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Am J Physiol Cell Physiol 279: C981-C989, 2000;
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Vol. 279, Issue 4, C981-C989, October 2000

Oxidants and regulation of K+-Clminus cotransport in equine red blood cells

M. C. Muzyamba, P. F. Speake, and J. S. Gibson

Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 7ZJ, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of oxidants on K+-Cl- cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O2. The substituted benzaldehyde, 12C79 (5 mM), markedly increased O2 affinity. In N2, however, O2 saturation was low (<10%) but KCC remained active. Nitrite (NO2-) oxidized heme to methemoglobin (metHb). High concentrations of NO2- (1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O2 independent but remained sensitive to other stimuli. 1-Chloro-2,4-dinitrobenzene (1-3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O2-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.

oxygen; nitrite; 1-chloro-2,4-dinitrobenzene; erythrocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

POTASSIUM-CHLORIDE COTRANSPORTERS (KCCs) are present in a variety of tissues, including red blood cells (19). A number have been cloned and share homology with the family of electroneutral cation-Cl- cotransporters, whose members include the Na+-K+-Cl- cotransporter (NKCC) and the Na+-Cl- cotransporter (30). Four KCCs have been sequenced to date from human and other tissues; that of red blood cells is probably KCC1 (15, 18, 31, 35).

KCC in vertebrate red blood cells responds to a number of potential physiological stimuli, including cell swelling, H+, and urea (19, 27). In normal high-K+-containing red blood cells, which have an outwardly directed chemical gradient for the transported ions, the activated cotransporter will mediate net KCl efflux, with water following osmotically. In some cells, it contributes to cell shrinkage following swelling and has therefore been implicated in regulatory volume decrease (19). In addition, inappropriate activity of KCC will result in excessive KCl loss, red blood cell shrinkage, elevation of Hb concentration, and also cytoplasmic viscosity (38). Such events will eventually cause deleterious rheological effects, including increased vascular resistance. They may also elevate plasma K+ concentration. KCC activity is inappropriately elevated in certain hemoglobinopathies [notably, in cells containing hemoglobin S (HbS) and in beta -thalassaemics; Refs. 23 and 34] and certain enzyme deficiencies and treatment with oxidants such as nitrite (NO2-; Refs. 1 and 33), diamide (27), and hydrogen peroxide (H2O2; Refs. 4 and 33). Understanding regulation of KCC in red blood cells is therefore important both physiologically and pathologically.

Recently, it has become apparent that physiological O2 tension (PO2) represents an important regulator of KCC (9, 12). In red blood cells from many vertebrate species, KCC is inhibited at low PO2 values and then becomes largely refractory to other stimuli such as cell swelling and low pH (5, 7, 13, 32), although species differences are apparent (see Ref. 12 for a review). A widely held hypothesis suggests that the cotransporter is controlled by the conformation of Hb, being activated by the oxy or relaxed form assumed on combination with O2 (5). A similar explanation has been proposed for the effect of carbon monoxide (CO) and NO2-, which support KCC activity in deoxygenated fish red blood cells, because CO-Hb and methemoglobin (metHb), respectively, assume the same conformation as oxyhemoglobin (oxyHb) (22, 21).

The action of various reagents on KCC activity has also been correlated with their redox potential. NO2- is a powerful oxidant that will oxidize Hb to metHb and deplete the red blood cells of reduced glutathione (GSH). Other oxidants (e.g., acetylphenylhydrazine or H2O2; Refs. 4 and 33) and reagents such as 1-chloro-2,4-dinitrobenzene (CDNB), which removes GSH nonoxidatively (26), also stimulate KCC. The high-KCC activity and abnormal O2 dependence, which is characteristic of sickle cells, may be due to their low content of GSH (1, 25, 33). Because GSH represents a major component of the antioxidant mechanisms of the red blood cell, its depletion represents another form of oxidative stress and will allow the accumulation of metHb secondarily. How these oxidative stresses affect the O2 dependency of the cotransporter has not been established.

