Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

Isabel Bize, Samara Taher, and Carlo Brugnara

Departments of Cell Biology, Harvard Medical School, and Department of Laboratory Medicine, Children's Hospital Boston, Boston, Massachusetts 02115

Submitted 27 September 2002 ; accepted in final form 22 February 2003


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d ~33.4 g/dl) has only about ~5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ~24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r2 = 0.995) and sickle erythrocytes (r2 = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity.

phosphorylation; protein phosphatase; urea; cell size; density


IT HAS BEEN POSTULATED that the elevated activity of K-Cl cotransport (KCC) in sickle erythrocytes contributes to their dehydration, their short life-span, and the pathology associated with this disease (7). Reticulocyte maturation is associated with a decrease in cell volume mediated by KCC (23), followed by a sharp decrease in KCC activity (2, 7, 8, 15, 16, 24). The functional inactivation of KCC in denser cells can be reversed by a variety of stimuli except in the 5% densest cells, indicating that in mature erythrocytes a significant amount of KCC-protein remains in the membrane in a latent state (9, 14). In normal (AA) and sickle (SS) erythrocytes of increasing densities, a slight decrease in membrane-associated KCC-protein has been reported (31). Thus it appears that the large decrease in KCC activity with increasing cell densities is due almost exclusively to functional inactivation. The functional state of KCC is determined by phosphorylation/dephosphorylation, either directly or via an associated protein. In mature erythrocytes, the low activity of KCC in isosmotic media appears to be due to high levels of phosphorylation of the transporter (or an associated protein) and is thought to be the result of the high activity of a kinase relative to the activity of a phosphatase, the substrate of which is the KCC transporter (19, 13, 17), although direct phosphorylation of the KCC-protein has yet to be demonstrated.

In erythrocytes, KCC dephosphorylation appears to be mediated by membrane-associated protein phosphatase(s) (4, 28). The activating phosphatase(s) belongs to a family of Ser/Thr phosphatases sensitive to okadaic acid and calyculin A (18). Type 1 phosphatases (PP1) have been proposed to activate KCC (4, 22, 26, 30), although more recent evidence indicates that type 2A protein phosphatases (PP2A) may also activate KCC (3, 4). Membrane-associated PP1 and PP2A (mb-PP1, mb-PP2A) appear to be specific for two functionally distinct phosphorylation sites. Dephosphorylation of one site by mb-PP1 appears to result in KCC activation in response to low ionic strength, whereas dephosphorylation of the other site (by mb-PP2A) appears to result from an increase in cell size (4). To determine whether the age/density-dependent decrease in basal KCC activity in AA erythrocytes is related to an age/density-dependent decrease in the basal activity of mb-PP1 and/or mb-PP2A, or on cellular redistribution of either phosphatase during reticulocyte maturation, we determined PP1 and PP2A activities in the soluble and membrane-associated compartments of density-fractionated human AA and SS erythrocytes and in red blood cells of control and anemic rabbits.

To determine whether the elevated activity of KCC in SS erythrocytes is due to a defect in its inactivation, we compared KCC activity in reticulocyte-rich fractions of AA and SS erythrocytes. To determine the role of the activating phosphatases in the defective regulation of KCC in SS cells, we compared phosphatase activity in density-matched populations of AA and SS erythrocytes.

Urea activates KCC in dog, human, sheep, and horse erythrocytes (12, 21, 27, 29). It has been postulated that urea may influence dehydration of SS cells passing through the kidney (11, 21). Even though KCC activity is reduced in the hypertonic conditions found in this organ, in equine red cells urea stimulation of KCC is not abolished by hypertonic shrinkage (29). Stimulation of KCC by urea is inhibited by okadaic acid (27) and by calyculin A (21, 29), potent inhibitors of PP1 and PP2A (18), indicating that urea stimulation is mediated by phosphorylation/dephosphorylation. Stimulation of KCC by urea could be mediated by inhibition of the KCC inhibitory kinase (12, 29). However, the absence of a lag time in urea-induced KCC stimulation in SS cells (11) suggests that urea may also stimulate an activating reaction, possibly a phosphatase (19). Unpublished preliminary experiments carried out in our laboratory demonstrated that urea stimulates erythrocyte mb-PP1 activity and does not stimulate the purified catalytic subunit of PP1 (PP1c, from rabbit muscle), suggesting that urea may relieve inhibition of mb-PP1c by an inhibitory subunit. We therefore set out to determine the degree of KCC stimulation and mb-PP1 stimulation by urea in AA erythrocytes of increasing density to understand the mechanism of urea-induced KCC activation.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Human Erythrocytes

