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Received 25 July 1997; accepted after revision 2 October 1997.
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ABSTRACT |
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INTRODUCTION |
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KCl cotransport in the low potassium-containing red cell of sheep (LK sheep red cells) has been particularly well investigated (e.g. Dunham & Ellory, 1981; Lauf et al. 1992; Flatman, Adragna & Lauf, 1996; Kelley & Dunham, 1996). Despite a considerable amount of information on many features of the sheep system, the O2 dependence of KCl cotransport has not been defined. There are a number of reasons why the response of LK sheep red cells to O2 may differ from that of other vertebrate species. First, as their name indicates, the cells contain a low concentration of K+, about 10 mM as opposed to about 100 mM in the more usual high K+-containing red cells (Evans, 1954). Second, the cells have a different content of organic phosphate compounds; in particular, mature sheep red cells have little or no glyceric acid 2,3-biphosphate (DPG) (see Nikinmaa, 1990, for references). Third, the structure of sheep haemoglobin reduces its ability to bind organic phosphates (Perutz, 1970; Bunn, 1971). As a consequence, the variation in free intracellular Mg2+ concentration ([Mg2+]i) upon oxygenation-deoxygenation may differ. The latter is important because fluctuations in free [Mg2+]i have been proposed as the intracellular messenger coupling changes in oxygen tension to transport activity (Canessa et al. 1987; Apovo, Beuzard, Galacteros, Bachir & Giraud, 1994; Parker, 1994), perhaps via the regulatory protein kinase/phosphatase (PP/PK) enzymes which are involved in many responses of the cotransporter (Flatman et al. 1996; Kelley & Dunham, 1996).
In this paper, we studied the effects of PO2 upon K+ influx in LK and HK sheep red cells. Results show that the response of these cells to volume and H+ is highly sensitive to PO2. Fluctuations in free [Mg2+]i upon oxygenation/deoxygenation could not account for the coupling of cotransporter activity to PO2. Our findings emphasize the importance of controlling PO2 when making quantitative comparisons between different experiments involving K+ flux in sheep red cells and also when modelling the regulatory transduction system.
A preliminary account of some of these findings has been published in abstract form (Campbell & Gibson, 1997a,b).
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METHODS |
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Materials
Bovine serum albumin fraction V (BSA), dimethylsulphoxide (DMSO), Drabkins reagent, EDTA, EGTA, MOPS, ouabain, KCl, KNO3, NaNO3, trichloroacetic acid (TCA) and Triton X-100 were purchased from Sigma. Glucose, KCl, NaCl and sucrose were purchased from Merck (Poole, UK), and A23187 from Calbiochem-Novabiochem (Nottingham, UK).
Saline solutions
The standard Mops-buffered saline (MBS) used for washing and storing the cells and for most experiments contained (mM): 145 NaCl, 5 glucose and 10 Mops (pH 7·4 at 37°C). For experiments in which Cl- dependence of K+ influx was examined, Cl- was substituted with NO3- - it is widely assumed that Cl--dependent K+ fluxes are mediated via a KCl cotransporter (e.g. Dunham & Ellory, 1981) and this interpretation is followed here. The possibility of a Cl--activated K+ transporter, however, cannot be excluded. Saline solutions had an osmolality of 290 ± 5 mosmol kg-1. During K+ influx measurement, K+ was added with the isotope to give a final [K+] of 7·5 mM. Stock solutions of A23187 (10 mM) were prepared in DMSO, stored at -20°C, and used at a final concentration of 20 µM. Stock solutions of ouabain (10 mM) were prepared with water, stored at 4°C and used at a final concentration of 100 µM.
