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Am J Physiol Cell Physiol 291: C710-C717, 2006. First published May 24, 2006; doi:10.1152/ajpcell.00631.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Tobias Hermle,1 Ekaterina Shumilina,1 Philipp Attanasio,1 Ahmad Akel,1 Daniela S. Kempe,1 Philipp A. Lang,1 Marlies Podolski,1 Sabine Gatz,1 Robert Bachmann,2 Cornelia Bachmann,2 Harald Abele,2 Stephan Huber,1 Thomas Wieder,1 and Florian Lang2

1Department of Physiology, and 2Department of Obstetrics and Gynecology, University of Tübingen, Tübingen, Germany

Submitted 16 December 2005 ; accepted in final form 16 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca2+-permeable cation channels with subsequent entry of Ca2+ and stimulation of Ca2+-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca2+ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E2 (PGE2) formation, cation channel activity, Ca2+ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca2+ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes.

annexin V; osmotic cell shrinkage; calcium; apoptosis

AS SHOWN RECENTLY, mature circulating erythrocytes may, like apoptotic nucleated cells, undergo suicidal death (9, 16, 28, 40). Suicidal erythrocyte death may be triggered by an increase of cytosolic Ca2+ activity (6, 12, 16). Treatment of erythrocytes with the Ca2+ ionophore ionomycin is followed by cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry (6, 12, 16), all typical features of apoptosis in nucleated cells (27). For this type of suicidal erythrocyte death the term "eryptosis" has been coined recently (28). The breakdown of phosphatidylserine asymmetry is at least partially due to Ca2+-sensitive scrambling of the cell membrane (47). In part, oxidative stress, hypertonic shock, removal of extracellular Cl–, and energy depletion stimulate eryptosis by stimulating Ca2+ entry (11, 15, 17, 18, 29, 32). Besides injury, aging of erythrocytes is paralleled by an increase in cytosolic Ca2+ activity (26, 41). The erythrocytes can further be sensitized to the eryptotic effects of Ca2+ by ceramide, a proapoptotic lipid-derived second messenger, which is formed by an erythrocyte sphingomyelinase (30). The enzyme is activated by exposure to hyperosmotic shock (30) or to platelet-activating factor (PAF) (35).

Phosphatidylserine-exposing cells, including mature red blood cells, are cleared by macrophages (7, 19, 21, 41). Accordingly, enhanced eryptosis shortens the life span of erythrocytes and thus favors the development of anemia. Phosphatidylserine-exposing erythrocytes could further adhere to the vascular wall and thus interfere with microcirculation (8, 13, 19, 22, 37, 44, 45). Because of the delicate balance between hematopoiesis and red blood cell removal, suicidal death of circulating erythrocytes is supposed to be a tightly regulated mechanism (9, 16, 28, 40).

The susceptibility to eryptosis may be influenced by the type of expressed hemoglobin. For instance, erythrocytes from patients with sickle cell anemia and thalassemia are more sensitive to apoptotic stimuli (32), a property correlating with the shortened erythrocyte life span in those disorders (5, 14, 42, 46). Fetal erythrocytes mainly contain fetal hemoglobin (20) and have been shown to differ from adult erythrocytes in their K+ transport properties (23). On the other hand, K+ exit from the cells is not only necessary for eryptotic shrinkage but also favors Ca2+-induced phosphatidylserine exposure at the erythrocyte membrane (33). The present study has thus been performed to explore the sensitivity of neonatal erythrocytes to different stimuli of eryptosis. To this end, neonatal erythrocytes have been compared with erythrocytes from adult volunteers, and their cation channel activities, their intracellular Ca2+ concentrations, phosphatidylserine exposure after different stressors, and their ability to generate prostaglandin E2 (PGE2) after Cl– removal have been determined.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and solutions. Erythrocytes were drawn from healthy volunteers or harvested from the placenta immediately after birth. The study was approved by the ethics committee of the University of Tübingen (project no. 184/2003V). All volunteers gave their written informed consent. Experiments were performed at 37°C in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 HEPES/NaOH (pH 7.4), 5 glucose, and 1 CaCl2. For chloride-free conditions, a 0 Cl– bath solution was used containing (in mM) 125 Na gluconate, 5 K gluconate, 32 HEPES/NaOH (pH 7.4), 5 glucose, 1 Ca(gluconate)2, and 1 MgSO4. Hyperosmotic solution was prepared by addition of 400 mM sucrose to standard Ringer solution. Where indicated, PGE2 and tert-butylhydroperoxide (both from Sigma, Taufkirchen, Germany), and PAF [1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-16) from Calbiochem, Schwalbach, Germany] have been added at the indicated concentrations. PGE2 and PAF were dissolved in ethanol and DMSO, respectively. Comparisons were made to controls containing the respective solvent concentrations.