In this paper, we examine the responses of the equine red blood cell KCC. This species was chosen because equine red blood cells have a high capacity for KCC and minimal K+ fluxes through other pathways and because their O2 dependence has been characterized in detail (6, 20, 37). We have compared the effects of several agents that cause Hb to assume an oxy shape, O2 per se, CO, and 12C79, a left-shift reagent acting at a site away from the heme group (2), together with NO2-, which directly generates metHb, and CDNB, an agent that removes GSH and thereby indirectly results in formation of metHb. The effect of these manipulations on the activity and O2 dependence of KCC was examined and correlated with changes in GSH and metHb. We found that oxy conformation of Hb was a prerequisite for KCC activity and that oxidative stress at an unidentified site amplified this activity. Findings are discussed in relation to the pathology of oxidant toxicity, red blood cell defects affecting GSH metabolism (e.g., glucose 6-phosphate dehydrogenase deficiency and sickle cell disease), and the mechanism by which O2 regulates membrane transporters.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. CDNB, MOPS, N-ethylmaleimide (NEM), ouabain, salts, and staurosporine were purchased from Sigma Chemical (Poole, Dorset, UK). Calyculin A was purchased from Calbiochem (Nottingham, UK), 86Rb was from DuPont-NEN (Stevenage, UK), and CO and N2 were obtained from BOC (Guilford, UK). 12C79 was a gift from Glaxo Wellcome (Stevenage, UK).

Solutions. The standard saline solution was composed of (in mM) 145 NaCl, 5 glucose, and 10 MOPS (pH 7.4 at 37°C; 290 ± 5 mosmol/kgH2O). For experiments in which Cl- dependence of K+ influx was examined, Cl- was substituted with NO3-. To investigate the effects of anisotonic saline, osmolality was adjusted by addition of distilled water or hypertonic sucrose; when required, pH was altered by addition of HNO3 or NaOH. Stock solutions of ouabain (10 mM) were prepared in distilled water and used at a final concentration of 100 µM. Stock solutions of NEM (100 mM) were prepared daily in distilled water; those of calyculin A and staurosporine were prepared in DMSO and frozen until required. Finally, CDNB was dissolved in methanol (100 mM). In all cases, controls and cells treated with inhibitors or other reagents were exposed to the same concentrations of solvents (methanol or DMSO, whose final concentrations did not exceed 0.5%).

Sample collection and handling. Blood samples were obtained from horses kept at the Department of Veterinary Clinical Sciences and Animal Husbandry (Leahurst, UK) by jugular venepuncture into heparinized vacutainers and prepared as previously described (37).

Tonometry. Before influx or O2 saturation measurements were made, red blood cell suspensions were incubated at about 40% hematocrit in glass tonometers (Eschweiler, Kiel, Germany) flushed with gas mixtures of the appropriate O2 tension (air replaced with N2 using a Wösthoff gas mixing pump), warmed to 37°C, and fully humidified through three humidifiers before delivery. For experiments with CO, cells were treated with CO for 5 min (after which O2 saturation was reduced to <1%) before incubation in the tonometers.

metHb, GSH, and O2 saturation. metHb content was determined colorimetrically by following the method of Hegesh et al. (17) and expressed as a percentage of total Hb. GSH (expressed in mM) was assayed following the procedure described by Beutler (3). O2 saturation was determined using the method of Tucker (39).

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 they were placed in tonometers to adjust PO2. Measurement of both O2 saturation and K+ influx were made in the presence of 12C79 (5 mM). Control samples and those with 12C79 had the same extracellular and intracellular pH.

K+ influx. K+ influx was measured at 37°C using 86Rb as a tracer for K+ (11). 86Rb was added in 150 mM KNO3 to give a final K+ concentration of 7.5 mM. Ouabain (100 µM) was present in all experiments, obviating any K+ influx through the Na+-K+-ATPase. Hematocrit was measured either by the cyanomethemoglobin method or by microhematocrit determination. Influxes are expressed as millimoles of K+ per liter of cells per hour. In horse red blood cells, in the presence of ouabain, KCC represents the predominant K+ transport pathway; however, in several experiments, Cl- dependence (substituted with NO3-) of K+ influx was determined. Also, although in these experiments K+ influx was measured because of the outwardly facing chemical gradient for KCl, a net loss of ions will occur through this pathway (as indicated by the cell volume measurements). Because this concept often causes confusion, in much of the text influx has been replaced with transport.