Sample collection and handling
Blood samples were obtained from sheep kept at the Department of Veterinary Clinical Sciences and Animal Husbandry, Leahurst, UK. All samples were taken by jugular venepuncture into heparinized vacutainers and kept on ice or refrigerated (4°C) until used (within 50 h of being taken). Prior to experimentation, samples were centrifuged at 10°C for 5 min at 1500 g to separate red cells from the buffy coat and plasma. Red cells were washed with the MBS a further three times (in 20 times the volume of cells). In some experiments, cells were either shrunken or swollen anisosmotically by addition of hypertonic sucrose or distilled water to the saline. The K+ phenotype of each sheep was established by lysing packed red cells in 10 times their volume of distilled water and measuring the [K+]: [K+] was < 2 mM for LK sheep and > 7 mM for HK sheep. The genotype of the sheep was not established.
Tonometry
Before flux measurement, red cell suspensions were incubated at about 50 % haematocrit in glass tonometers (Eschweiler & Co, Keil, Germany). Gas mixtures with variable concentrations of O2 and N2 were made using a calibrated gas mixing pump (Wösthoff, Bochum, Germany), warmed to 37°C and fully humidified through three humidifiers (Eschweiler & Co, Keil, Germany) prior to delivery to the tonometers. 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. When determining the PO2 required for half-maximal activation (P50) of K+ influx, samples were incubated in tonometers for at least 15 min at each PO2 before flux measurement - control experiments showed that cell samples were fully equilibrated with the new PO2 within 10 min.
K+ influx
K+ influx was measured at 37°C by conventional radioactive tracer techniques using 86Rb+ (DuPont-NEN, Bad Homburg, Germany) as a tracer for K+ (Dunham & Ellory, 1981; Gibson, Ellory, Culliford & Fincham, 1993). Ouabain (final concentration, 100 µM) was present in all experiments, obviating any K+ influx through the Na+,K+-ATPase. 86Rb+ (0·04-0·06 MBq ml-1 final) in 150 mM KCl or KNO3 (final [K+] = 7·5 mM) was added to the cell suspension and influx measured over 15 min, during which time uptake was linear. Fluxes were terminated and samples processed as described previously (Gibson et al. 1993). Haematocrit was measured either by the cyanomethaemoglobin method or by microhaematocrit determination (Gibson et al. 1993). Influxes are expressed as millimoles of K+ per litre of cells per hour.
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) using a Mettler Delta 340 meter in combination with an Mettler Inlab 423 micro pH probe. For measurement of intracellular pH (pHi), 1 ml of the same cell suspension used for Mg2+ determination was centrifuged in 1·5 ml Eppendorf tubes through 0·5 ml dibutylphalate oil (10 s at 15000 g) to separate the red cells from the incubation medium. The cell pellet (approximately 50 µl) was then transferred to 0·25 ml Eppendorf tubes, again under oil (50 µl), and then subjected to two cycles of freeze-thawing. Use of the smaller Eppendorf tubes allowed pHi to be measured directly with the Mettler micro pH probe despite the low volume of the red cell lysate. pH was 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. pHi and pHo determinations were used to calculate the Donnan ratio of the cells, r, given by [H+]i/[H+]o (= [Cl-]o/[Cl-]i).
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 (both fully deoxygenated and oxygenated) of about 5 % haematocrit were incubated for 20 min at 37°C in MBS containing A23187 (20 µM final), the required extracellular Mg2+ concentration ([Mg2+]o) and 50 µM EGTA to chelate contaminant calcium. Cell aliquots were then diluted into 1 ml ice-cold MBS containing 2 mM EDTA layered on 0·4 ml dibutylphthalate oil and centrifuged. The resultant cell pellet was lysed with Triton X-100, deproteinated with TCA and diluted for determination of the Mg2+ absorbance. This was converted into [Mg2+] by means of Mg2+ standards prepared in the same way as red cell samples. Control experiments showed that, for both loading and depletion of Mg2+, cells reached a new stable [Mg2+]i within 10 min. In addition, although no correction was made for trapped extracellular fluid, cells prepared in solutions of different [Mg2+]o, but in the absence of ionophore, showed that, over the range of [Mg2+]o tested (0-5 mM), the value obtained for total [Mg2+]i remained constant. Free [Mg2+]i was calculated using the equation: free [Mg2+]i = [Mg2+]o × r2. Physiological free [Mg2+]i was then estimated for oxygenated and deoxygenated cells by the null point method of Flatman & Lew (1980) by finding the [Mg2+]o at which total [Mg2+]i was unchanged in the presence of A23187. Finally, in the experiments shown in Fig. 8, following treatment of cells with A23187, ionophore was removed before determination of K+ influx by washing cells five times in MBS containing 25-50 mg ml-1 BSA.