Patch-clamp experiments. Patch pipettes made of borosilicate glass (model 150 TF-10; Clark Medical Instruments, Pangbourne, UK) were pulled using a horizontal DMZ puller (Zeitz, Augsburg, Germany). Pipettes with high resistance from 8 to 12 M{Omega} were connected via an Ag-AgCl wire to the headstage of a patch-clamp amplifier (model EPC 9; HEKA, Lambrecht, Germany). Data acquisition and analysis were controlled by a computer equipped with an ITC 16 interface (Instrutech, Port Washington, NY) and with the use of Pulse software (HEKA), as described earlier (18). For current measurements (room temperature), red blood cells were held at a holding potential of –20 to –30 mV and 200- or 400-ms pulses from –100 to +80 mV were applied in increments of +20 mV. The original whole cell current traces are depicted without filtering (acquisition frequency of 5 kHz). The currents were analyzed by averaging the current values measured between 350 and 375 ms of each square pulse current-voltage (I-V relationship). The applied voltages refer to the cytoplasmic face of the membrane with respect to the extracellular space. The offset potentials between both electrodes were zeroed before sealing. The liquid junction potentials between the bath and pipette solutions and between the bath solutions and the salt bridge (filled with NaCl bath solution) were calculated according to Barry and Lynch (3). Data were corrected for liquid junction potentials.

The pipette solution contained (in mM) 115 Na gluconate, 10 NaCl, 1 MgATP, 1 EGTA, and 5 HEPES/NaOH (pH 7.4). In the bath, NaCl and Cl–-free Ringer solutions (see above) were used.

Measurement of intracellular Ca2+. Intracellular Ca2+ measurements were performed as described (2). Briefly, erythrocytes were loaded with fluo-3 AM (Calbiochem, Bad Soden, Germany) by addition of 2 µl of a fluo-3 AM stock solution (2 mM in DMSO) to 1 ml erythrocyte suspension (3% hematocrit in NaCl Ringer solution). The cells were incubated at 37°C for 15 min. An additional 2-µl aliquot of fluo-3 AM was added, with incubation carried out for 25 min. Fluo-3-loaded erythrocytes were centrifuged at 1,000 g for 5 min at 22°C and washed two times with NaCl Ringer solution containing 1% bovine serum albumin (Sigma) and one time with albumin-free Ringer solution. For flow cytometry, fluo-3-loaded erythrocytes were resuspended in 1 ml of Ringer solution (3% hematocrit), or Cl–-free Ringer solution either in the presence or absence of the Ca2+ ionophore ionomycin (1 µM; Sigma) or vehicle alone. Cells were incubated for 5 min (ionomycin-treated cells that were used as a fluo-3 loading control, data not shown) or 6 h at 37°C. Ca2+-dependent fluorescence intensity of 20,000 cells was then measured by flow cytometric analysis on a fluorescence-activated cell sorter (FACS-Calibur; Becton Dickinson, Heidelberg, Germany) in the fluorescence channel FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

FACS analysis. FACS analysis was performed as described in detail elsewhere (1, 30). After incubation in the respective solutions, cells were washed in annexin-binding buffer containing (in mM) 125 NaCl, 10 HEPES/NaOH (pH 7.4), and 5 CaCl2. Erythrocytes were stained with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) at a 1:100 dilution. After 15 min, samples were diluted 1 to 5 and measured by flow cytometric analysis. Cells (20,000) were analyzed by forward scatter, and the annexin V fluorescence intensity of those cells was measured in FL-1.