Human red blood cells were obtained from AA healthy adult volunteers and patients with homozygous SS cell anemia after informed consent. Cells were washed by centrifugation in cold isotonic "NaCl wash" containing 150 mM NaCl, 10 mM Tris-MOPS, or Tris-Cl, pH 7.4, at 4°C.

Rabbit Erythrocytes

New Zealand White rabbits, weighing 3–5 kg, were used in this study. Blood was collected into heparinized tubes by peripheral ear vein puncture according to Brugnara and De Franceschi (6). Twenty percent of total blood volume was drawn on 5 consecutive days. Blood from the first day of bleeding (day 1) was used as control. Young red cells were collected on day 8 when the reticulocyte count reaches 40%. The bleeding protocol reduced the hematocrit to <20%. Studies were performed under Children's Hospital animal study protocol no. A01-08-077R.

Isosmotic Gradient Centrifugation (Human Erythrocytes)

Isotonic Stractan (arabino-galactan) was used to prepare density gradients (10). Densities ranged from 1.077 to 1.101 g/dl. Stractan was dissolved in buffer containing 134 mM NaCl, 5 mM KCl, 11.1 mM glucose, and 10 mM NaPO4 buffer, pH 7.4, osmolarity 290–295 mosM gradients were prepared in 15-ml tubes, with the addition of 0.5 ml of density 1.157 g/dl (as a cushion), 1 ml of 1.101, 0.75 ml of 1.095, 1 ml of 1.089, and 2 ml each of 1.086, 1.083, and 1.080 g/dl, followed by 0.75 ml of density 1.077 g/dl. Two milliliters of 50% cell suspension were added to the Stractan gradients, and the tubes were centrifuged for 35 min at 25°C at 72,000 g in a swinging bucket rotor. Fractions were collected, and the percentage of reticulocytes, the mean cell volume, and the mean cell hemoglobin concentration were determined using an automated system (ADVIA 120; Bayer Diagnostics, Tarrytown, NY). Mean cell hemoglobin concentration (MCHC, g/dl) is directly related to mean cell density (d). The density distribution of the whole blood cell population ranges from 22 to 40 g/dl. The mean density of whole blood is ~31 g/dl, and ~90% of the cells have values between 26 and 34 g/dl.

K-Cl Cotransport Activity

KCC activity was determined using 86Rb+ influx calculated from a single time point (20 or 30 min) or from 86Rb+ uptake over a 22.5-min period, as previously described (4). Washed red cells were incubated at 10–20% hematocrit at 37°C in flux medium containing 145 mM NaCl, 2 mM KCl, 5 mM glucose, 1 mM MgCl2, 10 mM Tris-MOPS, pH 7.4, at 37°C, 0.1 mM ouabain, and 10 µM bumetanide. For experiments presented in Fig. 4, the flux medium contained 5 mM K+. Isotonic Cl-free media contained 145 mM NaSfa (Sfa = sulfamate) and 1 mM MgNO3 instead of the respective chloride salts. Hypertonic media contained 200 mM NaCl or NaSfa. The flux media contained 10–20 µCi/ml 86Rb+. Fluxes are expressed in millimoles per liter of cells x hour, where liter corresponds to the original average volume and density of washed cells (~31 g/dl).



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Fig. 4. Urea stimulates 86Rb+ influx in human red cells suspended in hypertonic media. Cells of density (d) ~25 g/dl were incubated in isotonic and hypertonic media containing Cl, and in hypertonic media containing Sfa in the presence and absence of 500 mM urea. Flux media contained 5 mM K+. *P = 0.003; **P = 0.02, different from respective controls.