Statistics
Data are expressed as means ± standard error of the mean (
Volume-sensitive K+ influx and oxygenation
Figure 1 shows the effects of alteration of PO2 upon K+ influx in LK sheep red cells, shrunken and swollen anisosmotically by 10 %. At time 0, a control aliquot of cells (at approximately 40 % haematocrit) was taken from storage on ice, diluted 10-fold in saline of the required osmolality, but of unknown PO2, and K+ influx measured. At the same time, the remainder of the cell suspension (40 % haematocrit) was placed in a tonometer at 37°C and flushed with N2. Thereafter K+ influx measurements were made as for the control cells but using saline pre-equilibrated with N2, at the time intervals shown in Fig. 1. After 55 min, the majority of the deoxygenated cell suspension was taken from the first tonometer, placed in a second tonometer, and equilibrated with O2. Serial K+ influx measurements were repeated at this new PO2. At 110 min, an aliquot of cells from the first tonometer, which had remained deoxygenated throughout the experiment, was taken and the K+ influx measured in N2 along with the last measurement in O2.
Under deoxygenated conditions, the absolute magnitude of K+ influx and the volume-sensitive component (calculated as the difference in influx between shrunken and swollen cells) were low. In this experiment both values increased by approximately 2-fold on oxygenation; usually, K+ influx in shrunken cells was much less stimulated by oxygenation than that in swollen cells (see below). Upon changing gas tension, new and stable magnitudes of K+ influx were reached within 15-30 min, which represents the minimal time to alter PO2 and measure an influx under the experimental procedure used. After changing PO2, the new magnitude of K+ influx measured after 15 min was not significantly different from those made subsequently. In the experiment shown in Fig. 1, K+ influx measurements in cells held in saline of unknown PO2 exhibited low fluxes comparable to that of cells pre-equilibrated with N2. This finding was not consistent from one experiment to another, thus stressing the importance of controlling PO2.
Figure 1. Time course for the effect of oxygen tension on K+ influx in LK sheep red cells
K+ influx (mmol (l cells)-1 h-1) was measured at the times given in cells shrunken (
Similar experiments were repeated on samples from sixteen sheep of the LK phenotype. Mean K+ influxes in shrunken cells were 0·20 ± 0·03 (n = 16) and 0·26 ± 0·05 (n = 16) in N2 and O2 respectively; in swollen cells, influxes were 0·37 ± 0·07 (n = 16) in N2 and 0·61 ± 0·10 (n = 16) in O2. The volume-sensitive component of the K+ influx approximately doubled upon oxygenation (mean percentage change was 203 ± 23 % (n = 16)). Most of this change resulted from the stimulation of K+ influx in swollen cells, whilst that of shrunken cells was largely O2 independent. Nevertheless, cells maintained in N2 exhibited considerable volume sensitivity: the ratio of K+ influx in swollen cells to that in shrunken cells was 1·83 ± 0·10 (n = 16) in N2 and 2·35 ± 0·20 (n = 16) in O2.
In other species (Canessa et al. 1987; Borgese et al. 1991; Nielsen et al. 1992; Honess et al. 1996), the volume-sensitive K+ influx, mediated via the KCl cotransporter, is entirely O2 dependent. The apparent difference in behaviour of LK sheep red cells was therefore investigated further.
