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J Am Soc Nephrol 14:2750-2757, 2003
© 2003 American Society of Nephrology

BASIC SCIENCE

Inhibition of Erythrocyte Cation Channels by Erythropoietin

Svetlana Myssina*, Stephan M. Huber*, Christina Birka*, Philipp A. Lang*, Karl S. Lang*, Björn Friedrich{dagger}, Teut Risler{dagger}, Thomas Wieder* and Florian Lang*

*Department of Physiology, University of Tübingen, and {dagger}Department of Internal Medicine, University Medical Center, University of Tübingen, Tübingen, Germany.

Correspondence to: Dr. Florian Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. Phone: +49-7071-29-72194; Fax: +49-7071-29-5618;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Recombinant human erythropoietin therapy is used to counteract anemia that is the result of renal insufficiency. It stimulates the formation of peripheral blood erythrocytes by inhibiting apoptosis of erythrocyte precursor cells. Mature erythrocytes have similarly been shown to undergo apoptosis. Hyperosmotic shock and Cl- removal activate a Ca2+-permeable, ethylisopropylamiloride-inhibitable cation channel. The subsequent increase of cytosolic Ca2+ activates a scramblase that breaks down cell membrane phosphatidylserine asymmetry, leading to annexin binding. Studied was whether channel activity and erythrocyte cell death are regulated by erythropoietin. Scatchard plot analysis disclosed low-abundance, high-affinity binding of 125I-erythropoietin to erythrocytes. Whole cell patch clamp experiments revealed significant inhibition of the ethylisopropylamiloride-sensitive current by 1 U/ml erythropoietin. Cl- removal triggered annexin binding, an effect abrogated by erythropoietin (1 U/ml) but not by GM-CSF (10 ng/ml). Osmotic shock (700 mOsm) stimulated annexin binding within 24 h in the majority of the erythrocytes, an effect blunted by erythropoietin (1 U/ml) but not by GM-CSF (10 ng/ml). In the nominal absence of Ca2+, the effect of osmotic shock was blunted and the effect of erythropoietin abolished. In hemodialysis patients, intravenous administration of erythropoietin (50 IU/kg) within 4 h decreased the number of annexin binding circulating erythrocytes. Erythropoietin binds to erythrocytes and inhibits volume-sensitive erythrocyte cation channels and thus the breakdown of phosphatidylserine asymmetry after activation of this channel. The effect could prolong the erythrocyte lifespan and may contribute to the enhancement of the erythrocyte number during erythropoietin therapy in dialysis patients. E-mail: florian.lang@uni-tuebingen.de


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Erythropoietin, a hormone released on hypoxia in kidney (1) and extrarenal tissues (2,3), is the most potent stimulator of erythropoiesis (1). By increasing the number of circulating erythrocytes, it enhances the oxygen transport capacity of blood (1). Recombinant human erythropoietin is widely used for the treatment of renal anemia in dialysis patients (4–6). Clinical data further revealed that erythropoietin improved the viability of red blood cells, indicating that the hormone is a survival factor for those cells (7). Erythropoietin stimulates erythrocyte formation at least in part by inhibiting apoptosis of EryP progenitor cells (8–11). However, recent experiments reveal that apoptosis may occur not only in nucleated cells but also in differentiated erythrocytes (12). Thus, at least in theory, erythropoietin could enhance the number of circulating erythrocytes by stimulation of production and by inhibiting apoptosis of mature cells.