 

Subcellular Preparation

Human erythrocytes. Red cells were lysed in 10 volumes of isotonic solutions (290–300 mosM). Isotonic lysis buffer contained 140 mM KCl, 10 mM NaCl, 10 mM Tris-MOPS, pH 7.4, 0.1% {beta}-mercaptoethanol, 10 µM phenylmethylsulfonyl fluoride, and 25 µg/ml each of leupeptin and aprotinin. Sonic cell disruption was performed by one or two bursts (5 s each) at 0.05 W of power output. Nonruptured cells were removed by pelleting at low speed (500 g for 3 min) before the centrifugation at 30,000 g to separate the membranes from the cytosol.

Rabbit Erythrocytes

Red cells were lysed in 15 volumes of hypotonic lysis buffer containing 10 mM Tris-Cl, pH 7.4, at 4°C, 0.1% {beta}-mercaptoethanol, 10 µM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Red blood cell lysis in hypotonic buffer produces "ghosts," cellular structures devoid of soluble components.

All preparations were performed at 4°C. Cell lysates were centrifuged (30,000 g for 10 min). The supernatant (cytosol) was saved and the membrane pellet was washed (3–4 times) by centrifugation and resuspension in hypotonic lysis buffer. Other details are as previously published (3, 4). Protein was determined with the Lowry assay (25).

Phosphatase Activity

Ser/Thr protein phosphatase activity was determined using 32P-labeled glycogen phosphorylase-a. 32P-labeled substrate was prepared as previously described (3). Protein concentration in the phosphatase assay was 50–100 µg/ml for the membrane and 100–250 µg/ml for the cytosol preparations. Phosphatase activity is expressed as units per milligram of protein, where one unit (U) equals 1 nmol of phosphate released per minute.

Media and Reagents

Stractan (CellSep) was obtained from Cardinal Associates (Santa Fe, NM). Okadaic acid was obtained from LC Laboratories (Woburn, MA) or from Sigma Chemical (St. Louis, MO) and dissolved in either ethanol or dimethylsulfoxide (DMSO) at 100 µM and stored at –20°C. [{gamma}-32P]ATP (3,000 Ci/mmol) was obtained from Dupont New England Nuclear (Boston, MA). In some experiments, phosphatase assay was performed with a kit from GIBCO BRL (Gaithersburg, MD). Otherwise, phosphorylase-b was obtained from RBI (Natick, MA), and phosphorylase kinase was from Sigma Chemical. The purified catalytic subunit of PP1 (from rabbit skeletal muscle) and PP2A (human erythrocytes) were purchased from Upstate Biotechnology (Lake Placid, NY).

Statistics

The results were analyzed for differences between means using Student's t-test for paired comparisons and by ANOVA for unpaired comparisons. All error bars represent SE.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human Erythrocyte KCC Activity and mb-PP1 Activity Are Dependent on Cell Density in AA and SS Erythrocytes

Cl-dependent 86Rb+ influx was determined in density-separated human AA and SS red blood cell populations suspended in isotonic media. We have determined that Stractan density-gradient centrifugation does not affect KCC activity (data not shown). Figure 1A shows the expected decrease in "basal" (i.e., in isotonic conditions) Cl-dependent 86Rb+ influx (KCC activity) as the average density of AA and SS erythrocyte populations increased. Figure 1B shows that mb-PP1 activity also decreased as the average density of AA and SS erythrocyte populations increase. To illustrate the relative abundance of each population studied in relation to the total red blood cell population, Fig. 1C shows the average density distribution (whole blood) of washed red cells from AA and SS individuals, obtained with the Bayer ADVIA 120 Hematology analyzer,



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Fig. 1. Cl-dependent 86Rb+ influx and membrane (mb)-PP1 activity decrease in normal (AA) and sickle (SS) red cells of increasing density. A: cells were separated in isotonic Stractan density gradients, and 86Rb+ influx was determined in Cl-containing and Cl-free (Sfa) media. Flux media contained 2 mM K+. At least 4 fractions were collected in each experiment. Ten experiments were performed in AA cells and 6 in SS cells. B: PP1 activity was measured in the membrane fraction of density-separated AA and SS cells. Twenty-one experiments were performed in the membranes obtained from 5 AA subjects and 12 in membranes from 8 SS subjects. C: density distribution of AA and SS washed red cells in whole blood (shown for comparison).

