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首页医源资料库在线期刊美国生理学杂志2004年第287卷第6期

Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl -

来源:《美国生理学杂志》
摘要:【摘要】OsmoticshockbyadditionofsucrosetothemediumstimulateserythrocytesphingomyelinasewithsubsequentceramideformationandtriggersCa2+entrythroughstimulationofcationchannels。BothceramideandCa2+activateanerythrocytescramblase,leadingtobreakdownofphospha......

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【摘要】  Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca 2+ entry through stimulation of cation channels. Both ceramide and Ca 2+ activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 ± 4% ( n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl - with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl - and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl - is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide.

【关键词】  cation channels sphingomyelinase ceramide apoptosis cell volume osmolarity phosphatidylserine


ERYTHROCYTES LACK MITOCHONDRIA and nuclei, intracellular organelles involved in the induction of apoptosis ( 22, 24 ). Nevertheless, treatment of erythrocytes with the Ca 2+ ionophore ionomycin has recently been shown to induce erythrocyte shrinkage, membrane blebbing, and breakdown of cell membrane phosphatidylserine asymmetry, all typical features of apoptosis in other cell types ( 5, 10, 15, 33 ). Cells exposing phosphatidylserine at their surface are recognized by phosphatidylserine receptor-carrying macrophages ( 20 ), which bind, phagocytose, and thus clear those cells from circulating blood.


The breakdown of phosphatidylserine asymmetry results from the stimulation of a scramblase ( 63 ), which is activated by an increase of cytosolic Ca 2+ activity ( 16, 58, 59 ). Cytosolic Ca 2+ activity is increased during exposure of erythrocytes to osmotic shock by an increase of extracellular osmolarity, which opens Ca 2+ -permeable cation conductance ( 33 ). Moreover, osmotic shock stimulates a sphingomyelinase (Smase) with subsequent formation of ceramide ( 36 ), which potentiates the effect of cytosolic Ca 2+ activity on erythrocyte scramblase ( 33, 36 ).


Because osmotic shock may trigger Ca 2+ entry and ceramide formation, the question arises whether those events occur during passage of erythrocytes through kidney medulla with the extraordinary NaCl and urea required to concentrate the urine ( 49 ). The present study has been performed to test whether hypertonic NaCl and urea similarly induce annexin binding as hypertonic sucrose and to elucidate the related mechanisms.


METHODS


Solutions. Erythrocytes were drawn from healthy volunteer donors who gave informed consent, and the study was approved by the Ethical Committee of the Medical Faculty. Experiments were performed at 37°C in a standard Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1 MgSO 4, 32 HEPES/NaOH, 5 glucose, and 1 CaCl 2 (pH = 7.4). Where indicated, osmolarity was increased by adding sucrose, NaCl, or urea on top of isotonic Ringer. In some experiments, extracellular Cl - was removed by replacing NaCl, KCl, and CaCl 2 from the standard Ringer solution with equimolar amounts of sodium- D -gluconate (125 mM), potassium- D -gluconate (5 mM), and calcium-( D -gluconate) 2 (1 mM) or by NaNO 3 (125 mM), KNO 3 (5 mM), and Ca(NO 3 ) 2 (1 mM).


Smase from Staphylococcus aureus was purchased from Biomol and used at a final concentration of 0.005 U/ml by serial dilution from a 100 U/ml stock solution. Externally added Smase has been shown to be capable of degrading cell membrane sphingomyelin with the formation of ceramide ( 27, 43, 46, 51 ). D -Erythro- N -hexanoylsphingosine (C 6 -ceramide) was purchased from Biomol (Hamburg, Germany). D -Erythro- N -hexanoylsphingosine was dissolved in DMSO to give a 50 mM stock solution and further diluted to a final concentration of 50 µM in standard Ringer solution containing 0.1% BSA. The maximum concentration of DMSO was in all cases 0.1%, a concentration that did not induce annexin binding (data not shown).


A monoclonal anti-ceramide antibody (clone MID 15B4; isotype IgM) was purchased from Alexis (Grünberg, Germany).


