, we address the possibility that the physiological fluctuation in free [Mg2+]i, which occurs as cells are oxygenated and deoxygenated, is sufficient to account for the O2 dependence of the K+-Cl- cotransporter.
In the following experiments, red cell aliquots were first fully oxygenated or deoxygenated, and held at these PO2 values for all subsequent procedures. Cells were exposed to A23187 and different [Mg2+]o values to clamp their free [Mg2+]i at the requisite value, and then A23187 was removed by repeated washing with saline containing BSA. The effect of these procedures on K+ influx is shown in Fig. 3. In both oxygenated and deoxygenated cells, Mg2+ depletion stimulated K+ influx whilst loading with Mg2+ inhibited it. At all values of free [Mg2+]i, K+ influxes in oxygenated cells were substantially greater than those in deoxygenated cells. The effects of oxygenation and deoxygenation were reversible in Mg2+-clamped red cells (data not shown), as they are in control cells (Honess et al. 1996). Changing free [Mg2+]i from 0·45 to 0·75 mM in oxygenated cells resulted in a 6 ± 3 % (mean ±
All procedures were carried out on cell aliquots either fully oxygenated or fully deoxygenated throughout. Cells suspended in saline at 290 mosmol kg-1 and pHo 7·4 were treated with A23187 (20 µM) and various [Mg2+]o values to clamp free [Mg2+]i at 0·15 to 5 mM. A23187 was subsequently removed by washing with saline containing BSA (25 mg ml-1). K+ influxes (mmol (l cells)-1 h-1) were then measured in cells incubated at an osmolality of 260 mosmol kg-1. Data are given as means ±
Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped equine red cells
In these experiments, free [Mg2+]i was clamped using A23187 and [Mg2+]o of 0·1 mM. A23187 was then removed (see above) before volume or pH perturbation. Aliquots of the cells were then exposed either to different osmolalities to swell and shrink them anisotonically, or to different pHos, ranging from pH 6 to 8. Finally, K+ influx was measured (Figs 4 and 5). Again, all procedures were carried out in either O2 or N2. Mg2+-clamped equine red cells were about 6-fold more sensitive to changes in cell volume when oxygenated, compared with those held in N2, over the range of osmolalities tested (Fig. 4). Cell water contents were also measured under the same conditions and at all osmolalities, deoxygenated red cells were larger (e.g. 1·93 ± 0·03 and 2·15 ± 0·02 ml g-1 dry cell solids in O2 and N2, respectively, at 230 mosmol kg-1 (n = 3)). The higher K+ influx in Mg2+-clamped red cells when oxygenated cannot therefore be a result of greater cell swelling. When pHo was altered to values above 6, pHi was decreased in oxygenated red cells, although the difference from deoxygenated cells was small (< 0·1 pH unit). K+ influx in both oxygenated and deoxygenated cells was increased as pHi decreased, and, over the range examined, H+ ion sensitivity was about 2-fold higher in O2 than in N2 (Fig. 5). At the lowest pH, there was a substantial K+ influx even in deoxygenated cells. Nevertheless, at every pH, oxygenation stimulated K+ influx and this was not due to any difference in pHi between oxygenated and deoxygenated cells.
Again, all procedures were carried out on cell aliquots either fully oxygenated or fully deoxygenated throughout. Cells suspended in saline at 290 mosmol kg-1 and pHo 7·4 were treated with A23187 (20 µM) and [Mg2+]o of 0·1 mM to clamp free [Mg2+]i. A23187 was subsequently removed by washing with saline containing BSA (25 mg ml-1). K+ influxes (mmol (l cells)-1 h-1) were then measured in cell aliquots whose volume was altered anisotonically by incubation in salines (all pH 7·4) of different osmolalities (by addition of distilled water).
Again, all procedures were carried out on cell aliquots either fully oxygenated or fully deoxygenated throughout. Cells suspended in saline at 290 mosmol kg-1 and pHo 7·4 were treated with A23187 (20 µM) and [Mg2+]o of 0·1 mM to clamp free [Mg2+]i. A23187 was subsequently removed by washing with saline containing BSA (25 mg ml-1). K+ influxes (mmol (l cells)-1 h-1) were then measured in cell aliquots whose pHi was altered by incubation in salines at different pHos. Data are given as means ±
The present results demonstrate that Cl--dependent K+ influxes, usually taken as evidence for K+-Cl- cotransport, remained O2 stimulated in equine red cells despite clamping free [Mg2+]i. The higher fluxes in O2 were not caused by a greater sensitivity to changes in volume and [H+] ions. Together with our recent findings of O2-dependent cotransport in sheep red cells (Campbell & Gibson, 1998a), these observations demand re-evaluation of the role of changes in free [Mg2+]i in the activation of K+-Cl- cotransport by O2. They also imply that important differences occur between human sickle cells (Joiner et al. 1998) and red cells from other mammalian species.
Total [Mg2+]i in equine red cells was 2·55 mM, and, using the null point method of Flatman & Lew (1980), we estimated free [Mg2+]i as 0·45 and 0·68 mM in oxygenated and deoxygenated cells, respectively. Given that equine red cells contain levels of 2,3-diphosphoglycerate (2,3-DPG) and ATP similar to those in human red cells, and that equine Hb, like its human counterpart, has a higher affinity for organic phosphate compounds when deoxygenated, similar values for [Mg2+]i in equine and human red cells (Flatman, 1980) would be expected.