Microscopy. Erythrocytes were exposed for 24 h to Ringer solution or Cl–-free Ringer solution (Cl– replaced by gluconate) and subsequently placed on polylysine-coated cover slips. Cells were analyzed under a fluorescence microscope with 440/480-nm excitation and 535/550-nm emission wavelength [Q505LP beam splitter; AHF Analysentechnik (Tübingen, Germany)] combined with a Nikon microscope (Düsseldorf, Germany), and digital pictures were taken using a digital imaging system (Visitron Systems, Puchheim, Germany) equipped with Metaview software.

Determination of PGE2 in the supernatant. One billion erythrocytes were exposed to Cl–-free Ringer solution (Cl– replaced by gluconate) for the indicated time periods. After incubation, the cells were pelleted by centrifugation at 4°C, 450 g for 5 min. The supernatant was removed and stored at –20°C. PGE2 concentrations in the supernatant were determined using the Correlate-EIA Prostaglandin E2 Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI) according to the manufacturer's instructions as described elsewhere (34). Briefly, the samples were diluted 1:2.5 with assay buffer, and 100 µl of diluted sample, 50 µl of alkaline phosphatase PGE2 conjugate, and 50 µl of monoclonal anti-PGE2 EIA antibody were then applied to goat anti-mouse IgG microtiter plates and incubated at room temperature for 2 h. After being washed, 200 µl of p-nitrophenyl phosphate substrate solution was added, incubated at room temperature for 45 min, and the optical density at 405 nm was measured in a microplate reader. PGE2 concentrations in the samples were calculated from a PGE2 standard curve, which was run in parallel.

Statistics. Data are expressed as arithmetic means ± SE, and statistical analysis was made by paired or unpaired two-tailed t-test or ANOVA, where appropriate. Differences in means were considered statistically significant if P < 0.05. The number of experiments was adjusted to the variable scatter of the data for each experimental procedure.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Erythrocytes express cation channels, which are usually silent but could be activated by Cl– removal or cell injury, such as osmotic shock or oxidative stress (18, 24, 31). To explore whether the activity of those cation channels was different between adult and neonatal erythrocytes, whole cell currents were recorded with the patch-clamp technique. Replacing NaCl bath solution with a Cl–-free solution (Na gluconate) induced large increases in the outward and the inward currents (Fig. 1A). Interestingly, the currents observed under NaCl Ringer (Fig. 1, B and C, left) as well as under Cl– removal (Fig. 1, B and C, right) conditions were significantly larger in adult compared with neonatal erythrocytes. The corresponding I-V curves are depicted in Fig. 1B and the calculated conductances in Fig. 1C. In the presence of Cl–, the cation channels are superimposed upon by the large Cl– conductance of erythrocytes and thus cannot be resolved.


Figure 1
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  		Fig. 1. Cl–-sensitive nonselective cation channel activity is decreased in neonatal erythrocytes. A: whole cell current traces from an adult erythrocyte recorded successively with NaCl or Na gluconate (Cl– free) bath solutions. The pipette contained a Na gluconate solution. The gray line indicates the zero current value. Membrane voltage was held at –30 mV. B: mean current-voltage relationships (±SE; n = 5, each) recorded with Na gluconate pipette solution and NaCl (left) or Cl–-free bath solution (Na gluconate, right) of adult ({circ}) and neonatal erythrocytes ({blacktriangleup}). C: mean whole-cell conductance (±SE; n = 5, each) in NaCl (left) and Na gluconate (right) bath solutions as calculated from (B) by linear regression of neonatal erythrocytes (solid bars) and adult erythrocytes (open bars). *P < 0.05, significant difference between adult and neonatal erythrocytes, unpaired t-test.

 
The cation channels have previously been shown to allow permeation of Ca2+ (11, 15, 17, 18, 29, 32). Therefore, cytosolic Ca2+ activity was determined by fluo-3-dependent fluorescence to test whether the decreased cation channel activity of neonatal erythrocytes leads to decreased intracellular Ca2+ activity. To this end, erythrocytes were loaded with the Ca2+-sensitive fluorescent dye fluo-3. Fluo-3 fluorescence measurements revealed an increase in the percentage of cells with increased cytosolic Ca2+ activity following Cl– removal, an effect that was significantly blunted in neonatal erythrocytes (Fig. 2, A and B). Thus the blunted cation channel activity in neonatal red blood cells indeed leads to a decrease in their cytosolic Ca2+ activity.