FACS analysis. FACS analysis was performed essentially as described earlier ( 2, 31 ). After incubation, cells were washed in annexin-binding buffer containing 125 mM NaCl, 10 mM HEPES/NaOH (pH = 7.4), and 5 mM CaCl 2. Erythrocytes were stained with annexin-FLUOS (Böhringer Mannheim, Mannheim, Germany) at a 1:100 dilution. After 15 min, samples were diluted 1:5 and measured by flow cytometric analysis on a FACS-Calibur (Becton-Dickinson, Heidelberg, Germany). Cells were analyzed by forward and side scatter, and annexin-fluorescence intensity was measured in FL-1. Control experiments revealed that urea (600 mM) did not interfere with the annexin-binding assay of hypertonically stressed erythrocytes (Ringer + 650 mM sucrose for 8 h) when added 5 min before the end of incubation. In those experiments, the percentage of annexin-binding cells was 63 ± 6% ( n = 6) in the absence and 62 ± 5% ( n = 6) in the presence of urea.


For determination of ceramide formation, cells were stained for 1 h at 4°C with 1 µg/ml anti-ceramide antibody in PBS containing 1% FCS at a dilution of 1:5, as described recently ( 36 ). After three washes with PBS/1% FCS, cells were stained with polyclonal FITC-conjugated goat anti-mouse Ig-specific antibody (Pharmingen, Hamburg, Germany) in PBS/1% FCS at a dilution of 1:50 for 30 min. Unbound secondary antibody was removed by washing the cells two times with PBS/1% FCS, and samples were analyzed by flow cytometric analysis on a FACS-Calibur. FITC-fluorescence intensity was measured in FL-1.


Immunofluorescence. Control or ischemic (30 min) kidneys were quickly frozen in liquid nitrogen and transferred to a cryostat. Experiments were performed according to the German Animal Protection Law and were approved by the local authorities. Sections were taken at 20 µm and stained unfixed with annexin-V-FLUOS (Roche) diluted 1:100 in buffer containing (in mM) 140 NaCl, 10 HEPES/NaOH, and 5 CaCl 2 (pH 7.4). After being rinsed two times with the same buffer, sections were covered with a coverslip and analyzed immediately. Sections were examined on a Zeiss LSM510 confocal microscope using an Argon laser with excitation at 488 nm, with appropriate emission filters, and a x 40 oil immersion lens (numerical aperture 1.3). Images or small z-stacks of images from large blood vessels in the outer medulla were scanned, and the number of annexin-V-FLUOS-positive cells was counted. The confocal microscopy software supplied by the manufacturer was used to measure the cross-sectioned area of the blood vessels. Together with the optical section thickness or z-dimension of the stack (ranging from 3 to 6 µm), we calculated the number of stained cells per volume.


Measurement of intracellular ATP concentrations. The intracellular ATP concentration of human erythrocytes was quantified by a luciferin-luciferase assay kit (Roche Diagnostics, Mannheim, Germany) using a luminometer (Berthold Biolumat LB9500, Bad Wildbad, Germany). Briefly, fresh erythrocytes were washed (3 x 5 min) in PBS Ca 2+ -free medium and centrifuged, and 100 µl of blood pellet (hematocrit 100%) were incubated for 8 h at 37°C with standard Ringer solution or Ringer supplemented with 650 mM urea or 650 mM sucrose (final hematocrit 5-7%). After incubation, cells were lysed in distillated water, and proteins were precipitated by HClO 4 (5%). After centrifugation, an aliquot of the supernatant (400 µl) was adjusted to pH 7.7 by addition of saturated KHCO 3 solution. All of the manipulations were performed at 4°C to avoid ATP degradation. After dilution of the supernatant, the ATP concentration of the aliquots was determined by luciferin/luciferase reaction according to the manufacturer's protocol. The given values are representative of the average ATP concentration measured for 100 µl pelleted blood (hematocrit 100%).