Measurement of cell volume. Cell water content was determined by the wet weight-to-dry weight method of Borgese et al. (5) and expressed as milliliter per gram of dry cell solids.

Statistics. Data are presented as means ± SD for n replicates for single experiments representative of at least two others on samples from different animals or as means ± SE for n experiments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of CO. We have demonstrated previously that KCC in equine red blood cells is O2 dependent: stimulation by swelling, H+, and moderate (but not high) concentrations of urea only occurs if PO2 is sufficiently high. This is confirmed in the experiments shown in Figs. 1 and 2. Equine red blood cells swollen anisosmotically by suspension in hypotonic saline (260 mosmol/kgH2O) had a high K+ transport in air (Fig. 1) and shrank with time (Fig. 2); when equilibrated with N2, K+ transport was minimal (<10% of that at high PO2), and there was no decrease in cell water content.


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  		Fig. 1.   Effect of carbon monoxide (CO) on K+ transport in equine red blood cells. K+ influx was measured in cells equilibrated at an O2 tension of 0 or 100 mmHg (air replaced with N2), in control cells and in cells treated for 15 min with CO in aliquots shrunken or swollen anisotonically by 10% (by addition of hypertonic sucrose or distilled water). A: N2. B: air. Histograms represent means ± SD (n = 3) for a single experiment representative of 2 others.



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  		Fig. 2.   Effect of nitrite (NO2-) and CO on cell volume of equine red blood cells. Control cells were equilibrated with air or N2; other aliquots were treated with 5 mM NO2- or CO and then fully deoxygenated in N2. All samples were then swollen anisotonically by 10%, and cell water content was measured at the times indicated. Symbols represent means ± SD (n = 3) for a single experiment representative of 2 others.

Figure 1 also shows the effect of pretreating the cells with CO. In this case, the magnitude of K+ transport was similar in both air and N2 and not significantly different from that in oxygenated control cells. The effect of CO on cell water content is shown in Fig. 2: cells pretreated with CO, but subsequently deoxygenated by incubation in N2, shrank to a similar extent as those in O2. Finally, the interaction of CO with inhibitors of the regulatory phosphorylation cascade controlling KCC was investigated. Deoxygenated or oxygenated control cells, and cells pretreated with CO, were exposed to staurosporine (2 µM), NEM (1 mM), or calyculin A (100 nM) or incubated in the absence of inhibitors. The two kinase inhibitors, NEM and staurosporine, stimulated K+ transport; calyculin A inhibited transport. Again, CO-treated cells showed a similar response to oxygenated control cells (data not shown).

These findings imply that CO prevented the inactivation of KCC in equine red blood cells by low PO2, and, in all responses studied, they are consistent with CO mimicking O2.

Effect of 12C79. 12C79 is a substituted benzaldehyde developed to increase the O2 affinity of human Hb, thereby protecting against sickling. The effect of 5 mM 12C79 on O2 saturation of equine red blood cells is shown in Fig. 3. The relationship of O2 saturation against PO2 was shifted to the left; the PO2 required for half-maximal O2 saturation fell from 25 ± 2 to 8 ± 2 mmHg (mean ± SD, n = 4). This marked increase in O2 affinity was very similar to the effect of 12C79 on human red blood cells (14). Notwithstanding the increase in O2 affinity, at very low PO2 values, O2 saturation was low (< 10%) and cells were dark in color, implying that Hb was in the deoxy form.


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  		Fig. 3.   Effect of the substituted benzaldehyde 12C79 on O2 saturation of equine red blood cells. Cells (~40% hematocrit) were exposed to 12C79 (5 mM) or left untreated for 15 min before equilibration in tonometers for a further 15 min at the O2 tensions indicated. O2 saturation was then determined following the method of Tucker (39). Symbols represent means ± SD (n = 4).