Erythrocyte apoptosis is triggered by increase of cytosolic Ca2+ activity. Treatment of erythrocytes with the Ca2+ ionophore ionomycin leads to cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all typical features of apoptosis in nucleated cells (12–14). The breakdown of phosphatidylserine asymmetry, which is apparent from annexin binding at the cell surface, is the result of activation of a Ca2+-sensitive scramblase (15). Further studies revealed that hypertonic shock, oxidative stress, removal of extracellular Cl-, and energy depletion lead to the activation of a Ca2+-permeable cation channel (16,17). Ca2+ then activates the scramblase and triggers annexin binding of the affected erythrocytes (16,17). The channel is inhibited by ethylisopropylamiloride (EIPA, 10 µM), which similarly blunts the annexin binding after osmotic shock (18). Cells exposing phosphatidylserine at the cell surface are prone to be cleared by macrophages (19–21). Thus, triggering of the scramblase is expected to shorten the lifespan of affected erythrocytes. Along those lines, erythrocyte aging is paralleled by increase of cytosolic Ca2+ activity (22,23). Oxidative stress or defects of antioxidative defense (24) enhance Ca2+ entry via the cation channels leading to higher intracellular Ca2+ concentrations and acceleration of erythrocyte cell death and clearance. Indeed, erythrocytes from patients with sickle cell anemia, thalassemia, and glucose phosphate dehydrogenase deficiency are more sensitive to apoptotic stimuli (16), a property correlating with the shortened erythrocyte lifespan in those disorders (25–28).

The study presented here was performed to test whether the cation channel activity and erythrocyte annexin binding could be influenced by erythropoietin. To this end, erythrocytes have been exposed to hyperosmotic shock or Cl--free extracellular fluid. Patch clamp experiments were performed to determine cation channel activity, and annexin binding was taken as a measure of scramblase activation. Erythropoietin indeed inhibited the cation channel and blunted the annexin binding after exposure of erythrocytes to hypertonic shock and Cl- depletion. In the nominal absence of extracellular Ca2+, the annexin binding was blunted, and the antiapoptotic effect of erythropoietin was abolished.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Solutions
Erythrocytes were drawn from healthy volunteers or from patients suffering from chronic renal failure. Patients underwent a 4-h hemodialysis treatment either without or with intravenous administration of 50 IU/kg body weight erythropoietin (NeoRecormon [epoetin beta]; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) at the beginning of dialysis. Erythropoietin plasma concentration increased 10- to 30-fold after administration of erythropoietin and remained low in untreated patients (data not shown). Informed consent was obtained from all patients, and the study was approved by the Ethical Committee of the Medical Faculty of the University of Tübingen. Erythrocytes were either used without purification or after separation by centrifugation for 25 min (2000 x g over Ficoll; Biochrom KG, Berlin, Germany). Experiments with nonpurified erythrocytes or experiments with Ficoll-separated erythrocytes yielded the same results (data not shown). Experiments were performed at 37°C in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2, pH 7.4. For the nominally calcium-free solution, CaCl2 was replaced by 1 mM ethylene glycol-bis ({beta}-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Cl- was replaced by appropriate amounts of Na, K, and Ca gluconate. Osmolarity was increased to 700 mM by adding sucrose. EIPA was purchased from Sigma (Taufkirchen, Germany) and was used at a concentration of 10 µM. The final concentration of the solvent DMSO was 0.1% and was added to controls. Erythropoietin was purchased from Sigma. When not otherwise indicated, erythropoietin was applied at a concentration of 1 U/ml. Erythropoietin vehicle contained 40 mM sodium citrate, pH 7.0, and 170 mM NaCl. Appropriate amounts of vehicle were added to controls. The vehicle only marginally influenced annexin binding after hyperosmotic shock or Cl- removal (data not shown). GM-CSF (100 µg/ml) was purchased from Pepro-Tech (London, UK) and was diluted 1:10,000 in the appropriate solutions (final concentration, 10 ng/ml).