In vitro Smase assay. In vitro measurements of Smase activity were performed essentially as described earlier ( 36 ) using total lipid extracts from erythrocytes supplemented with [ choline - methyl - 3 H]sphingomyelin (with a specific radioactivity of 2.96 TBq/mmol; purchased from American Radiolabeled Chemicals, St. Louis, MO) as substrate. Erythrocytes (2 x 10 9 ) were washed two times with 1 ml standard Ringer solution. Next, total lipids were extracted by resuspending the cell pellets in 500 µl methanol, 250 µl chloroform, and 200 µl water. Samples were stirred for 10 min on a vortex mixer and centrifuged at 13,000 g for 2 min. Phase separation was accomplished by the addition of 250 µl chloroform and 250 µl water. The suspension and centrifugation steps were repeated. [ choline - methyl - 3 H]sphingomyelin (55.5 kBq) was added to 150 µl of the chloroform phase and stirred for 1 min. The radioactive substrate solution (10 µl) was dried under nitrogen and used for the in vitro Smase assay. The radioactive substrate was resuspended in 100 µl of assay buffer (100 mM Tris·HCl, pH 7.4, 6 mM MgCl 2, and 0.1% Triton X-100 in the presence or absence of 600 mM urea). Samples were sonicated for 5 min, and 0.2 U/ml Smase from Staphylococcus aureus (Biomol) or Streptomyces species (Sigma, Taufkirchen, Germany) were added. Reaction mixtures were incubated for different times at 37°C. After 5, 10, and 30 min, 20 µl of the samples were removed, and the reactions were stopped by addition of 133 µl chloroform and 67 µl methanol. Phase separation was completed by addition of 20 µl water. Smase-catalyzed release of [ 3 H]phosphocholine was quantitated by counting 20 µl of the upper, aqueous phase. Blank reactions contained no Smase, and values were subtracted from the sample values.


Patch clamp. Patch-clamp experiments have been performed as described earlier ( 26, 36 ). Erythrocytes were recorded at 21°C. A continuous superfusion was applied through a flow system inserted in the dish. The bath was grounded via an NaCl bridge filled with bath NaCl solution (see below). Borosilicate glass pipettes (8-14 M 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 an MS314 electrical micromanipulator (MW; Märzhäuser, Wetzlar, Germany). The currents were recorded in voltage-clamp mode in fast whole cell configuration by an EPC-9 amplifier (Heka, Lambrecht, Germany) using 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 -10 mV holding potential to potentials between -100 and +100 mV.


The potentials were corrected for liquid junction potentials ( 3 ). The original whole cell current traces are depicted after 500 Hz low-pass filtering, and currents of the individual voltage square pulses are superimposed. 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 and depicted as downward deflections of the original current traces.


Whole cell currents were recorded in standard isotonic bath solution containing (in mM) 115 NaCl, 10 HEPES, 5 CaCl 2, and 10 MgCl 2, titrated with NaOH to pH 7.4, in combination with a sodium- D -gluconate or potassium- D -gluconate pipette solution containing (in mM) 115 sodium- D -gluconate or potassium- D -gluconate, 10 NaCl, 1 EGTA, 1 MgCl 2, 1 Mg-ATP, and 5 HEPES, titrated to pH 7.35 with NaOH. A residual Cl - concentration of 10 mM was used in these pipette solutions to avoid shifts in offset potential.


Where indicated, NaCl bath solution was replaced by sodium- D -gluconate solution containing (in mM): 140 sodium gluconate, 5 HEPES, 1 CaCl 2, and 1 MgCl 2. The removal of Cl - eliminates the outward currents generated by Cl - influx and upregulates the cation conductance ( 18 ). For some experiments, the osmolarity of the sodium- D -gluconate bath solution was increased by addition of 250 mM sucrose or 250 mM urea.


Statistics. Data are expressed as arithmetic means ± SE. Statistical analysis was made by paired or unpaired t -test, where appropriate.


RESULTS


Effects of hypertonic sucrose, NaCl, and urea on erythrocyte annexin binding. In isotonic extracellular fluid, only a small fraction of human erythrocytes (4 ± 1%, n = 12) showed detectable annexin binding, reflecting the virtual absence of phosphatidylserine at the extracellular face of the erythrocyte cell membrane ( Fig. 1 A ). Increase of osmolarity by addition of sucrose, however, led to a sharp increase in annexin binding, reflecting breakdown of the phosphatidylserine asymmetry ( Fig. 1 A ); after addition of 650 mM sucrose, 80 ± 4% ( n = 4) of the human erythrocytes bound annexin within 8 h ( Fig. 1, A and B ). The same increase in osmolarity by addition of 325 mM NaCl (650 mosmol/l) induced annexin binding only in 18 ± 3% ( n = 4) of the cells ( Fig. 1, A and B ). In sharp contrast, addition of 650 mM urea did not significantly enhance the percentage of annexin-binding cells (4 ± 1%, n = 4; Fig. 1, A and B ). As shown in Fig. 1 B, a correlation is observed between the number of annexin-binding cells and the hyperosmolarity by increasing concentrations of sucrose (varying from 300 to 650 mM) or NaCl (varying from 150 to 325 mM) but not urea (varying from 300 to 650 mM). The annexin binding after osmotic shock was not significantly blunted in the presence of the Na + -K + -2Cl - cotransport inhibitor bumetanide. In this series of experiments, after 8 h of osmotic shock (Ringer + 650 mM sucrose), the percentage of annexin-binding cells was 45 ± 2 ( n = 4) in the absence and 46 ± 2 ( n = 4) in the presence of bumetanide (100 µM).