The effect of 12C79 on KCC activity was also determined (Fig. 4). In the absence of the reagent, KCC activity in control equine red blood cells was completely O2 dependent. In the presence of 12C79, however, K+ transport remained at levels observed in oxygenated red blood cells regardless of the PO2. Transport did not depend on O2 saturation. Thus in N2, when O2 saturation fell to <10% maximal, KCC activity remained high.


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  		Fig. 4.   Effect of the substituted benzaldehyde 12C79 on K+ transport in equine red blood cells. Cells were handled as described in the legend to Fig. 3. On removal from the tonometers, aliquots were diluted 10-fold into saline (260 mosmol/kgH2O, preequilibrated at indicated O2 tension) for determination of K+ influx. Influxes are expressed as a percentage of those measured at 150 mmHg, which were 0.53 ± 0.03 and 0.42 ± 0.01 mmol · liter cells-1 · h-1 in the presence and absence of 12C79, respectively. Symbols represent means ± SD (n = 3) for a single experiment representative of 2 others.

Effect of nitrite. In the next series of experiments, we examined the effect of NO2- on K+ transport in equine red blood cells. It has been suggested that NO2- acts by oxidizing Hb to metHb, which has a conformation like that of oxyHb. If this is the case, its action should be identical with that of CO.

Untreated cells or cells treated for 30 min with various concentrations of NO2- were equilibrated with different PO2 levels to examine the effect of K+ transport on O2 dependence (Fig. 5). Control (untreated cells) had the lowest K+ transport, which decreased with PO2 so that, in N2, K+ transport was abolished, as expected for an O2-dependent KCC (compare with Fig. 1). As the concentration of NO2- was raised from 0.5 to 5 mM, K+ transport was stimulated above those of control cells and also became progressively independent of PO2. For example, at 5 mM NO2-, K+ transport in air was stimulated fivefold with respect to that in control cells; its magnitude in N2 was 94% that in air. The effect of NO2- on cell volume is shown in Fig. 2. Cell water content in deoxygenated red blood cells treated with 5 mM NO2- decreased progressively with time and at a faster rate than that observed for oxygenated control red blood cells or deoxygenated ones pretreated with CO. In cells treated with 5 mM NO2- but in the absence of Cl-, K+ transport was <0.20 mmol · liter cells-1 · h-1 and cell water content declined by 1% after 2 h, indicative of an action of NO2- mainly on KCC.


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  		Fig. 5.   Effect of NO2- on the O2 dependence of K+ transport in equine red blood cells. Cells were washed into Cl--free saline (~40% hematocrit) and then treated with various concentrations of NO2- (0, 0.5, 1, and 5 mM) for 30 min before equilibration in tonometers at the indicated O2 tensions for 15 min. Aliquots were then removed from the tonometers and diluted 10-fold into saline ± Cl- (260 mosmol/kgH2O) for determination of K+ influx. Cl--dependent K+ influx was calculated as the difference in influx ± Cl-. Symbols represent means ± SD (n = 3) for a single experiment representative of 2 others.

The effect of NO2- on KCC in red blood cells in vivo (e.g., NO2- toxicity in farm animals) will also depend on the extent to which the transporter remains susceptible to the physiological stimuli that act on control cells. We therefore examined in some detail the extent to which NO2--treated cells could respond to other stimuli of KCC. The effect of combining treatment with NO2- and exposure to anisotonic saline is shown in Fig. 6, A and B, for oxygenated and deoxygenated red blood cells, respectively. In air, K+ transport in control cells increased progressively as osmolality was lowered. Oxygenated NO2--treated cells had the same pattern of response; however, in all cases, K+ transport was higher than that in control cells (5-fold and 2.5-fold at 290 and 230 mosmol/kgH2O). In deoxygenated red blood cells, K+ transport in NO2--treated cells was also volume sensitive and similar in magnitude to that observed in air; in deoxygenated, control cells, transport was low and unresponsive to volume. Similar results were obtained if the stimulus was urea or H+, rather than volume (Fig. 7).