 

Figure 1A shows that in AA erythrocytes, a 50% decrease (compared with the least-dense cells) in KCC activity (d50) was observed at d ~26 g/dl. A second-order linear regression of KCC activity vs. d in each experiment yielded a d50 = 26.8 ± 0.3 g/dl (AA, n = 10). Because <5% of the AA cell population had d <= 26.8 g/dl, and because the majority of cells in population of average d <= 26.8 g/dl were presumably very young, the data are consistent with a fast decrease in KCC activity associated with, or following soon after, reticulocyte maturation (7, 8, 15, 16). The decrease in basal KCC activity in AA erythrocytes was associated with a nonsignificant decrease in the percentage of reticulocytes (5.3 ± 1% reticulocytes at d = 23.4 g/dl and 3.9 ± 0.8% reticulocytes at d = 26.3 g/dl). In fractions of d >= 29 g/dl, the percentage of reticulocytes was <1%.

Figure 1A also shows a decrease in KCC activity in dense SS cells. A 50% decrease was observed at d ~30 g/dl. A second-order linear regression of KCC activity vs. d in each experiment yielded a d50 = 31.4 ± 0.9 g/dl. (SS, n = 8). The calculated d50,sickle value appears higher than shown in Fig. 1A because two experiments had high KCC activity in the low-density fractions. The calculated values for each experiment were 27.5, 28.7, 31.6, 32.1, 35.1, 32.2, 29.5, and 34.6. The d50 for KCC inactivation as a function of cell density in SS cells (n = 8) was significantly higher than in AA cells (31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, P = 0.004), suggesting an abnormal KCC regulation in less dense SS erythrocytes and a failure to inactivate KCC when d reaches ~28 g/dl. KCC activity in least-dense SS cells was not different (0.5 ± 0.12 mmol/l x h, n = 6) from KCC activity in least dense AA cells (0.31 ± 0.06 mmol/l x h, n = 10).

Figure 1B shows a decrease in mb-PP1 activity as the density of AA and SS cell populations increased. Density-separated AA and SS erythrocytes were lysed (by sonication) in isotonic media, and the cytosol and membranes (fragments) were separated (5). No discernible difference in the rate of mb-PP1 activity decrease between AA and SS cells, as the cell density increased, was detected. A 50% decrease in mb-PP1 activity (second order linear regression) in AA cells was calculated at 27.2 ± 0.2 g/dl and in SS cells at 28.1 ± 0.4 g/dl.

Interestingly, soluble PP1 (sol-PP1) appeared to decrease at the same rate as mb-PP1 in AA and SS cells (not shown), indicating that the percentage of PP1 associated to the membrane fraction is maintained relatively unchanged as cell density increases (i.e., the affinity of PP1 for the membrane does not depend on cell age/density).

Figure 1C shows the density distribution of the AA and SS cell population studied. These data are similar to previously published results. Because the life-span of SS erythrocytes is only ~20 days compared with 120 days of AA erythrocytes, and because the density increases as reticulocytes mature and erythrocytes age, the least-dense AA and SS erythrocytes must be of a similar age (i.e., 0–10 days old), whereas denser AA and SS erythrocytes (i.e., >30 g/dl) must have large age differences. For example, most AA cells of density >30 g/dl are probably 60 days or older, whereas most SS erythrocytes of density >30 g/dl are probably only 2–20 days old.