Fig. 1. Effect of hypertonic sucrose, NaCl, and urea on erythrocyte annexin binding and intracellular ATP. A : histograms showing the percentage of annexin-binding erythrocytes after incubation for 8 h in isotonic Ringer solution ( top left ), Ringer solution + 650 mM sucrose ( top right ), Ringer solution + 325 mM NaCl ( bottom left ), or Ringer solution + 650 mM urea ( bottom right ). Nos. refer to the calculated percentage of one single experiment. B : percentage of annexin-binding erythrocytes after 8 h of treatment with urea, NaCl, and sucrose as a function of osmolarity (arithmetic means ± SE, n = 4). The cells were suspended in Ringer NaCl solution (300 mosmol/l) with additional increasing concentrations of sucrose (from 300 to 650 mM), NaCl (from 150 to 325 mM), or urea (from 300 to 650 mM). C : histograms showing the concentrations of intracellular ATP after incubation for 8 h in isotonic Ringer solution, Ringer solution + 650 mM sucrose, or Ringer solution + 650 mM urea (arithmetic means ± SE, n = 15). Data are expressed as mM intracellular ATP/l blood (hematocrit 100%, n = 10).


ATP measurements using luciferin-luciferase assay revealed that an 8-h incubation in Ringer supplemented with 650 mM urea or 650 mM sucrose induced a moderate decrease (8 and 32%, respectively, n = 15) of the intracellular ATP concentration compared with the control condition (isosmotic Ringer, n = 15, Fig. 1 C ).


Inhibitory effect of urea on annexin binding in erythrocytes exposed to osmotic shock. The inability of hypertonic urea to induce annexin binding prompted us to explore whether urea could inhibit the annexin binding of erythrocytes exposed to hypertonic sucrose concentrations. In the absence of urea, the addition of sucrose (Ringer + 650 mM) led within 8 h to annexin binding in 73 ± 5% of the cells ( n = 4; Fig. 2 A ). The addition of urea (600 mM) blunted the sucrose-induced annexin binding despite a further increase of osmolarity ( Fig. 2 A ). In contrast, an increase to the same osmolarity by addition of sucrose (1,250 mM) induced annexin binding in 80 ± 3% ( n = 6) of the cells. Figure 2 B shows the dose-response curve for urea-dependent inhibition of erythrocyte annexin binding at a constant concentration of sucrose (650 mM on top of isotonic Ringer) and increasing concentrations of urea (added on top of isotonic Ringer and sucrose). The additional presence of 600 mM urea decreased the percentage of annexin-binding cells to 20 ± 5% ( n = 4, Fig. 2, A and B ) despite the presence of 650 mM sucrose on top of isotonic Ringer. Although the addition of up to 300 mM sucrose on top of isotonic Ringer does not significantly increase the number of annexin-binding cells ( Fig. 1 B ), the addition of 250 mM sucrose and replacement of NaCl with sodium gluconate significantly enhanced the number of annexin-binding cells, an effect partially reversed by further addition of urea (600 mM, Fig. 2 C ).


Fig. 2. Inhibition of sucrose-induced annexin binding by urea. A : histograms of annexin binding in a representative experiment of erythrocytes incubated in isotonic Ringer solution ( left ), in Ringer solution + 650 mM sucrose (suc; middle ), and in Ringer solution + 650 mM sucrose + 600 mM urea ( right ) for 8 h. B : percentage of annexin-binding erythrocytes after 8 h of incubation with a Ringer solution + 650 mM sucrose as a function of the concentration of urea added on top of 650 mM sucrose (arithmetic means ± SE, n = 4). C : percentage of annexin-binding erythrocytes after 8 h of incubation with Cl - -free Ringer solution (NaCl replaced by sodium gluconate) + 250 mM sucrose as a function of the concentration of urea added on top of 250 mM sucrose (arithmetic means ± SE, n = 4).