Figure 2
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  		Fig. 2. Increase in cytosolic Ca2+ activity following Cl– removal is blunted in neonatal erythrocytes. A: original flow cytometry histograms of fluo3-dependent fluorescence in adult (top) or neonatal (bottom) erythrocytes exposed for 6 h to solutions with (NaCl, left) or without (Cl–-free, right) Cl–. The numbers indicate the mean fluorescence of the respective erythrocyte population. B: arithmetic means (± SE, n = 5) of the mean fluorescence of adult (open bars) or neonatal (solid bars) erythrocytes after exposure to Cl–-containing (control) or Cl–-free Ringer solution for 6 h. By comparison, exposure of erythrocytes to the Ca2+ ionophore ionomycin (1 µM for 5 min) increased the mean fluorescence of the erythrocytes to 75 ± 8 (n = 6, data not shown). *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 
The decreased cation channel activity and Ca2+ entry following Cl– removal were expected to blunt the triggering of eryptosis, i.e., phosphatidylserine exposure and cellular shrinkage of the cells. To explore whether neonatal erythrocytes are relatively resistant to the eryptotic effect of Cl– removal, we investigated the death of adult and neonatal erythrocytes by fluorescence microscopy. Indeed, inspection of the respective cells by fluorescence microscopy revealed that neonatal erythrocytes seemed to be less susceptible to cellular stress, i.e., Cl– removal (Fig. 3, A and B). Next, phosphatidylserine exposure of erythrocytes was quantitatively determined by annexin V binding, and cell volume by forward scatter in FACS analysis (Fig. 3, C and D). Before Cl– removal, only a small percentage of neonatal (3.85 ± 1.06%, n = 20) and adult (2.10 ± 0.36%, n = 20) erythrocytes bound FITC-coupled annexin V. The annexin V binding was slightly but significantly (P < 0.05) larger in neonatal than in adult erythrocytes. However, removal of Cl– from the bath solution (replacement of Cl– by gluconate) for 24 h markedly increased the percentage of annexin V binding cells, an effect significantly smaller in neonatal erythrocytes (Fig. 3, C and D).


Figure 3
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  		Fig. 3. Increase of erythrocyte annexin V-binding following Cl– removal is blunted in neonatal erythrocytes. A and B: transmission light micrographs (left) and corresponding annexin V fluorescence images (right) of two adult (A) and two neonatal (B) erythrocytes following a 24-h exposure to Cl–-free solution. C: original flow cytometry histograms of annexin V binding adult (top) or neonatal (bottom) erythrocytes exposed for 24 h to solutions with (NaCl, left) or without (Cl– free, right) Cl–. D: arithmetic means (±SE, n = 6) of annexin V binding in adult (open bars) and neonatal (solid bars) cells after exposure to Cl–-free or Cl–-containing (control) bath solution for 24 h. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 
The annexin V binding following Cl– removal was paralleled by a decrease in forward scatter, pointing to erythrocyte shrinkage. The decrease in forward scatter following Cl– removal was significantly blunted in neonatal erythrocytes (Fig. 4, A and B). Thus, compared with adult erythrocytes, neonatal erythrocytes are relatively resistant to the shrinking effects of Cl– removal.


Figure 4
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  		Fig. 4. Decrease in forward scatter following Cl– removal is blunted in neonatal erythrocytes. A: original flow cytometry histograms of forward scatter of adult (top) or neonatal (bottom) erythrocytes after exposure of 24 h to solutions with (NaCl, left) or without (Cl– free, right) Cl–. B: arithmetic means (±SE, n = 6) of forward scatter of adult (open bars) or neonatal (solid bars) erythrocytes after exposure to Cl–-free or Cl–-containing bath solution (control) for 36 h. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 
The effects of Cl– removal on forward scatter and annexin V binding were mimicked by osmotic cell shrinkage. As illustrated in Fig. 5, A and B, increase of extracellular osmolarity by addition of 400 mM sucrose to standard Ringer solution (i.e., an increase to ~700 mosM) was followed by a marked increase of the percentage of annexin V-binding erythrocytes. The increase was paralleled by a sharp decrease in forward scatter, reflecting erythrocyte shrinkage (Fig. 6, A and B). The effect of hyperosmotic shock on phosphatidylserine exposure was again significantly blunted (Fig. 5, A and B) and the forward scatter was significantly higher in neonatal erythrocytes (Fig. 6, A and B).