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  		Fig. 6.   Effect of NO2- on volume sensitivity of K+ transport in equine red blood cells. Cells (40% hematocrit) were treated with 5 mM NO2- or left untreated and then placed in tonometers for 15 min for equilibration with air (A) or N2 (B). These gas tensions were maintained for the remainder of the experiment. Aliquots were then removed from the tonometers and diluted 10-fold into saline at a range of osmolalities (230-360 mosmol/kgH2O, again fully equilibrated with air or N2) to anisotonically swell and shrink the cells. K+ influx was determined immediately. Symbols represent means ± SD (n = 3) for a single experiment representative of 2 others.



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  		Fig. 7.   Effect of NO2- on H+- and urea-stimulated K+ transport in equine red blood cells. Cells (40% hematocrit) were treated with 5 mM NO2- or left untreated and then placed in tonometers for 15 min for equilibration with air (A) or N2 (B). On removal from the tonometers, aliquots were then diluted 10-fold into saline to give a final pH of either 7 or 7.4 (both 290 mosmol/kgH2O) or exposed to saline to which 500 mM urea had been added (all salines fully equilibrated with air or N2). K+ influx was determined 10 min later. Symbols represent means ± SD (n = 3) for a single experiment representative of 2 others.

Untreated cells or cells pretreated with NO2- were also exposed to protein phosphatase (PP) inhibitors (calyculin A) and protein kinase (PK) inhibitors (staurosporine and NEM). Results are shown in Fig. 8. The two kinase inhibitors, NEM and staurosporine, stimulated K+ transport. Treatment with NO2- had little effect on the responses to these inhibitors, and the effects of PK inhibition and NO2- were not additive. Calyculin A inhibited NO2--stimulated K+ transport by 79% when added before NO2- (as shown in Fig. 8B) and 58 ± 6% (mean ± SE, n = 5) when added afterward.


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  		Fig. 8.   Effect of NO2- and kinase and phosphatase inhibitors on K+ transport in equine red blood cells. Cell samples (40% hematocrit) were left untreated or exposed to various protein kinase or phosphatase inhibitors [1 mM N-ethylmaleimide (NEM), 2 µM staurosporine, or 100 nM calyculin A] for 15 min. Samples were divided into 2, one was treated with NO2- (5 mM; B) and the other (control) aliquot was not (A). Samples were then placed in tonometers and equilibrated with N2 for 45 min and then air for a further 15 min, after which they were then diluted 10-fold into saline for measurement of K+ influx. Histograms represent means ± SD (n = 3) for a single experiment representative of 2 others.

Similar to CO, treatment with NO2- was associated with a KCC activity unresponsive to PO2. It appeared to act via the regulatory PP-PK enzymes. In marked contrast to CO, however, after treatment with NO2-, K+ transport was stimulated above the levels observed in oxygenated control cells.

Effect of CDNB. CDNB reacts with GSH in the presence of glutathione-S-transferase, thereby depleting the red blood cell of GSH. The effect of various concentrations of CDNB on K+ transport in equine red blood cells is shown in Figs. 9 and 10. In Fig. 9, oxygenated or deoxygenated cells were treated with 1 mM CDNB at a hematocrit of 40% for 30 min and then K+ transport was measured in the absence of CDNB. In these experiments, the magnitude of K+ transport was similar in control and CDNB-treated cells in both air and in N2. After a 3-h exposure to 1 mM CDNB, however, KCC activity was stimulated over oxygenated control levels and only inhibited about 50% by deoxygenation (data not shown). In the second series of experiments, cells at 40% hematocrit were treated with progressively higher concentrations of CDNB (1, 2, and 3 mM) for 30 min, and transport was measured at one-tenth this hematocrit and CDNB concentration. As the concentration of CDNB was increased, K+ transport was stimulated over oxygenated control levels (Fig. 10). K+ transport (expressed as a percentage) in N2 compared with that in air increased from 7% in control cells to 19, 56, and 66% in cells treated with 1, 2, and 3 mM CDNB, respectively, so that the transport also became progressively less dependent on O2.