Because urea, unlike sucrose, rapidly enters cells ( 32 ), it does not create an osmotic gradient across the cell membrane. We thus explored whether phloretin, a nonspecific inhibitor of urea transport ( 41 ), modifies the effect of urea on annexin binding. Phloretin (700 µM) led to a slight increase of annexin binding in isotonic Ringer (9 ± 2%, n = 9) but did not significantly interfere with the inhibitory effect of urea. Urea (600 mM) reduced the annexin binding induced by hyperosmotic shock (Ringer plus 650 mM sucrose for 8 h) by 44 ± 8% ( n = 9) and 52 ± 8% ( n = 9) in the presence and absence of phloretin, respectively.


In the absence of extracellular Ca 2+, the effect of exposure to hypertonic shock (Ringer + 650 mM sucrose) on annexin binding was significantly blunted by 17 ± 6% ( n = 5), but the inhibitory effect of urea was preserved. The percentage of inhibition was 57 ± 5% ( n = 5) in the absence of Ca 2+ compared with 58 ± 5% ( n = 5) in the presence of Ca 2+.


Activation of cation conductance by Cl - removal and hyperosmolarity. Patch-clamp experiments have been performed to elucidate the role of osmolarity, Cl -, and urea in the regulation of the nonselective cation conductance. As shown in Fig. 3 A, the cation conductance was activated by replacement of NaCl with sodium gluconate, even in isotonic extracellular fluid. Upon removal of external Cl -, the conductance ( G; as calculated between +40 and +100 mV) was 223 ± 41 pS ( n = 17) and 251 ± 51 pS ( n = 8) when recorded with sodium gluconate and potassium gluconate pipette solution, respectively.


Fig. 3. Activation of cation channels by Cl - removal and hyperosmolarity. A : original whole cell current traces (sodium gluconate pipette solution) recorded with isotonic bath solution (NaCl), after removal of Cl - (sodium gluconate), addition of 250 mM sucrose (sodium gluconate + sucrose), replacement of sucrose by 250 mM urea (sodium gluconate + urea), and washout of urea by isotonic bath solution (NaCl). B : current ( I )-voltage ( V ) relationships recorded as in A with isotonic ( ) and hypertonic sodium gluconate bath solution (sodium gluconate + 250 mM sucrose; ) and after isosmotic replacement of sucrose by urea (sodium gluconate + 250 mM urea, ). Data are means ± SE ( n = 4). C : comparison of the mean conductance ± SE ( n = 4) calculated from the outward current (+40 to +100 mV) in the presence of isotonic sodium gluconate bath solution or under hypertonic conditions after addition of 250 mM sucrose or 250 mM urea to the sodium gluconate bath solution. D : annexin binding of erythrocytes after a 20-h treatment in isotonic extracellular fluid without and with isosmotic replacement of NaCl by sodium gluconate (arithmetic means ± SE, n = 4).


Addition of either sucrose (250 mM) or urea (250 mM) on top of isotonic sodium gluconate further stimulated the channel. Figure 3 B depicts the corresponding current-voltage relationships recorded with isosmotic sodium gluconate ( n = 16), sodium gluconate + 250 mM sucrose ( n = 4), or sodium gluconate + 250 mM urea ( n = 4). Figure 3 C shows the mean slope conductance (calculated between +40 and +100 mV) recorded with isosmotic sodium gluconate bath solution ( G = 0.31 ± 0.06 nS, n = 16) and after increasing the osmolarity of the sodium gluconate bath solution by addition of 250 mM sucrose ( G = 0.52 ± 0.04 nS, n = 4) or 250 mM urea ( G = 0.47 ± 0.06 nS, n = 4). Thus, similar to hypertonic sucrose, hypertonic urea activates the cation conductance.


Because removal of extracellular Cl - activates the cation conductance even without an increase of osmolarity, the effect of Cl - removal on annexin binding was tested. As shown in Fig. 3 D, replacement of isotonic Cl - by isotonic gluconate within 20 h significantly increased the number of annexin-binding cells. Similarly, replacement of extracellular Cl - by isotonic NO 3 - significantly increased the number of annexin-binding cells from 5 ± 1% ( n = 10) to 11 ± 2% ( n = 10).