Patch Clamp
Patch clamp experiments have been performed as previously described (29,30). Red blood cells were recorded at 35°C. A continuous superfusion was applied through a flow system inserted into the dish. The bath was grounded via a 2% agarose bridge filled with NaCl bath solution (see below). Borosilicate glass pipettes (9 M{Omega} tip resistance; GC 150 TF-10, Clark Medical Instruments, Pangbourne, UK) manufactured by a microprocessor-driven DMZ puller (Zeitz, Augsburg, Germany) were used in combination with a MS314 electrical micromanipulator (MW, Märzhäuser, Wetzlar, Germany). The currents were recorded in voltage clamp, fast whole cell mode by an EPC-9 amplifier (Heka, Lambrecht, Germany) by Pulse software (Heka) and an ITC-16 Interface (Instrutech, Port Washington, NY). The whole cell currents were evoked by a pulse protocol, clamping the voltage in 11 successive 400-ms square pulses from the -30 mV holding potential to potentials between -100 mV and +80 mV. The potentials were corrected for liquid junction potentials (31). The applied voltages refer to the cytoplasmic face of the cell membrane with respect to the extracellular space. The inward currents, defined as flow of positive charge from the extracellular to the cytoplasmic membrane face, are negative currents.

Whole cell currents were recorded first in standard NaCl bath solution containing (in mM) 115 NaCl, 10 HEPES, 5 KCl, 5 CaCl2, 10 MgCl2, titrated with NaOH to pH 7.4 in combination with a pipette solution containing (in mM) 115 Na-gluconate, 10 NaCl, 1 EGTA, 1 MgATP, 10 HEPES titrated to pH 7.4 with NaOH. The whole cell currents were further recorded with a low Cl- bath solution containing (in mM) 140 Na-gluconate, 10 HEPES 1 CaCl2, 1 MgCl2 titrated to pH 7.4 with NaOH in the absence and presence of 10 µM EIPA. Experiments were performed both in control erythrocytes or erythrocytes preincubated (for 3 h at 37°) and coincubated with erythropoietin (1 U/ml).

FACS Analysis
FACS analysis was performed essentially as described earlier (16,32). After incubation, cells were washed in annexin-binding buffer containing (in mM) 125 NaCl, 10 HEPES, pH 7.4, and 5 CaCl2. Erythrocytes were stained with Annexin-Fluos (Böhringer Mannheim, Germany) at a 1:100 dilution. After 15 min, samples were diluted 1:5 and measured by flow cytometric analysis (FACS-Calibur; Becton Dickinson, Heidelberg, Germany). Cells were analyzed by forward and sideward scatter, and annexin fluorescence intensity was measured in FL-1.

Erythropoietin Binding Studies
Erythropoietin binding studies were performed on whole cells as previously described (33) by use of human recombinant (3-[125I]iodotyrosyl)erythropoietin (molecular weight, 34,000 D; specific activity, 92.5 TBq/mmol) from Amersham Biosciences (Freiburg, Germany). Briefly, 1 x 108 erythrocytes were incubated with selected concentrations of (3-[125I]iodotyrosyl)erythropoietin in a final volume of 100 µl Ringer solution containing 0.1% BSA on ice for 2 h. For determination of nonspecific binding, a 55- to 220-fold excess of unlabeled erythropoietin (R&D Systems, Wiesbaden, Germany) was added simultaneously to selected tubes. At the end of the incubation period, cells were centrifuged at 250 x g at 4°C for 3 min, and medium was removed. Cells were washed three times with Ringer/0.1% BSA, and cell-bound radioactivity was determined in a gamma scintillation counter.

Determination of Cell Numbers
Erythrocytes were suspended at 0.2% (hyperosmotic shock) or 0.4% (Cl- removal) hematocrit and incubated under different control and stress conditions (osmotic stress, Cl- removal). After incubation, the cell number was determined with a hemocytometer as described previously (34).