Mb-PP1 Activity Correlates With KCC Activity in Density-Separated Cells

Figure 2 shows the correlation between basal KCC activity and basal mb-PP1 activity in AA and SS cells. Pooled data of mb-PP1 and KCC activities for AA and SS erythrocytes were grouped in such a way that the average density for mb-PP1 data was equal to the average density of KCC data. Between d = 26.2 and d = 33.7 g/dl, the correlation (r2 of average data) between mb-PP1 and KCC activity in AA cells was 0.995. In the least-dense AA cell population, however, the slope of KCC decrease was higher than the slope of mb-PP1 decrease, indicating that additional factors determine basal KCC inactivation between d ~24 and d ~26 g/dl. In density-separated SS erythrocytes, a good correlation was found between mb-PP1 and KCC activities (r2 = 0.93), but the relationship seems to be qualitatively different from that seen in AA cells. For example, at 0.2 U/mg protein of mb-PP1 activity, KCC activity was ~0.03 mmol/l cells x h in AA cells and ~0.2 mmol/l cells x h in SS cells. The data, therefore, suggest that abnormal regulation of KCC activity during maturation of SS erythrocytes is not explained by abnormal regulation of mb-PP1.



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Fig. 2. Correlation between K-Cl cotransport (KCC) activity and mb-PP1 activity in AA and SS cells. KCC and mb-PP1 activities data from Fig. 1, A and B, were compared in groups of the same average density. At the origin are cells with high density (low mb-PP1 and low KCC). KCC activity (average, SE) was plotted against mb-PP1 activity (average, SE).

 

A Marked Decrease in PP1 Activity is Associated With Reticulocyte Maturation in Rabbit Red Cells

Erythrocytes obtained from control rabbits (mature cells) and from bled rabbits (~40% reticulocytes, young cells) were lysed in hypotonic lysis buffer (50 mosM), and membranes and soluble fractions were separated. In bled rabbits, erythropoietic activity increases to compensate for the anemia induced by blood loss, resulting in a younger red blood cell population. Table 1 shows that PP1 activity was lower in ghosts and cytosol of mature cells than of young cells. In mature cells, the activity of PP1 was only 15% of the activity in young cells, indicating effective regulation of this phosphatase during reticulocyte maturation. In analogy to density-separated human erythrocytes, the percentage of PP1 associated to the membrane was the same in young and mature cells, suggesting a single mechanism of PP1 degradation and/or inactivation during reticulocyte maturation. Table 1 also shows results of PP2A activity determinations in rabbit erythrocytes. Mb-PP2A activity increased slightly with rabbit reticulocyte maturation, whereas cytosolic PP2A activity in mature cells was ~70% of the activity in young cells. The percentage of PP2A associated to the membrane was ~2% in young and mature cells. The similarity in results obtained in rabbits (reticulocyte maturation) and in humans (increases in cell density) indicates that increases in human red cell density are in fact associated with increases in cell age. However, an increase in mb-PP2A activity was not observed in AA dense human cells (not shown).


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Table 1. Activity of PP1 and PP2A in the membrane and cytosol of rabbit young and mature red cells

 

Urea-Stimulation of KCC Is Decreased in Dense Cells

Urea stimulates KCC activity in human and sheep, young and mature red blood cells of AA cell volume (11, 12, 21, 27). Figure 3 shows KCC activity in media with and without 500 mM urea in AA human red cell populations of increasing density. Urea-stimulation of KCC activity was appreciable in all but the densest cells. Average stimulation by urea was of similar magnitude (two- to fourfold) in fractions of d = 24.3 and 30.5 g/dl and was therefore markedly different in its density dependence from that of basal KCC activity (see Fig. 1). We also studied the time course of urea stimulation in whole blood and in the least-dense cell fraction. In unfractionated AA human red cells (whole blood cell population), urea stimulated 86Rb+ influx from 0.08 ± 0.08 to 0.17 ± 0.03 (mmol/l cells x h), with an average delay of 5.2 ± 0.3 min (n = 3, not shown). In the least-dense cell fraction, urea stimulated influx from 0.32 ± 0.05 to 0.73 ± 0.03 (mmol/l cells x h), and stimulation proceeded without a delay (not shown), suggesting that urea stimulates an activating phosphatase (19). The observed absence of a delay in urea-stimulation of KCC activity in young cells has been detected in SS erythrocytes (11).