Inhibitory effect of urea on Smase-induced annexin binding. Because urea inhibited annexin binding despite the activation of the nonselective cation conductance, experiments were performed to test for a putative effect of urea on Smase-mediated annexin binding. As shown in Fig. 4 A, exposure of erythrocytes to purified Smase (0.005 U/ml) led within 8 h to annexin binding in 68 ± 2% (n = 4) of the cells. A similar increase of the percentage of annexin-binding cells (62 ± 11%, n = 4) was observed after C 6 -ceramide exposure (50 µM). Figure 4 B shows the concentration-dependent effect of urea on Smase-induced annexin binding. In these experiments, addition of 600 mM urea virtually abolished (3 ± 2%, n = 4) the breakdown of phosphatidylserine asymmetry after addition of Smase. In contrast, ceramide-induced annexin binding was further enhanced in the presence of urea, approaching 95 ± 3% (n = 4) of the cells for 600 mM urea ( Fig. 4 B ).


Fig. 4. Inhibition of sphingomyelinase (Smase)-induced annexin binding by urea. A : representative histograms of erythrocytes incubated for 8 h in isotonic Ringer solution with purified Smase (0.005 U/ml, top left ), Smase (0.005 U/ml) + urea (600 mM, top right ), ceramide (50 µM, bottom left ), or ceramide (50 µM) + urea (600 mM, bottom right ). B : arithmetic means ± SE ( n = 4) of the percentage of annexin-binding cells stimulated with Smase (0.005 U/ml) or ceramide (50 µM) for 8 h in the presence of different urea concentrations. Control refers to cells stimulated with Smase (0.005 U/ml) or ceramide (50 µM) in the absence of urea.


Inhibition of ceramide formation and Smase activity by urea. The observation that urea inhibits annexin binding after addition of Smase and hypertonicity, but not after addition of ceramide, suggested that urea directly influences Smase. Further experiments have been performed to test for an effect of urea on Smase activity. As shown in Fig. 5, osmotic shock induced by addition of sucrose (650 mM sucrose) stimulated ceramide formation in erythrocytes (the levels of ceramide formation start to increase after 30 min and reach a plateau after 4 h; data not shown). After 4 h, ceramide levels reached 181 ± 30% of control ( n = 4), an effect completely reversed to 106 ± 15% of control ( n = 4) in the presence of urea (650 mM sucrose + 600 mM urea). As a positive control for ceramide detection, erythrocytes were treated with purified Smase (1 U/ml during 5 min). As expected, this maneuver led to increased ceramide formation, approaching 303 ± 48% ( n = 4) of control ( Fig. 5 B ).


Fig. 5. Inhibition of ceramide formation by urea. A : representative histograms of ceramide detection in control cells (isotonic Ringer, black, all histograms) and cells exposed for 4 h to hypertonic sucrose solution (Ringer + 650 mM sucrose) in the absence (gray, left ) or in the presence (gray, middle ) of 600 mM urea. Cells exposed for 5 min to purified Smase in isotonic Ringer (1 U/ml) served as a positive control for ceramide production (gray, right ). B : arithmetic means ± SE ( n = 4) of the mean ceramide-related fluorescence after 4 h of incubation with an isotonic Ringer NaCl solution (control), after 4 h in hypertonic sucrose solution (suc; Ringer + 650 mM sucrose), after 4 h in hypertonic sucrose solution in the presence of urea (suc + urea, Ringer + 650 mM sucrose + 600 mM urea), and after 5 min in the presence of 1 U/ml purified Smase in isotonic Ringer (Smase) as a positive control. *Significant difference from control cells and #significant difference from cells in hypertonic sucrose solution in the absence of urea (2-tailed t -test, P

In vitro assays were performed in addition to test for the influence of urea on Smase activity. For this, total erythrocyte lipid extract supplemented with [ choline - methyl - 3 H]sphingomyelin was used as substrate. As shown in Fig. 6 A, production of [ 3 H]phosphocholine increased within 10 min of incubation with Smase and could thus serve as a direct measure of Smase activity. Urea significantly reduced the activity of Smase from Staphylococcus aureus and Streptomyces species by 44 ± 4% ( n = 3) and 35 ± 2% ( n = 3), respectively ( Fig. 6 B ).