First, experiments carried out on equine red cells, under identical conditions to those described for LK sheep, demonstrated that the volume-sensitive component of K+ influx in the horse was indeed entirely O2 dependent (data not shown). Second, K+ influx measurements on LK sheep red cells, incubated in N2 for up to 180 min, retained volume sensitivity (data not shown). A third possibility, that the volume-sensitive K+ influx in deoxygenated LK sheep red cells was mediated through a K+ transport pathway other than KCl cotransport, was investigated in the experiment shown in Fig. 2. Again, K+ influxes were measured in N2 and O2, in cells shrunken and swollen anisosmotically. Fluxes were carried out in normal Cl--containing saline and in a saline in which Cl- was replaced with NO3-. (These experiments test for Cl- dependence of the K+ flux and, as explained in Methods, although not definitive, this is often taken as evidence for KCl cotransport.) Cl- controls showed the same pattern as before: volume-sensitive K+ influx in both N2 and O2, notwithstanding the higher influxes in O2. In NO3- media, however, volume-sensitive K+ influxes were very small (approximately 0·03 mmol (l cells)-1 h-1) and unaffected by oxygenation. Considerable Cl--dependent volume-sensitive K+ influx remained in N2, indicative of a residual component of KCl cotransport at this gas tension.
Figure 2. Effect of anion replacement on volume-sensitive K+ influx in LK sheep red cells
K+ influx was measured in N2 or O2 in the presence or absence of Cl- (Cl- replaced with NO3-). Samples were shrunken (
H+-sensitive K+ influx and oxygenation
Physiologically, [H+] is potentially a more important modulator of red cell KCl cotransport than cell volume (Ellory, Hall, Ody, deFigueiredos, Chalder & Stuart, 1991). We therefore examined the effects of oxygenation upon H+-stimulated K+ influx in LK sheep red cells. Cell suspensions were incubated at pH 7·4 in tonometers flushed with N2 or O2. Aliquots were then diluted 10-fold into saline, equilibrated at the same PO2, to give a final pH of 7·4 or 7·0. Extracellular pH was checked in the presence of red cells at the final haematocrit, prior to the addition of 86Rb+. Results are shown in Fig. 3. At both gas tensions, K+ influx was stimulated upon lowering the pH from 7·4 to 7·0. K+ influx at pH 7·0 in O2 was considerably higher than in N2; the influx at pH 7·4 was also stimulated by oxygenation but to a lesser extent.
Figure 3. Effect of oxygen tension on H+-sensitive K+ influx in LK sheep red cells
K+ influx was measured in cells at pH 7·4 (
H+-sensitive K+ influx was also measured at pH 7·0 in the presence and absence of Cl-, with Cl- substituted by NO3-. Figure 4 shows that, as for volume-sensitive K+ influxes, at low pH, oxygenation stimulated only the Cl--dependent component of K+ influx. Again there was considerable H+-stimulated Cl--dependent K+ influx in N2, consistent with a substantial component of KCl cotransport present in the absence of O2.
Figure 4. Effect of anion replacement on H+-sensitive K+ influx in LK sheep red cells
K+ influx was measured at pH 7·0 in N2 or O2 in the presence (
We have previously characterized the O2 dependence of both volume- and H+-stimulated K+ influx in equine red cells and shown that the PO2 required for half-maximum activation (P50) of K+ influx is similar for both stimuli, approximately 30 mmHg. (The P50 at normal volume and pH 7·4 was also similar but technically more difficult to measure because of the small magnitude of K+ influx.) Thus in red cells from the horse, physiological variations in PO2 will affect cotransport. In the experiment shown in Fig. 5, K+ influx in LK sheep red cells was stimulated by reduction in pH to pH 7 and measured over a range of PO2 values from 0 to 713 mmHg. The effect of PO2 on K+ influx was pronounced over the physiological range (0-100 mmHg). The P50 for O2-dependent K+ influx was 56 ± 1 mmHg (3 sheep).