We went on to look at the response of K+ influx in the equine red cell to changes in free [Mg2+]i. As shown for other species, notably sheep, Mg2+ depletion stimulated the cotransporter whereas Mg2+ loading inhibited it (Delpire & Lauf, 1991). The K+ influx stimulated by Mg2+ depletion was abolished by Cl- substitution and also by treatment with calyculin A. It is highly likely therefore that the flux was mediated via the K+-Cl- cotransporter. Subsequently, however, we found that changes in free [Mg2+]i of a magnitude similar to that occurring physiologically during oxygenation-deoxygenation cycles (a few hundred micromolar), had very little effect on the cotransporter. Significantly, K+ influx was stimulated by oxygenation at all clamped [Mg2+]i values.
It is difficult to reconcile these findings with a primary role for [Mg2+]i in the regulation of K+-Cl- cotransport by O2 (cf. Canessa et al. 1987). In fact, examination of the literature shows that pharmacological changes in free [Mg2+]i required to affect the K+-Cl- cotransporter are also well outside the physiological range (e.g. Delpire & Lauf, 1991). O2-dependent K+-Cl- cotransport was also observed by Motais and coworkers after Mg2+ clamping trout red cells (Borgese et al. 1991), and we have previously shown that the same occurs in LK sheep red cells (Campbell & Gibson, 1998a), a system commonly used to study K+-Cl- cotransport. Trout red cells, however, are nucleated, aerobic and lack 2,3-DPG; those in sheep contain a low [K+]i and sheep Hb also lacks the capacity to bind organic phosphates. It was important, therefore, to study a more typical mammalian red cell.
Trivial explanations for an apparent O2-dependent K+-Cl- cotransporter in Mg2+-clamped red cells could be secondary or non-specific effects of A23187, either following prolonged perturbation of [Mg2+]i or via some other unknown action. We believe that this is unlikely in the present work. The effect of Mg2+ loading or depletion was fully reversible, as were those following changes in PO2. During our Mg2+ depletion, which was not total, K+ influx remained calyculin sensitive. This is similar to the situation in sheep following partial Mg2+ depletion (Flatman et al. 1996), whereas subsequent addition of calyculin A was without effect following total Mg2+ depletion (Bize & Dunham, 1994). We also excluded the possible caveat that changes in cell volume, volume sensitivity or H+ ion sensitivity of the K+-Cl- cotransporter could artificially produce an apparent O2 dependence. Finally, similar results were obtained if A23187 was present throughout the experiments, rather than removed prior to influx measurement (data not shown).
Inhibition of K+-Cl- cotransport by increases in [Mg2+]i has recently been observed for human sickle cells (Joiner et al. 1998). In sickle cells, however, Joiner et al. (1998) found that deoxygenation (not oxygenation) stimulated K+-Cl- cotransport, in Mg2+-clamped sickle cells, albeit modestly. Many membrane proteins of human red cells become dephosphorylated on deoxygenation (Fathallah et al. 1995). It is suggested that dephosphorylation is stimulatory to the K+-Cl- cotransporter, whereas the concomitant rise in free [Mg2+]i is inhibitory, and therefore obscures this deoxygenation activation: Mg2+ clamping removes the inhibitory component of this system, revealing a deoxygenation-stimulated K+-Cl- cotransporter. The apparent differences between sickle cells and red cells from horse and sheep may be methodological - an obvious experiment would be a study of normal human (HbA) red cells, though this is complicated by the low activity of the cotransporter in these cells. More interestingly, there may be a difference in the control pathways for K+-Cl- cotransport in sickle cells and other red cells. Certainly, many properties of sickle cells, not least the ability of deoxygenated HbS to form polymers, responsible for the characteristic sickled shape, may underlie the difference.
Our findings make it imperative to examine other mechanisms besides fluctuations in [Mg2+]i to account for the O2 dependence of K+-Cl- cotransport in red cells. Previous results indicate a role for protein kinases and phosphatases since cells treated with inhibitors of these cannot respond to O2 (Cossins et al. 1994; Honess et al. 1996). The sigmoidal relationship between activity of the cotransporter and PO2 (Speake et al. 1997; Gibson et al. 1998), together with observations that carbon monoxide mimics O2 (Borgese et al. 1991) and that methaemoglobin mimics oxyhaemoglobin (Jensen, 1992), indicate a role for Hb, or at least a haem-containing compound. The present data with Mg2+-clamped red cells implies that the property of bulk cytoplasmic Hb, through changes in free [Mg2+]i, is not involved. A specific fraction of the total cell Hb may be responsible, for example that portion of the Hb found at the membrane, which binds to the cytoplasmic tail of band 3 with higher affinity when deoxygenated (Chétrite & Cassoly, 1985). This region of band 3 can be phosphorylated, thereby altering its affinity for Hb, and is also the attachment site at which several glycolytic enzymes may compete with Hb (Low, 1986). It is therefore potentially involved with coupling Hb and changes in O2 tension to the protein kinase/phosphatase enzymes which ultimately regulate K+-Cl- cotransport.
We thank the Wellcome Trust for financial support. E. H. C. is funded jointly by the University and the Veterinary Faculty of Liverpool.
Corresponding author
J. S. Gibson: Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 3BX, UK.
Email: jsg{at}liverpool.ac.uk
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RESULTS
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Figure 1. The change in Mg2+ content with extracellular Mg2+ concentration in equine red cells permeabilized with a divalent cation ionophore
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Figure 2. The effect of A23187 and different inhibitors on the K+ influx in deoxygenated equine red cells
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Figure 3. O2-dependent K+ influxes in Mg2+-clamped equine red cells
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Figure 4. Volume dependence of K+ influx in Mg2+-clamped equine red cells
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Figure 5. H+ ion dependence of K+ influx in Mg2+-clamped equine red cells
DISCUSSION
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Abstract
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References
REFERENCES
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Abstract
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Methods
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References
Acknowledgements
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M. C. Muzyamba, P. F. Speake, and J. S. Gibson
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