Fig. 6. Inhibition of in vitro Smase activity by urea. A : time-dependent release of [ 3 H]phosphocholine from [ choline - methyl - 3 H]sphingomyelin by Smase was measured in control assay buffer as described in METHODS. Arithmetic means ± SE ( n = 3) of liberated [ 3 H]phosphocholine are given in dpm. B : arithmetic means ± SE ( n = 3) of liberated [ 3 H]phosphocholine/min after incubation in assay buffer in the absence (control) or in the presence of 600 mM urea (urea). Smase either from Staphylococcus aureus or Streptomyces species was used as an enzyme source. *Significant difference from control assay buffer (2-tailed t -test, P

Induction of erythrocyte phosphatidylserine exposure in ischemic mouse kidney. To test for a possible role in acute renal failure, erythrocyte phosphatidylserine exposure and vessel wall adherence were estimated in control and ischemic (30 min) mouse kidney by fluorescence microscopy. As shown in Fig. 7, A and B, ischemia induced a moderate but significant ( P

Fig. 7. Annexin-binding erythrocytes in ischemic kidney. A and B : confocal laser scan images from sections of control ( A ) and ischemic (30 min; B ) mouse kidneys stained with annexin-V-FLUOS to detect phosphatidylserine-exposing erythrocytes. Shown are fluorescence images ( left ) and superimposed fluorescence and transmission light images ( right ) from veins at the cortex medulla border (as indicated by scheme on top right ). Arrowheads indicate phosphatidylserine-exposing erythrocytes adhering at the vessel wall. C : relative number of phosphatidylserine-exposing erythrocytes in sections of control (open bar) and ischemic (closed bar) mouse kidneys. Data are means ± SE of 6-8 z-stacks of vessel images from 2 kidneys for each condition. OM, outer medulla; IM, inner medulla.


DISCUSSION


The present study confirms the previous observations that hyperosmotic shock stimulates breakdown of the phosphatidylserine asymmetry in erythrocytes ( 33, 35 ). In vivo, nucleated cells ( 20 ) and erythrocytes ( 6, 19 ) exposing phosphatidylserine will be eliminated by macrophages. As shown previously, hyperosmotic shock and oxidative stress open a nonselective cation conductance allowing the passage of Ca 2+ ( 18, 26 ). Volume-regulatory cation conductances are not only expressed in erythrocytes but also in a wide variety of nucleated cells ( 11, 12, 21, 30, 32, 54 - 56 ). The cation conductance in erythrocytes has been characterized previously ( 18, 26 ). In those cells, the cation conductance probably does not serve cell volume regulation. In contrast, Ca 2+ entry through the cation conductance ( 33 ) activates the Gardos channel ( 35 ), leading to hyperpolarization, loss of cellular KCl, and cell shrinkage. Increase of extracellular K + rather blunts cell shrinkage and erythrocyte annexin binding after an increase of cytosolic Ca 2+ ( 34 ).


Increase of cytosolic Ca 2+ in the erythrocytes may result not only from enhanced entry but also from impaired extrusion. Osmotic shock leads to a decrease of ATP concentrations, which may at least in part result from enhanced ATP hydrolysis ( 60 ). A decrease of ATP concentration may interfere with Ca 2+ extrusion by the Ca 2+ -ATPase and thus increase cytosolic Ca 2+ concentration.


Beyond the activation of nonspecific cation conductance, hyperosmotic cell shrinkage activates in erythrocytes an endogeneous Smase, leading within 30 min to the formation of ceramide and with a slight delay to stimulation of annexin binding ( 36 ). The formation of ceramide is paralleled by the decrease of sphingomyelin ( 36 ). External application of ceramide triggers annexin binding in erythrocytes ( 36 ) and nucleated cells ( 17, 38 ). Ceramide sensitizes erythrocytes for the action of Ca 2+ ( 36 ). Along those lines, the increase of annexin binding after osmotic shock was blunted in the presence of the putative Smase inhibitor dichloroisocoumarin ( 36 ). In nucleated cells ( 27, 43 ) and erythrocytes, addition of exogenous Smase similarly stimulates ceramide formation and subsequent increase of annexin binding. Involvement of ceramide in signaling of apoptosis has been shown for nucleated cells, such as CD95-induced lymphocyte death ( 23, 25 ) and treatment of cells with various chemotherapeutic agents, e.g., hexadecylphosphocholine ( 57 ) or daunorubicin ( 9 ). The ability of C 6 -ceramide to induce erythrocyte phosphatidylserine exposure is surprising, since erythrocytes lack mitochondria, crucial elements in the signaling cascade triggered by C 6 -ceramide in nucleated cells. C 6 -ceramide must utilize a pathway distinct from that described in nucleated cells. Thus the capacity of C 6 -ceramide to trigger phosphatidylserine exposure in erythrocytes discloses the presence of an alternative pathway that may exist in nucleated cells as well. C 6 -ceramide triggers erythrocyte phosphatidylserine exposure even though it does not enhance Ca 2+ uptake ( 36 ).