Statistical Analyses
Data are expressed as arithmetic means ± SEM, and statistical analysis was made by paired or unpaired t test, where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Erythropoietin Binding to Erythrocytes
As illustrated in Figure 1, recombinant 125I-erythropoietin binds to human erythrocytes. The binding is reduced in the presence of excess unlabeled erythropoietin at low (38 pM) and high (760 pM) erythropoietin concentrations. Figure 1B illustrates the equilibrium binding isotherm for erythrocytes and Scatchard analysis (Figure 1C) reveals a binding affinity (kD) of 290 ± 60 pM and the expression of about six erythropoietin binding sites per cell. Thus, we conclude that erythrocytes are able to specifically bind erythropoietin, presumably via the erythropoietin receptor.



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  		Figure 1. Binding of 125I-erythropoietin to erythrocytes. (A) Erythrocytes were exposed to 38 pM or 760 pM of 125I-erythropoietin (molecular weight, 34,000 D; specific activity, 92.5 TBq/mmol) for 2 h at 4°C. Arithmetic means ± SEM (n = 3) of total binding of 125I-erythropoietin to erythrocytes either in the presence (open columns) or in the absence (solid columns) of excess unlabeled erythropoietin are shown. (B) Erythrocytes were exposed to varying concentrations of 125I-erythropoietin for 2 h at 4°C. Specific binding of 125I-erythropoietin to erythrocytes is shown. SEM for each data point was less than 10% (n = 3). (C) Scatchard analysis of the data shown in Panel B.

 
Erythropoietin Inhibits the Cl--Sensitive Cation Channel
As shown in Figure 2A, erythrocytes exposed to Cl--free bath solution display an almost linear conductance, which is significantly inhibited by addition of EIPA. In erythrocytes stimulated with erythropoietin (1 U/ml), the Cl--dependent conductance is significantly decreased (Figure 2B).



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  		Figure 2. The Cl--sensitive nonselective cation channel in human erythrocytes is inhibited by erythropoietin. (A) Current voltage relation of the whole cell current (Na-gluconate pipette solution) recorded with NaCl bath solution (open triangles) or after replacement of bath Cl- by gluconate, either in the absence (open circles) or in the presence (solid circles) of 10 µM ethylisopropylamiloride (EIPA). Data are means ± SEM (n = 4). (B) Cl--dependent conductance in control erythrocytes (control) or erythrocytes preincubated (3 h at 37°C) and coincubated with 1 U/ml erythropoietin (EPO). Control refers to erythrocytes exposed to the respective vehicle-containing buffer solution. Data are mean ± SEM (n = 4).

 
Erythropoietin Blunts the Annexin Binding of Erythrocytes Exposed to Osmotic Shock
As illustrated in Figure 3A, an increase of extracellular osmolarity to 700 mOsm by addition of sucrose leads to a marked increase of annexin binding within 24 h. This effect is blunted by the presence of 1 U/ml erythropoietin. Upon an increase to 700 mOsm, the number of annexin binding cells approached 66 ± 11% (n = 6) in the absence and 27 ± 8% (n = 6) in the presence of erythropoietin, values significantly different (Figure 3B). As shown in Figure 3C, little annexin binding is observed up to 500 mOsm, but a further increase of osmolarity leads to a sharp increase of annexin binding. Erythropoietin significantly reduces osmotic shock–induced annexin binding at all osmolarities tested (Figure 3C). Furthermore, we demonstrate that increase of osmolarity to 700 mOsm for 24 h leads to a reduction of the cell number, an effect that is again counteracted by 1 U/ml erythropoietin (Figure 3D). The dose-response curve of erythropoietin reveals a half maximal inhibition of osmotic shock–induced annexin binding at 0.3 U/ml erythropoietin (Figure 4). Thus, erythropoietin is effective at concentrations well below 1 U/ml.