Figure 5. Effect of oxygen tension on H+-stimulated K+ influx in LK sheep red cells
Cells were incubated for 15 min at pH 7·4 at the PO2 indicated before measurement of K+ influx in saline of pH 7, pre-equilibrated with the same PO2. Points represent means ±
KCl cotransport and oxygenation in HK sheep red cells
From the preceding sections, it would appear that Cl--dependent K+ influx in LK sheep red cells was substantially O2 dependent but that the response showed important differences from that shown by the KCl cotransporter of trout and horse red cells. The cotransporter in all these species is regulated by cellular PP/PK enzymes. Cation concentration has a considerable effect on enzyme activity and part of the difference in response of LK sheep red cells to O2 may be attributable to their high content of Na+ and low content of K+, i.e. the reverse to that found in red cells from other species. HK sheep red cells, whose [K+] is high like that in red cells from species such as human, horse and trout, have a much reduced KCl cotransport activity but it is still present (Lauf, 1988). Figure 6 shows the O2 dependence of volume-sensitive K+ influxes in red cells from the HK phenotype. K+ influxes were again measured in N2 and O2 for cells anisosmotically shrunken and swollen by 10 %. The response of K+ influx to oxygenation was very similar to that in LK sheep red cells. Mean volume sensitivity (given by the ratio of influx in swollen and shrunken cells) of the KCl cotransporter in cells of the HK phenotype was 1·50 ± 0·17 (mean ±
Figure 6. Effect of oxygen tension on volume-sensitive K+ influx in HK sheep red cells
K+ influx was measured in cells shrunken (
Changes in intracellular magnesium concentration and O2 dependence of K+ influx
A number of studies have shown that the free intracellular magnesium concentration (free [Mg2+]i) of human red cells increases and decreases upon deoxygenation and oxygenation, respectively (Gupta, Benovic & Rose, 1978; Flatman, 1980). It has been proposed that these changes alter the activity of cellular regulating PP/PKs and hence affect the regulatory pathway of the KCl cotransporter (Parker, 1994). Variations of free [Mg2+]i in sheep red cells under oxygenated and deoxygenated conditions have not been measured, however, and it therefore remains uncertain whether modulation of [Mg2+]i over the physiological range can alter cotransport activity. We addressed this possibility in the experiments shown in Figs 7 and 8.
Free and total [Mg2+]i of oxygenated and deoxygenated LK sheep red cells was altered using the ionophore A23187 (20 µM final) in combination with different concentrations of [Mg2+]o (0·1-1 mM). Total [Mg2+]i was determined by atomic absorption spectroscopy. The Donnan ratio, r, used to calculate free [Mg2+]i was 1·91 ± 0·03 (n = 5) and 1·87 ± 0·13 (n = 5) in N2 and O2, respectively. r was unaffected by changes in [Mg2+]o over the range used for experiments shown in Figs 7 and 8 (0·1-1·0 mM), although it declined at higher concentrations (to 1·69 ± 0·04, n = 9, at 5 mM). Cell volumes for control cells and for cells treated with A23187 and [Mg2+]o of 0·1, 1 and 5 mM were 1·97 ± 0·03, 1·94 ± 0·07, 1·95 ± 0·05 and 1·94 ± 0·06 ml (g dry cell solids)-1, respectively, and were thus unaffected by Mg2+ loading and depletion.
Figure 7 shows the relationship between total [Mg2+]i and free [Mg2+]i in oxygenated and deoxygenated LK sheep red cells. Total [Mg2+]i in control cells, in the absence of A23187, was 1·29 ± 0·08 (n = 5), as shown by the dashed line in Fig. 7. Physiological values of free [Mg2+]i, estimated by the null point method of Flatman & Lew (1980), increased significantly from 0·39 ± 0·05 mM (n = 5) in O2 to 0·52 ± 0·04 mM (n = 5) in N2 (P < 0·05).