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  		Fig. 9.   Effect of 1-chloro-2,4-dinitrobenzene (CDNB) on O2-dependent K+ transport in equine red blood cells. Cells (40% hematocrit) were left untreated (A) or exposed to 1 mM CDNB (B) and then placed in tonometers for 30 min for equilibration with air or N2. These gas tensions were maintained for the remainder of the experiment. Aliquots were then diluted 10-fold into saline (290 or 260 mosmol/kgH2O) for determination of K+ influx. Histograms represent means ± SD (n = 3) for a single experiment representative of 2 others.



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  		Fig. 10.   Effect of different concentrations of CDNB on O2-dependent K+ transport in equine red blood cells. Cells were handled as described in the legend to Fig. 9, except that 3 concentrations of CDNB were used (1, 2, and 3 mM). K+ influx was then determined at 290 mosmol/kgH2O only with CDNB present during the measurement at one-tenth the concentration in the tonometers (0.1, 0.2, and 0.3 mM, respectively). Histograms represent means ± SD (n = 3) for a single experiment representative of 2 others.

Determination of GSH and metHb. Finally, we determined the effect of CO, NO2-, 12C79, and CDNB on levels of GSH and metHb under the conditions used for K+ transport measurements. Results are given in Table 1. In air and N2, and after treatment with CO and 12C79, metHb levels were low (<1%) and reduced GSH concentrations were high (about 3 mM). After treatment with NO2-, metHb levels increased progressively with NO2- concentration. At low concentrations of NO2-, the percentage of metHb was higher in air than in N2; the reverse occurred at higher NO2- concentrations. GSH levels were reduced by NO2- but, even at 5 mM, NO2- levels in deoxygenated cells (in N2) remained at about 50% those in control cells. NO2- therefore appeared to oxidize Hb before lowering the GSH concentration. With CDNB, GSH levels were most sensitive, being reduced by about 50% by 0.5 mM CDNB and reduced totally at higher levels, whereas metHb only increased at the highest CDNB concentration (15% in N2 with 3 mM CDNB). At 1 mM CDNB, metHb levels were 1.7, 9.9, and 16.4% after 10 min, 1.5 h, and 3 h, respectively, whereas at all time points GSH was depleted (<7%).

                              
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  		Table 1.   Effect of various manipulations on the reduced glutathione and methemoglobin contents of equine red cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we compared the effects of several manipulations that affect KCC. First, we looked at reagents that bind to the O2 site of Hb: O2 per se and CO. Second, we looked at the substituted benzaldehyde 12C79, which interacts with other sites in the Hb molecule but again increases O2 affinity and causes it to assume the oxy conformation. Third, we examined an oxidant, NO2-, which oxidizes the heme from Fe2+ to Fe3+ to produce metHb. Fourth, we looked at the reagent CDNB, which does not directly alter Hb but has removal of GSH as its primary action, although loss of the antioxidant capacity of the red blood cell will secondarily encourage the accumulation of metHb. We correlated the effects of these manipulations on the activity and O2 dependence of KCC and on GSH and metHb levels. Our findings show that an oxy conformation of Hb was required for KCC activation. An increase in KCC activity over control levels in oxygenated cells, however, did not correlate with either oxyHb conformation or depletion of GSH alone but rather required both accumulation of metHb and depletion of GSH, implying that oxidative damage was required at some as yet unidentified target. Finally, the lack of additivity between NO2- and staurosporine or NEM, and the continued sensitivity to calyculin A, may imply a common site of action on the regulatory phosphorylation cascade that controls KCC upstream of the calyculin-sensitive phosphatase.