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  		Figure 3. Erythropoietin inhibits osmotic shock–induced erythrocyte annexin binding and cell death. (A) Effects of an increase of osmolarity to 700 mOsm for 24 h on annexin binding in the absence (C 700) or in the presence (E 700) of 1 U/ml erythropoietin. Annexin binding at 300 mOsm in the absence (C 300) or in the presence of erythropoietin (E 300) is also shown. (B) Arithmetic means (± SEM) of annexin binding in cells after exposure to 300 mOsm or 700 mOsm for 24 h, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h. (C) Arithmetic means (± SEM) of annexin binding in cells after exposure to increasing osmolarities for 24 h, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h. (D) Arithmetic means ± SEM (n = 3) of erythrocyte number in percentage of control after exposure to 300 mOsm or 700 mOsm for 24 h, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h. Erythrocyte numbers under control conditions in the absence and presence of erythropoietin were (2.5 ± 0.2) x 107 cells/ml and (2.4 ± 0.2) x 107 cells/ml, respectively.

 


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  		Figure 4. Dose-response curve of erythropoietin-mediated inhibition of osmotic shock–induced erythrocyte annexin binding. Erythrocytes were exposed to 700 mOsm for 24 h in the presence of different concentrations of erythropoietin. Arithmetic means ± SEM (n = 3) of erythropoietin-mediated inhibition of annexin binding are provided. In these experiments, annexin binding of erythrocytes exposed to 700 mOsm in the absence of erythropoietin was 72.7 ± 1.5%.

 
Ca2+ Removal Blunts the Annexin Binding of Erythrocytes Exposed to Osmotic Shock and Abolishes the Protective Effect of Erythropoietin
The effect of hyperosmotic shock is blunted in the nominal absence of extracellular Ca2+ (Figure 5). Upon an increase to 700 mOsm, the number of annexin binding cells approaches 85 ± 2% (n = 3) in the presence and 59 ± 8% (n = 3) in the nominal absence of Ca2+ within 24 h, values that are significantly different. In the presence of 1 U/ml erythropoietin, the number of annexin binding cells approaches 60 ± 2% (n = 3) in the presence of Ca2+ and 58 ± 9% (n = 3) in the nominal absence of extracellular Ca2+. Thus, erythropoietin inhibits annexin binding in the presence of extracellular Ca2+ but does not exert an additional protective effect in the absence of extracellular Ca2+ (Figure 5B).



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  		Figure 5. Ca2+ removal abolishes the protective effect of erythropoietin. (A) Arithmetic means (± SEM) of annexin binding in cells after exposure to 300 mOsm or 700 mOsm for 24 h in Ringer solution containing 1 mM Ca2+, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h. (B) Arithmetic means (± SEM) of annexin binding in cells after exposure to 300 mOsm or 700 mOsm for 24 h in Ringer solution containing 1 mM EGTA (instead of 1 mM Ca2+), either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h.

 
Erythropoietin Blunts the Annexin Binding of Erythrocytes Exposed to Cl- Depletion
Replacement of extracellular Cl- with gluconate leads to a significant increase of annexin binding cells within 36 h, an effect efficiently reversed by 10 µM EIPA. The number of annexin binding cells approaches 39 ± 3% (n = 6) in the absence and 18 ± 4% (n = 6) in the presence of EIPA, values significantly different from each other. Similar to EIPA, exposure of the cells to 1 U/ml erythropoietin almost abolishes the annexin binding after Cl- removal (Figure 6A). The number of annexin binding cells approaches 56 ± 13% (n = 6) in the absence and 8 ± 6% (n = 6) in the presence of erythropoietin, values again significantly different (Figure 6B). Furthermore, removal of Cl- for 36 h leads to a reduction of the cell number, an effect that is partly counteracted by 1 U/ml erythropoietin (Figure 6C).