Figure 7. Effect of variations in free intracellular magnesium concentration ([Mg2+]i) on total [Mg2+]i
Total [Mg2+]i was determined in oxygenated (
The effect of changes in free [Mg2+]i on K+ influx in oxygenated and deoxygenated cells is shown in Fig. 8. Free [Mg2+]i was altered as described above but A23187 was then removed by repeated washing with saline containing BSA (50 mg ml-1) before K+ influxes were determined in cells swollen anisosmotically by 5 %. In the absence of Cl-, substituted with NO3-, K+ influxes following treatment with A23187 were unaffected by oxygenation (< 0·08 mmol (l cells)-1 h-1 difference between measurements in N2 or O2) or different [Mg2+]o (< 0·12 mmol (l cells)-1 h-1 difference between measurements in [Mg2+]o from 0·1 to 1·0 mM). In Cl--containing MBS, K+ influx in oxygenated cells remained significantly higher than in deoxygenated cells at all [Mg2+] tested (Fig. 8). The dashed lines show K+ influx in control cells, to which ionophore had not been applied, in O2 and N2. Although K+ influx in O2 was reduced by elevating [Mg2+]i, influx was not suppressed to that observed in deoxygenated controls even when free [Mg2+]i was elevated above the physiological concentration estimated for control cells in N2, (i.e. > 0·5 mM). From subsequent experiments, it was estimated that an [Mg2+]o of 1·2 ± 0·1 (n = 8) mM, corresponding to a free [Mg2+]i of > 2 mM, was required before influx in oxygenated cells approximated that in deoxygenated controls. Similarly, in deoxygenated cells, reducing free [Mg2+]i considerably below the physiological concentration in O2 (i.e. < 0·4 mM), was unable to stimulate K+ influx to that observed in oxygenated control cells. Finally, the data shown in Fig. 8 can also be used to estimate free [Mg2+]i in cells under O2 and N2: the free [Mg2+]i which correlated with the magnitude of K+ influx observed in the control cells was about 0·4 and 0·5 mM for oxygenated and deoxygenated cells, respectively, in agreement with the estimates obtained from Fig. 7.
Figure 8. Effect of variations in free intracellular magnesium concentration ([Mg2+]i) on K+ influx in LK sheep red cells
Free [Mg2+]i, calculated as described in Fig. 7, was altered using A23187 (20 µM) and different extracellular [Mg2+]. Ionophore was removed by washing cells with saline containing BSA (50 mg ml-1) and K+ influxes were then measured in O2 (
The results described in this paper represent the first demonstration that Cl--dependent K+ influxes in sheep red cells are affected by physiological PO2. We also present the first estimates of free [Mg2+]i in sheep red cells during controlled oxygenation and deoxygenation. Changes in Mg2+ buffering were small and were unlikely to account directly for the O2 dependence of the K+ influx.
Effect of oxygen on K+ influx in sheep red cells
K+ transport in sheep red cells has been extensively investigated. It has been shown that sheep present two phenotypes with respect to potassium concentration in their red cells (Evans, 1954), termed high potassium (HK) and low potassium (LK). LK sheep red cells are the more unusual in having a high passive K+ 'leak' coupled with a small active K+ uptake through the Na+,K+-ATPase (Tosteson & Hoffman, 1960); the reverse pertains for HK red cells. The large K+ 'leak' in LK red cells is, in fact, a very specific anion-dependent transporter, exquisitely sensitive to volume, pH and urea (Dunham & Ellory, 1981; Lauf et al. 1992 ; Dunham, 1995). It is almost certainly via the KCl cotransporter, recently cloned from human, rat and rabbit tissues (Gillen, Brill, Payne & Forbush, 1996).