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Fig. 3. Urea-stimulated KCC activity decreases only in dense cells. Cells were separated according to density, and KCC activity was determined in Cl and Sfa media ± 500 mM urea. The results show the means ± SE of 5 experiments in 4 subjects. *P < 0.02, different from control cells.

 

Urea Stimulates KCC Activity in Isotonic and Hypertonic Conditions

To simulate conditions similar to those encountered by red blood cells in the kidney medulla, we investigated whether urea can stimulate KCC activity in hypertonic conditions in vitro. Figure 4 shows significant stimulation of 86Rb+ influx in the least-dense fraction of cells suspended in isotonic and hypertonic (Cl-containing) media, and no stimulation in hypertonic Cl-free media (Sfa). As expected, hypertonic media inhibited KCC activity. However, in the presence of urea, there was a large increase in 86Rb+ influx in hypertonic, Cl-containing media. The results suggest that urea may induce dehydration in young erythrocytes exposed to hypertonic conditions like those present in the kidney (11, 21), suggesting a role for the kidney in erythrocyte volume regulation in general and in reticulocyte maturation in particular.

Density Dependence of Urea-Stimulated KCC and Urea-Stimulated mb-PP1 Activities

We studied the effect of urea on mb-PP1 in density-separated cells to test whether the susceptibility of mb-PP1 to urea decreases with cell age/density. Figure 5A shows the degree of stimulation by urea of KCC activity in density-separated cells. Average urea-stimulation was calculated from stimulation in each fraction of six separate experiments from data used in Fig. 3. Figure 5B shows average urea-stimulated mb-PP1 activity calculated in each fraction of nine separate experiments. There was a small (20–40%) but significant stimulation of mb-PP1 in all fractions except in the densest cells. The percentage of stimulation by urea was the same in the three least-dense fractions assayed, suggesting that the sensitivity of mb-PP1 to urea does not change considerably until the cells reach average density. The data suggest that the decrease in urea-sensitive KCC stimulation in dense cells is due to a decrease in urea-stimulated mb-PP1 activity. The correlation coefficient between urea-stimulated mb-PP1 activity and urea-stimulated KCC activity in cells of densities ranging from 25 to 31.5 g/dl was 0.99. This correlation supports the hypothesis that stimulation of KCC by urea is mediated by stimulation of mb-PP1. There was no effect of urea on mb-PP2A m activity or on the activity of the purified catalytic subunit of mb-PP2A (data not shown).



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Fig. 5. Urea stimulates mb-PP1 activity in all but the densest cells. Urea stimulation of KCC and mb-PP1 activities have similar density dependence. A: average stimulation of KCC activity by 500 mM urea. Values are means ± SE of 5 experiments in 4 subjects. B: membranes were isolated from cells separated according to their density in isotonic media, and stimulation of mb-PP1 by 500 mM urea was assayed in each fraction. Values are means ± SE. *P < 0.01; **P < 0.04 different from control; t-test, n = 9.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A distinguishing feature of KCC in human red blood cells is its rapid and selective deactivation with cell aging (2, 7, 8, 15, 16). We show here that in AA human red blood cells, the decrease in KCC activity observed in cells of increasing densities is highly correlated with a decrease in mb-PP1 activity and that maturation of rabbit reticulocytes is also accompanied by a decrease in mb-PP1 activity. These findings provide further support to the notion that mb-PP1 plays a central role in KCC regulation, not only after cell swelling (4, 5, 22, 30) but also during maturation of reticulocytes and aging of erythrocytes.