Figure 5
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  		Fig. 5. Increase of annexin V binding following hyperosmotic shock is blunted in neonatal erythrocytes. A: original flow cytometry histograms of annexin V binding adult (top) or neonatal (bottom) erythrocytes exposed for 24 h to isotonic solution (300 mosM; left) or hypertonic solution (700 mosM; right). B: arithmetic means (±SE, n = 9) of annexin V binding in adult (open bars) and neonatal (solid bars) cells after exposure to isotonic solution (300 mosM) or hypertonic solution (700 mosM) for 24 h. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 

Figure 6
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  		Fig. 6. Decrease in forward scatter following hyperosmotic shock is blunted in neonatal erythrocytes. A: original flow cytometry histograms of forward scatter of adult (top) or neonatal (bottom) erythrocytes exposed for 24 h to isotonic solution (300 mosM; left) or hypertonic solution (700 mosM; right). B: arithmetic means (±SE, n = 9) of forward scatter in adult (open bars) and neonatal (solid bars) cells after exposure to isotonic solution (300 mosM) or hypertonic solution (700 mosM) for 24 h. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 
The cation channels of erythrocytes from adult volunteers are activated by PGE2, which is released by Cl– removal (see Fig. 7A and Ref. 34). Thus we explored whether PGE2 release is altered in neonatal erythrocytes. As shown in Fig. 7A, the stimulation of PGE2 formation by Cl– removal is completely lacking in neonatal erythrocytes. Furthermore, there is a slight reduction in the sensitivity of neonatal erythrocytes to PGE2 (Fig. 7B). In addition, neonatal erythrocytes were less sensitive to the pro-eryptotic effect of platelet-activating factor PAF-16 (Fig. 7C), which has been described as another inducer of programmed death in adult red blood cells (35).


Figure 7
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  		Fig. 7. Reduced PGE2 formation and PAF sensitivity in neonatal erythrocytes. A: arithmetic means (±SE, n = 3) of prostaglandin E2 (PGE2) concentration in the supernatant (in % of control) in adult ({square}, control: 85 ± 2 pg/ml) and neonatal ({blacksquare}, control: 77 ± 4 pg/ml) cells following exposure to Ringer solutions without Cl– (Cl– replaced by gluconate). *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes. B: arithmetic means (±SE, n = 4) of annexin V binding in adult ({square}) and neonatal ({blacksquare}) cells after a 24-h exposure to PGE2. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes. C: arithmetic means (±SE, n = 4) of annexin V binding in adult ({square}) and neonatal ({blacksquare}) cells after a 24 h exposure to platelet-activating factor (PAF-16). *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 
In contrast to the relative insensitivity of neonatal erythrocytes to Cl– depletion, osmotic shock, PGE2, and PAF, their sensitivity toward oxidative stress was significantly enhanced (Fig. 8, A and B). Treatment of cells with the oxidant tert-butyl hydroperoxide (t-BOOH) led to annexin V binding in both adult and neonatal erythrocytes. The t-BOOH concentrations required, however, were significantly lower in neonatal than in adult erythrocytes. Specifically, 50 µM t-BOOH had no significant effect on annexin V binding in adult erythrocytes but triggered eryptosis in ~40% of the neonatal erythrocytes (Fig. 8B).