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  		Figure 6. Erythropoietin inhibits erythrocyte annexin binding and cell death after Cl- removal. (A) Effects of Cl- removal for 36 h on annexin binding in the absence (control 0 Cl-) or in the presence (E 0 Cl-) of 1 U/ml erythropoietin. Annexin binding of erythrocytes in normal Ringer solution, either in the absence (control + Cl-) or in the presence of erythropoietin (E + Cl-) is also shown. (B) Arithmetic means (± SEM) of annexin binding in cells after exposure to Cl--free (0 mM Cl-) or Cl--containing (125 mM Cl-) Ringer solution for 36 h, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 36 h. (C) Arithmetic means ± SEM (n = 3) of erythrocyte number in percentage of control after exposure to Cl- free (0 mM Cl-) or Cl- containing (125 mM NaCl) Ringer solution for 36 h, either in the presence or in the absence of 1 U/ml erythropoietin. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 36 h. Erythrocyte numbers under control conditions in the absence and presence of erythropoietin were (4.1 ± 0.3) x 107 cells/ml and (4.0 ± 0.3) x 107 cells/ml, respectively.

 
Lack of Protective Effects of GM-CSF
To test for the specificity of the erythropoietin-mediated effects, we used human recombinant GM-CSF, a cytokine that does not support the erythroid lineage. GM-CSF (10 ng/ml) did not influence annexin binding of erythrocytes after hyperosmotic shock (Figure 7A) or removal of extracellular Cl- (Figure 7B).



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  		Figure 7. GM-CSF does not inhibit annexin binding of erythrocytes after hyperosmotic shock or Cl- removal. (A) Arithmetic means ± SEM (n = 3) of annexin binding in cells after exposure to 300 mOsm or 700 mOsm for 24 h in Ringer solution, either in the presence or in the absence of 10 ng/ml GM-CSF. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 24 h. (B) Arithmetic means ± SEM (n = 3) of annexin binding in cells after exposure to Cl--free (0 mM Cl-) or Cl--containing (125 mM Cl-) Ringer solution for 36 h, either in the presence or in the absence of 10 ng/ml GM-CSF. Controls refer to erythrocytes exposed to the respective vehicle-containing buffer solutions for 36 h.

 
Erythropoietin Treatment of Dialysis Patients Decreases the Number of Annexin-Binding Erythrocytes
In dialysis patients receiving 50 IU/kg body weight erythropoietin immediately before a 4-h dialysis session, the number of annexin binding cells was significantly reduced from 2.3 ± 0.3% (n = 4) before the treatment to 1.3 ± 0.2 (n = 4, P < 0.01) after the treatment. In contrast, the number of annexin binding cells was not significantly altered when dialysis was performed without previous administration of erythropoietin. The respective values were 2.8 ± 0.4% (n = 4) before and 2.7 ± 0.5% (n = 4) after dialysis. Erythropoietin treatment further decreased the susceptibility of erythrocytes to hyperosmotic shock (700 mOsm, 12-h incubation). The number of annexin binding cells after hyperosmotic shock was significantly lower (by 33.5 ± 9%) in erythrocytes drawn 4 h after treatment with erythropoietin (7.9 ± 1.2%, n = 4) as compared with erythrocytes drawn immediately before the treatment (11.9 ± 0.7%, n = 4). In contrast, no significant difference in the susceptibility of erythrocytes to hyperosmotic shock was observed in erythrocytes drawn before (9.6 ± 1.0%, n = 4) and after (10.9 ± 1.3%, n = 4) dialysis without erythropoietin treatment before dialysis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study presented here confirms the previous observation that osmotic shock stimulates erythrocyte scramblase, leading to annexin binding (16,17), a process that is mediated in part by a Ca2+-permeable cation channel (17). It is further demonstrated that Cl- removal, a maneuver known to activate the same cation channel (18,30,35), similarly triggers annexin binding.

Most importantly, however, these data disclose the ability of erythropoietin, a hormone released by the kidney and used for management of renal anemia (4), to inhibit the cation channel and thus to interfere with the erythrocyte apoptosis. The dose-response curve (see Figure 4) revealed that erythropoietin inhibits erythrocyte death at concentrations below 1 U/ml. This direct effect of erythropoietin on mature erythrocytes is surprising because it has been shown that reticulocytes appear to be devoid of erythropoietin receptors (36,37). Indeed, Scatchard blot analysis discloses that the erythrocytes express very low but detectable numbers of erythropoietin binding sites. 125I-erythropoietin binding studies further revealed high affinity with half-maximal binding at an erythropoietin concentration (kD 290 ± 60 pM), which is similar to that for the erythropoietin receptor of burst-forming unit-erythroid cells (kD 220 pM) (37) or for the erythropoietin receptor expressed in Sf9 cells (kD 330 pM) (33).