The experiments presented in this manuscript do not provide evidence for a causal role of mb-PP1 in determining the inactivation of KCC in the least-dense cell fractions. Even though in AA cells the calculated d50% are not statistically different (26.8 ± 0.3 g/dl for KCC and 27.2 ± 0.2 g/dl for mb-PP1), in very young (least-dense) erythrocytes the decrease in mb-PP1 activity appears to be slightly delayed when compared with that of KCC activity. An increase of 1.77 g/dl in density (from 24.68 to 25.63) is associated with a 77% in KCC activity (~43% change per g/dl), whereas a decrease of 3.04 g/dl (from 23.76 to 26.8) is associated with a 41% decrease in mb-PP1 (~13% change per g/dl). A possible interpretation of the results is that the high-KCC activity in reticulocytes or very young erythrocytes causes an increase in cell density, which in turn causes a decrease in mb-PP1 activity, resulting in a negative feedback loop that results in further KCC inactivation. The results are consistent with the hypothesis that basal levels of KCC phosphorylation increase soon after (or during) reticulocyte maturation and that the initial fast decrease in KCC activity, likely associated with reticulocyte maturation, is independent of mb-PP1 activity. The results suggest that in very young cells, KCC inactivation may be due to activation of a yet unidentified density-dependent KCC inhibitory Ser/Thr kinase in this process. Transcription-dependent increase in a Ser/Thr kinase upon cell volume decrease has been observed in a rat tumor cell line (32).

Concomitantly to determinations of PP1 activity, we also determined PP2A activity. The ranges of mb-PP2A activity in AA and SS erythrocytes overlap, but in SS cells the values tend to be lower and have less variation. In cells of d > 30 g/dl, mb-PP2A activity is significantly reduced in SS cells (data not shown). Because lower mb-PP2A activity is expected to result in lower KCC activity (3), these data cannot explain the elevated KCC activity in dense SS cells, and it may reflect a compensatory mechanism to decrease KCC activity. Alternatively, based on the increase in mb-PP2A activity seen in more mature rabbit erythrocytes (Table 1), the higher mb-PP2A activity of AA dense cells may simply reflect their increased cell age compared with SS cells of similar density.

Density-dependent decreases in KCC and mb-PP1 activity in cells with d > 27 g/dl are highly correlated (r2 = 0.99 for AA cells and 0.93 for SS cells), suggesting that after the initial stages of reticulocyte maturation, the density-dependent decrease in mb-PP1 activity is the most likely cause of the observed progressive inactivation of KCC in erythrocytes. The large difference in mean cell age between AA and SS cells of increasing densities, together with the similarity in the density-dependent decrease in mb-PP1 activity between AA and SS cells, indicates that mb-PP1 activity is density dependent but age independent. Furthermore, given the short life-span of SS cells, the data indicate that mb-PP1 activity decreases sooner in SS cells than in AA cells.

In SS erythrocytes, inactivation of KCC has a different density dependence than in AA erythrocytes (Fig. 1). Clearly, a much larger percentage of SS cells than AA cells have undergone dehydration when KCC is inactivated. In SS erythrocytes, in which where mb-PP1 activity has the same density dependence than AA cells, the delay in KCC inactivation is possibly due to a delayed activation of a density-dependent, Ser/Thr KCC-inactivating kinase. Therefore, SS cells may continue to loose water in spite of a compensatory early decrease in mb-PP1 activity. It is unclear whether the defect in density-dependent KCC inactivation in SS cells is due to defective regulation of one or more of the KCC isoforms found in erythrocytes. Consistent differences between AA and SS cells in the relative abundance of KCC1 splicing variants have been reported (1).

To gain some insight into the mechanisms that govern basal PP1 activity in aging cells, we estimated the percentage of total PP1 associated to the membrane in rabbit and human red cells. This value is similar in cells of both species (about 30% in rabbits and about 20% in humans). These values are quite stable during maturation of reticulocytes and with the density increase associated with aging. In human red cells, the percentage of PP1 associated to the membrane was estimated to be 19% in least-dense cells and 21% in cells of average density (~31 g/dl). In young and mature rabbit red cells, these percentages were 32 and 39%, respectively. The parallel decrease in mb-PP1 and sol-PP1 suggests that there is a common mechanism controlling the decrease in PP1 activity in the membrane and cytosol during erythrocyte maturation. It is possible that the decrease in basal mb-PP1 and sol-PP1 activities may take place via a single mechanism, preferentially associated with the soluble enzyme; this event may be followed by equilibration of mb-PP1 and sol-PP1, thus maintaining the percentage bound to the membrane unchanged.