Figure 8
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  		Fig. 8. Enhanced sensitivity of neonatal erythrocytes to oxidative stress. A: original flow cytometry histograms of annexin V binding of adult (top) or neonatal (bottom) erythrocytes exposed for 24 h to isotonic solution without (left) or with (right) 50 µM tert-butylperoxide (t-BOOH). B: arithmetic means (±SE, n = 9) of annexin V binding in adult (open squares) and neonatal (solid squares) cells after exposure to 0–150 µM t-BOOH for 24 h. *P < 0.05 vs. control; #P < 0.05 vs. adult erythrocytes.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As reported previously, Cl– removal and osmotic cell shrinkage trigger phosphatidylserine exposure of adult erythrocytes via activation of nonselective, Ca2+-permeable cation channels and subsequent Ca2+-sensitive erythrocyte membrane scrambling (18, 24, 31). Cl– removal further decreases forward scatter, an indirect measure of cell volume. The decrease in forward scatter during eryptosis is paralleled by a decrease of packed cell volume and thus indeed reflects cell shrinkage (39). As shown previously, the decrease in forward scatter is reversed by increased extracellular K+ concentration, which dissipates the chemical driving force for K+ exit. The decrease in forward scatter is further blunted by inhibitors of Ca2+-sensitive K+ channels (33). Thus the decrease in forward scatter following entry of Ca2+ is most likely due to activation of Ca2+-sensitive K+ channels, with subsequent exit of K+, hyperpolarization, and exit of Cl– and H2O (33). As shown previously, Cl– removal triggers eryptosis exclusively by activating the erythrocyte nonselective cation conductance. The conductance has been characterized and shown to be inhibited by amiloride and ethylisopropylamiloride (18, 24, 31). The channels and/or Ca2+ entry could similarly be activated by hyperosmotic shock (11, 17, 18, 30, 32). However, in addition, osmotic shock triggers eryptosis by stimulation of ceramide formation (30).

The present paper demonstrates the relative insensitivity of neonatal erythrocytes to stimulation of eryptosis by Cl– removal or osmotic shock. The relative resistance of neonatal erythrocytes to the eryptotic effects of Cl– removal is at least partially due to lacking release of PGE2, which accounts for the activation of the cation channels following Cl– removal or osmotic shock (34). Notably, the seemingly complete lack of PGE2 formation does not lead to complete suppression of cation channel activity and eryptosis. Thus additional mechanisms may be involved in the triggering of channel activity and eryptosis of neonatal erythrocytes. Neonatal erythrocytes are further resistant to PAF, which in adult erythrocytes activates a sphingomyelinase (35) with subsequent ceramide formation and ceramide-induced scrambling of the cell membrane (30).

The relative resistance to different eryptotic stimuli counteracts premature death and clearance of neonatal erythrocytes. As shown most recently (25), phosphatidylserine-exposing erythrocytes are cleared in <1 h from circulating blood. However, eryptosis shares similarities with but may be distinct from erythrocyte senescence (4, 9, 28, 40, 43). Thus the life span of erythrocytes is not only determined by eryptosis but by additional mechanisms as well.

In contrast to cation channel-dependent eryptosis following Cl– removal or osmotic shock, spontaneous eryptosis appears to be slightly but significantly more rapid in neonatal than in adult erythrocytes. This observation points to additional differences between neonatal and adult erythrocytes, which may be relevant for intrauterine or extrauterine survival of neonatal or adult erythrocytes. Oxidative stress has previously been shown to be a major cause of erythrocyte injury (4, 10, 36, 38). Accordingly, the decreased cation channel activity in neonatal erythrocytes does not necessarily imply that their life span is enhanced, irrespective of their microenvironment. Instead, they may be less resistant to other mechanisms of erythrocyte clearance from circulating blood. The present observations indeed reveal that neonatal erythrocytes are more sensitive to oxidative stress. The exquisite sensitivity to oxidative stress may account for the rapid clearance of neonatal erythrocytes after birth, when the neonatal erythrocytes are exposed to alveolar oxygen.

In conclusion, erythrocytes from neonatal blood express less cation channel activity and are less sensitive to Cl– removal or osmotic shock than erythrocytes from adults. In contrast, the neonatal erythrocytes are more sensitive to oxidative stress. The altered sensitivity of neonatal erythrocytes to different eryptotic stimuli may thus contribute to the intrauterine survival and postnatal clearance of neonatal erythrocytes.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Deutsche Forschungsgemeinschaft No. 315/13-1 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 European Union (BMH4-CT96-0602). E. Shumilina has been supported by a grant from the Alexander von Humboldt Foundation, and P. A. Lang and D. S. Kempe have been recipients of stipends from the Federal Ministry of Education and Research, Interdisciplinary Center of Clinical Research of the University of Tübingen.


   ACKNOWLEDGMENTS
 
We thank K. Bauer of the Institute of Pharmacy, University of Tübingen, for technical assistance. We also thank Tanja Loch and Lejla Subasic for the meticulous preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstrasse 5, D72076 Tübingen, Germany (e-mail: florian.lang{at}uni-tuebingen.de)

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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 DISCUSSION
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