The inhibition of the cation channel provides an additional mechanistic explanation for the clinical observation of prolonged survival of red blood cells during erythropoietin treatment. According to a previous hypothesis, the erythropoietin effects on red cell survival were considered to be indirect—that is, secondary to an increased recruitment of reticulocytes under erythropoietin therapy (7). Mature red blood cells from patients with chronic renal failure have been shown to exhibit increased annexin binding compared with erythrocytes from healthy individuals (38). Reticulocytes, however, showed less annexin binding than mature red blood cells and were therefore considered less likely to undergo macrophage recognition in the spleen (38).

The observations in hemodialysis patients described here reveal that our in vitro observations may well reflect an in vivo effect of the hormone. The erythropoietin-induced inhibition of the erythrocyte cation channel with subsequent reduction of phosphatidylserine exposure thus contributes to the effect of erythropoietin on the number of circulating erythrocytes. However, the apoptosis after osmotic shock is only partially blunted by the hormone. Nominal absence of extracellular Ca2+ is similarly not able to fully suppress erythrocyte apoptosis. In a previous study, we were unable to completely abolish the apoptosis after osmotic shock by Ca2+ removal (17). Obviously, osmotic shock stimulates two independent pathways that both eventually trigger the scramblase. Only one of them, the Ca2+ entry through the volume-sensitive cation channel, is inhibited by erythropoietin.

In contrast to the apoptosis induced by osmotic shock, the apoptosis triggered by Cl- removal is almost fully inhibited by erythropoietin. Obviously, Cl- removal triggers apoptosis exclusively by activating the cation channel. The channels have been characterized and shown to be inhibited by amiloride and EIPA previously (18,35).

Osmotic shock is a well known trigger of apoptotic death for both erythrocytes and nucleated cells (39–43). Moreover, a variety of nucleated cells express volume-regulatory cation channels (44–50). An increase of cytosolic Ca2+ is similarly a trigger of apoptosis in nucleated cells (51,52). To the extent that the volume regulatory cation channels are in nucleated cells similarly permeant to Ca2+, they may participate in the triggering of apoptosis after osmotic shock. If so, altered channel activity may contribute to the antiapoptotic effect of erythropoietin in nucleated cells, such as EryP progenitor cells (8,10,53,54) and neurons (55).

In summary, we conclude from our results that erythrocyte apoptosis can be induced by different stimuli, such as hyperosmotic shock or Cl- removal, and that erythrocyte cell death is regulated by erythropoietin. Besides its effect on EryP progenitor cells and the number of presumably more resistant reticulocytes, erythropoietin has a direct antiapoptotic effect on mature erythrocytes, which significantly contributes to the enhanced erythrocyte survival observed in erythropoietin-treated patients. This direct regulatory effect of erythropoietin is at least partially due to inactivation of calcium-permeable cation channels. The data described here thus disclose a physiologic mechanism that may indeed be relevant for half-life and turnover of this highly specialized cell type.


   Acknowledgments
 
We acknowledge the technical assistance of E. Faber and the preparation of the manuscript by Tanja Loch. We thank Dr. T. Haug (University of Tübingen) for technical support in the erythropoietin binding assays. This study was supported by the Deutsche Forschungsgemeinschaft (La 315/4-3 and La 315/6-1), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) (01 KS 9602) and the Biomed program of the EU (BMH4-CT96-0602).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 13, 2003. Accepted for publication August 18, 2003.




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