In this manuscript, we have demonstrated that oxygen tension (PO2) represents a further physiological modulator of passive K+ flux in both LK and HK sheep red cells, and that PO2 affects the response of cells to both volume and pH. Only the Cl--dependent component of K+ transport was O2 sensitive, consistent with an effect solely on the KCl cotransporter. Further, the PO2 required to affect the cotransporter was within the physiological range: at pH 7, the P50 for O2-dependent K+ influx was about 50 mmHg. This value of P50 is similar to that for O2 saturation of sheep haemoglobin (Moraga, Monge, Riquelm & Llanos, 1996), which, like that from cattle, has a relatively low O2 affinity amongst mammalian haemoglobins (Bunn, 1971). In a similar way, P50 for K+ influx and O2 saturation for horse red cells are both about 30 mmHg (Clerbaux, Gustin, Detry, Cao & Frans, 1993, P50 for O2 saturation; Speake & Gibson, 1997, P50 for K+ influx). Taken together, these observations are indicative of a role for O2-haemoglobin interactions in the mechanism of O2 dependence of the cotransporter.
In both trout and horse, the Cl--dependent K+ influx is abolished in deoxygenated cells (Nielsen et al. 1992; Honess et al. 1996). This was not the case with LK sheep red cells, in which the Cl--dependent K+ influx in N2 comprised a substantial component of the residual K+ influx in deoxygenated cells. We have presented evidence that this residual flux in N2 was unlikely to be artefactual resulting from incomplete deoxygenation. Nor was the low [K+] of LK sheep red cells responsible, since similar results were obtained with both LK and HK cells. It is probable that this anomalous behaviour of sheep red cells is caused by subtle differences in the regulatory cascade of protein kinase/phosphatase enzymes which controls cotransporter activity (Flatman et al. 1996; Kelley & Dunham, 1996), but the mechanism awaits further investigation.
Intracellular magnesium and O2 dependency of K+ influxes
O2-sensitive Cl--dependent K+ fluxes have been observed in red cells from a number of different species, including human (Canessa et al. 1987), trout (Nielsen et al. 1992) and horse (Honess et al. 1996). Fluctuations in free [Mg2+]i are widely held to participate in the transduction pathway coupling PO2 to activity of the cotransporter (Canessa et al. 1987; Apovo et al. 1994). Thus on deoxgenation-oxygenation the change in haemoglobin binding of organic phosphate compounds especially DPG (mammals), or guanosine triphosphate and inosine pentaphosphate (fish), affects the Mg2+ buffering capacity of the cell (for references see Nikinmaa, 1990). The subsequent change in free [Mg2+]i may interact with the complex PP/PK cascade regulating cotransport activity at several points (Lauf, Erdmann & Adragna, 1994; Flatman et al. 1996). Certainly Mg2+ depletion or loading of red cells modulates cotransport activity (Delpire & Lauf, 1991; Lauf et al. 1992) and, furthermore, changes in PO2 are unable to alter cotransport activity in the absence of functioning PP/PK enzymes (trout - Cossins, Weaver, Lykkeboe & Nielsen, 1994; horse - Honess et al. 1996). Pharmacologically, however, the sensitivity of the cotransporter to magnesium is low (Delpire & Lauf, 1991; Lauf et al. 1992), and there is no definitive evidence that the magnitude of O2-dependent (physiological) flucations in free [Mg2+]i per se is sufficient to modulate cotransport activity.
Mature sheep red cells lack most organic phosphates (notably DPG) which modulate the O2 affinity of haemoglobin in red cells of other mammalian species and lower vertebrates (e.g. Nikinmaa, 1990). Their haemoglobin also has an N-terminal deletion of the
The final experiment described in this manuscript (Fig. 8) shows that pharmacological changes in free [Mg2+]i affect K+ influx in a manner consistent with reports in the literature (Delpire & Lauf, 1991) but that variations of free [Mg2+]i within the physiological range (about 100 µM) were far too low to account directly for the O2 dependence of the K+ influx. Similar observations have been obtained previously in equine red cells (Gibson, Scott & Cossins, 1995b). It remains possible that the rise in free [Mg2+]i upon deoxygenation, albeit small, may act synergistically with other cell parameters to modulate the cotransporter activity to a significant extent. In this context, the interaction in sheep red cells between pH, volume and Mg2+ has been investigated by Lauf et al. (1994), although the effect of PO2 tension was not addressed. On deoxygenation, changes in cell volume and/or pHi may sensitize the cell to rises in free [Mg2+]i sufficiently to inhibit the cotransporter. If this is the case, the synergism must be considerable because individually these parameters show little variation with PO2.