O2-dependent membrane transporters and oxidants. A number of membrane transporters in vertebrate red blood cells respond to PO2 (9, 12). The effect occurs over physiological PO2 levels and is selective. Most work has involved inorganic ion cotransporters and countertransporters (9, 12), but ion channels and other transport systems (e.g., amino acid transporters; Ref. 24) can also be O2 dependent. As a general rule, regulatory volume decrease pathways (e.g., KCC) are stimulated by O2 and regulatory volume increase pathways [e.g., NKCC or Na+/H+ exchanger (NHE)] are stimulated by deoxygenation (9). Although the effect is potentially significant both physiologically and pathologically (as reviewed in the introduction), little is known about its mechanism. A role for Hb has been proposed (29). The differential binding of oxyHb and deoxyHb to the cytoplasmic tail of band 3 (8) may modulate membrane permeability via a signaling pathway involving other parts of the cytoskeleton or via regulatory enzymes (glycolytic, kinases, phosphatases), which also associate with this site (28). Most evidence in support of this hypothesis comes from work with fish red blood cells. Thus, for trout NHE and KCC, CO mimics O2 (5, 29); alteration of Hb O2 affinity in carp red blood cells through modulation of intracellular pH causes changes in KCC activity that correlate with the conformation of Hb (21). NO2- activates KCC in deoxygenated carp red blood cells, which is explained because metHb has the oxy conformation (21, 22). In addition, other oxidants (e.g., acetyl-phenylhydrazine and H2O2; Refs. 4 and 33) and nonoxidizing agents, such as CDNB (26), increase KCC activity to levels greater than those in control cells. All are associated with a decrease in GSH and sometimes elevation in metHb, but their O2 dependence has not been investigated.

Several pathological conditions are relevant to this discussion. First, in sickle cell disease, red blood cells dehydrate rapidly partly because of an unusually active KCC (23). Although this is partly due to a younger population of red blood cells, it remains unclear why the transporter is so active in HbS-containing red blood cells. However, the cells are under oxidative stress because HbS breaks down faster than hemoglobin A (HbA) (16, 37). GSH levels are low (25, 40), and it has been suggested that this may in part account for the elevated rates of KCC in sickle cells compared with HbA-containing red blood cells (1, 33). A similar situation occurs in beta -thalassaemia and in certain red blood cell enzymopathies (such as glucose-6-phosphate dehydrogenase deficiency). Second, oxidant toxicity by NO2- occurs in fish exposed to this environmental pollutant and in herbivores (cattle, sheep, and horses) ingesting high levels of nitrate (e.g., in heavily fertilized grass or in Brassicas), which is subsequently reduced to NO2- in the rumen or large intestine. Third, a number of other hemolytic diseases are caused by foreign substances acting as oxidants, ranging from a variety of chemotherapeutic agents in humans (e.g., antimalarials and antipyretics) to onion toxicity in dogs and sheep (36, 41). Here, we compared several oxidants and other reagents that cause Hb to assume its oxy configuration, to lower GSH, or to apply oxidative stress, and we analyzed their similarities and differences with respect to control of KCC.

CO and 12C79. In the first series of experiments, we examined the effect of CO. This gas binds to heme with an affinity about 200-fold greater than O2, forming CO-Hb, which has the same conformation as oxyHb. As observed for fish, CO-treated equine red blood cells behaved like oxygenated ones with regard to K+ transport via KCC or the change in cell volume that it mediates, irrespective of PO2. Responses to a number of PP-PK inhibitors (NEM, staurosporine, calyculin A) were also unaffected. These observations are all consistent with CO mimicking O2 and agree well with previous work on fish red blood cells.

12C79 is one of a series of substituted benzaldehyde compounds, originally developed as antisickling agents (2). These compounds combine with Hb, forming Schiff bases with amino groups; those with the terminal amino groups of the alpha -chain force Hb into the oxy conformation. They were originally developed as antisickling agents because oxygenated HbS does not polymerize; however, their use clinically is now restricted mainly to neoplasia therapy; stabilizing Hb in the oxy form starves neoplastic tissue of O2. We demonstrated that 12C79 also caused a marked increase in O2 affinity in equine red blood cells. KCC activity in 12C79-treated cells was stimulated at low PO2 levels, and the transporter became largely independent of PO2. KCC activity, however, did not correlate with O2 saturation; at low PO2 levels, even with 12C79, O2 saturation was very low (<10%), but KCC activity remained at levels close to those of fully oxygenated red blood cells. A similar finding was observed in human HbA red blood cells (14). If oxyHb controls KCC (and if 12C79 acts predominantly on Hb and not at some other site), these results imply that only a small fraction of Hb is involved, not total Hb. As discussed above, Motais et al. (29) speculated that the Hb fraction that associates with band 3 is responsible, and these findings would support such a model.