We determined the effect of increasing concentrations of urea on mb-PP1 and mb-PP2A activity in the membrane of nonfractionated AA washed erythrocytes. Urea (500 mM) stimulated mb-PP1 activity (average 23 ± 5%) but did not reliably enhance mb-PP2A activity (data not shown). There was no significant effect of urea (500 mM) on the purified catalytic subunit of PP1 and PP2A. The effect of urea on mb-PP1 was not due to an increase in the assay medium osmolarity because sucrose (500 mM) had no effect. Because the purified catalytic subunit of PP1 (PP1c) was not stimulated by urea, the data suggest that a direct target of urea in stimulating KCC activity may be the release of an inhibitory subunit of mb-PP1.

This study reports novel findings on the possible mechanism of action of urea on KCC function. Urea has been shown to activate KCC activity in isotonic conditions in dog, human, sheep, and horse red blood cells (11, 12, 21, 27, 29). Because urea-stimulated KCC activity is inhibited by okadaic acid and calyculin A, it has been proposed that exposure to urea results in net dephosphorylation of the transporter (or a protein regulator), (12, 21, 27, 29). The finding that the time course for urea stimulation of KCC is slow in mature sheep erythrocytes (12) has been interpreted as an indication that urea activates KCC by inhibiting phosphorylation catalyzed by a KCC inhibitory kinase. The underlying concept is that the lag time is shortened by stimulation of a reaction and lengthened by inhibition of a reaction (19). However, the finding that urea stimulates KCC without a delay in SS cells (11) and in least-dense AA red blood cells (this report) suggested that urea may also activate KCC by stimulating mb-PP1. This second possibility is supported by the findings of this study, which show that 1) urea stimulates mb-PP1 (see Fig. 5B); and 2) urea stimulation of KCC and urea stimulation of mb-PP1 have similar density-dependence profiles (Fig. 5, A and B). However, the fact that a moderate stimulation of mb-PP1 by urea induces a much greater stimulation of KCC suggests either signal amplification between mb-PP1 and KCC or additional regulatory mechanisms. Interestingly, the density-dependent changes in basal KCC and mb-PP1 (Fig. 1, A and B) are markedly different from the density dependence of the urea stimulation of KCC and mb-PP1 activities (Fig. 5, A and B).

In hypertonic conditions, KCC is usually in an inactive state (7, 8, 12, 19). We have now established that urea can stimulate KCC in hypertonic conditions, an otherwise silent KCC (Fig. 3). This mechanism may be relevant in the pathophysiology of SS cell dehydration, because cells circulating through the hypertonic, acidic environment of the kidney could undergo K+ loss and shrinkage via KCC stimulation by urea (11, 21).

In summary, our results show that KCC activity is not abnormally elevated in least dense SS cells, but a defect in KCC regulation, not due to mb-PP1, results in delayed inactivation. In both AA and SS cells, a large part of the decrease in basal KCC activity associated with increases in cell age/density is due to a decrease in mb-PP1 activity. The results are consistent with activation of a KCC inhibitory kinase during reticulocyte maturation in AA cells but not in SS cells. The results also indicate that urea stimulates mb-PP1 activity and suggest that part of the stimulation of KCC by urea is mediated by stimulation of mb-PP1.


    ACKNOWLEDGMENTS
 
We acknowledge the help and contribution of the late Dr. Mitzy Canessa in the rabbit experiments and the help of Dr. Lillian Mc-Mahon from Boston University Medical Center in obtaining blood from patients with SS cell disease.

This work was supported by Fondecyt 1960879 and 1940339 from Conicyt, Chile, and by Fundación Andes, Chile, as well as by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50422 and National Heart, Lung, and Blood Institute Grant HL-15157.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. Bize, Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115 (E-mail: isabel_bize{at}hms.harvard.edu).

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.


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