In conclusion, K+ influx in sheep red cells is O2 dependent but, whilst the similarity between the P50 for O2-activated K+ influx and O2 saturation indicates a role for haemoglobin in this effect, the link may not be via simple alteration of free [Mg2+]i. Our results are relevant to red cells from other mammalian species, including human, and raise important questions concerning the mechanism by which red cell membrane transporters respond to changes in PO2.
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Acknowledgements
This work was supported by The Wellcome Trust and The Physiological Society. E.H.C. holds a University of Liverpool Research Studentship. We are grateful to Ms Helen Braid for assistance with blood sampling.
Corresponding author
J. S. Gibson: Department of Veterinary Preclinical Sciences, University of Liverpool, Brownlow Hill, Liverpool L69 3BX, UK.
Email: JSG@liverpool.ac.uk
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RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References
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) or swollen (
) by 10 %. Cells were incubated in tonometers equilibrated with N2 or O2, as indicated, and K+ influxes measured under this same gas atmosphere. *Cells in which K+ influx was measured in saline of unknown PO2 without pre-incubation in tonometers; **cells incubated in N2 for the entire duration of the experiment, including during influx measurement. Points represent means ±
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) or swollen (
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) in N2 or O2. Bars represent means ±
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) or absence (
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) or swollen (
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) and deoxygenated () cells, incubated with A23187 (20 µM) and different concentrations of extracellular Mg2+ ([Mg2+]o ). Free [Mg2+]i was calculated from the Donnan ratio and [Mg2+]o, as explained in the Methods. Total [Mg2+]i measured in control cells in the absence of ionophore was 1·29 ± 0·08 mM (n = 5) and is indicated by the dashed line. Points represent means ±
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) or N2 () in cells swollen by 5 %. K+ influxes in oxygenated and deoxygenated control cells, i.e. handled in the same way except that they were untreated with A23187, were 1·03 ± 0·02 (n = 4) and 0·45 ± 0·02 mmol (l cells)-1 h-1 (n = 4), respectively, and are indicated by the dashed lines. Points represent means ±
DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References
-chain, as well as other differences with higher affinity haemoglobins, and its structure precludes increased binding of these compounds on deoxygenation (e.g. Perutz, 1970; Bunn, 1971). Nevertheless, the results presented in this manuscript demonstrate considerable O2 dependence of Cl--dependent K+ flux, thus questioning the hypothesis that fluctuations in free [Mg2+]i affect cotransport. We therefore measured total, and estimated free, [Mg2+]i in oxygenated and deoxygenated LK sheep red cells. Total and free [Mg2+]i in oxygenated cells were both similar to those quoted in the literature (i.e. 1·3 and 0·4 mM, respectively; Delpire & Lauf, 1991 - assuming full oxygenation in their paper). Free [Mg2+]i did increase on deoxygenation (to 0·5 mM), and this change, although small, was statistically significant (P < 0·05). The O2-dependent fluctuation in free [Mg2+]i may have been due to alteration in the affinity of haemoglobin for organic phosphates other than DPG, and ATP represents one possible candidate. Overall, however, differences in total [Mg2+]i between oxygenated and deoxygenated cells, loaded or depleted with Mg2+, were low (Fig. 7), indicating a smaller change in intracellular Mg2+ buffering than human red cells (Flatman, 1980).
REFERENCES
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