NO2- and CDNB. NO2- oxidizes Fe2+ to Fe3+, forming metHb, which has an oxy conformation. If this is its only significant effect, NO2- should behave like CO. Indeed, like CO and 12C79, NO2- also stimulated KCC in deoxygenated cells, making it O2 independent. Unlike either of these, however, it increased KCC activity over the control levels observed in untreated, oxygenated cells. Cells dehydrated faster when treated with NO2- compared with oxygenated controls or CO-treated cells, and this effect was fully Cl- dependent, consistent with mediation via KCC. In addition to being stimulated by NO2-, the transporter also remained sensitive to other stimuli, such as H+, urea, and cell volume. These features will potentially exacerbate the deleterious effects of NO2- toxicity. MetHb, which cannot transport O2, will cause tissue hypoxia, anaerobic metabolism, and acidosis, further stimulating the cotransporter. These stimuli are thought to act via the phosphorylation cascade that regulates KCC1. Pharmacologically, enzymes of this cascade can be modulated by PK inhibitors (e.g., staurosporine and NEM) to stimulate KCC1 activity or by PP inhibitors (e.g., calyculin A) to inhibit it. The response to such stimuli in cells treated with NO2-, the lack of additivity between NO2- and staurosporine or NEM, and the continued sensitivity to calyculin A may imply a common site of action on the regulatory phosphorylation cascade that controls KCC (10). If this is the case, NO2- must act upstream of the calyculin A-sensitive phosphatase.

Unlike NO2-, the primary effect of CDNB is to deplete GSH, although this will eventually lead to accumulation of metHb. Low levels of CDNB, or shorter exposure, had little effect on KCC activity or O2 dependence. In contrast, at high concentrations or longer exposures, CDNB treatment stimulated KCC over the level observed in oxygenated control cells and its activity also became progressively independent of PO2.

OxyHb, GSH, and control of KCC. Table 2 summarizes the effects of the various manipulations on transporter activity together with their effects on metHb and GSH levels. KCC was activated in deoxygenated cells by CO and 12C79 and by the higher concentrations of NO2- and CDNB. CO and 12C79 directly cause adoption of the oxyHb conformation either through effects on heme or elsewhere. Nevertheless, in red blood cells treated with 12C79 and incubated in N2, most Hb is in the deoxy form. Low levels of CDNB, despite completely depleting cells of GSH, did not activate the transporter in N2 nor did they increase its activity in oxygenated red blood cells. At the higher levels of NO2- and CDNB, when KCC became O2 independent, significant accumulations of metHb were present, again indicative of Hb in the oxy form, and GSH was depleted. Higher concentrations of CDNB and NO2- also increased KCC activity above that observed in oxygenated controls. Similar results were obtained with prolonged exposure (3 h) to 1 mM CDNB. In agreement with our findings, for elevated KCC activity in LK sheep red blood cells treated with NO2-, both accumulation of metHb and depletion of GSH (1) are shown, whereas in human red blood cells, activity of KCC in response to a number of oxidants did not correlate with GSH depletion alone (33).

                              
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  		Table 2.   Correlation of KCC activity, indicators of red cell oxidation, and conformation of Hb in equine red cells in response to CO, the substituted benzaldehye 12C79, NO2-, and CDNB

In conclusion, our observations imply that the oxy form of Hb is a prerequisite for transporter activation but the oxy form of Hb or depletion of GSH alone does not increase KCC activity over the level found in oxygenated control cells. Oxidative damage must occur at some other unidentified site to produce such stimulation, presumably interacting with the phosphorylation cascade that controls the cotransporter.


    ACKNOWLEDGEMENTS

We thank J. E. Cox for provision of equine blood samples.


    FOOTNOTES

This work was supported by the Wellcome Trust.

M. C. Muzyamba holds a Beit Fellowship and ORS award.

Address for reprint requests and other correspondence: J. S. Gibson, Dept. of Physiology, St. George's Hospital Medical School, Univ. of London, Cranmer Terrace, Tooting, London, SW17 0RE, UK (E-mail:jsgibson{at}sghms.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 December 1999; accepted in final form 13 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(4):C981-C989
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