Excessive osmolarity, as it prevails in renal medulla, may trigger apoptosis of nucleated cells ( 7, 8, 32, 35, 40, 45, 47, 48 ). Moreover, it compromises the function of lymphocytes, which inhibits defense in kidney medulla ( 37 ). In cultured renal tubular epithelial cells, the apoptosis induced by NaCl could be reversed by urea ( 53, 61, 62 ). On the other hand, urea has been shown to sensitize cells to apoptosis ( 13 ) and lead to cell cycle delay ( 45 ). In the present study, we clearly show that urea (600 mM) inhibits hyperosmotic shock-induced erythrocyte annexin binding. Notably, erythrocytes exposed to 300 mM NaCl plus 600 mM urea for 8 h (which approximates the hyperosmotic condition in the medullary tip) only show marginal annexin binding of 11.4 ± 4.5% ( n = 8) compared with erythrocytes exposed to isotonic Ringer solution (2.9 ± 1.4%, n = 8). Thus the high Cl - and urea concentrations prevailing in renal medulla prevent erythrocyte phosphatidylserine exposure despite increased osmolarity. Under physiological conditions, the contact time of erythrocytes with renal medulla is too short to trigger significant phosphatidylserine exposure. However, the contact time may be considerably increased in acute renal failure, which typically leads to trapping of erythrocytes in the transition from medulla to cortex ( 42 ). In the present study, experimental renal ischemia, indeed, induced phosphatidylserine exposure of erythrocytes. Moreover, at least in theory, urea could similarly interfere with ceramide formation in renal medullary cells.


The mechanisms by which urea interferes with apoptotic signaling are poorly understood. Urea may activate ras ( 52 ) and increase phosphatidylinositol 3-kinase activity ( 62 ). In erythrocytes, urea has been shown to modify several transport processes, including the Na + -K + -ATPase, KCl cotransport, and Na + -K + -2Cl - cotransport ( 28, 29, 39, 50 ). Interestingly, hydroxyurea protects erythrocytes against oxidative damage ( 1 ) and prevents sickling ( 4, 14, 44 ). In this respect, it may be of interest that sickle cells are especially sensitive to phosphatidylserine exposure induced by osmotic shock, oxidative stress, and energy depletion ( 33 ). The direct ability of urea to inhibit Smase and ceramide formation may well participate in the regulation of phosphatidylserine exposure not only in erythrocytes but also in nucleated cells.


In summary, similar to nucleated cells, erythrocytes loose phosphatidylserine asymmetry, leading to annexin binding after exposure to hyperosmotic shock. The phosphatidylserine exposure is mediated by both activation of cation conductance and by Smase-related ceramide formation. Cl - directly inhibits the cation conductance, and urea decreases the Smase activity. Thus, at least in erythrocytes, both extraordinary Cl - and urea concentrations, as they prevail in renal medulla, partially abrogate the proapoptotic effects of hyperosmotic shock. The present observations in erythrocytes may thus shed light on the complex interplay of NaCl and urea in the regulation of medullary kidney cell apoptosis during osmotic shock.


GRANTS


This study was supported by Deutsche Forschungsgemeinschaft Grant Nos. La 315/4-3, La 315/6-1, and La 315/11-1, Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) Grant 01 KS 9602, and the Biomed program of the European Union (BMH4-CT96-0602).


ACKNOWLEDGMENTS


We acknowledge the technical assistance of E. Faber and the meticulous preparation of the manuscript by L. Subasic and T. Loch.

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作者单位:1 Physiology and 2 Anatomy, University of Tübingen, D-72076 Tübingen, Germany

作者: Karl S. Lang, Swetlana Myssina, Philipp A. Lang, V 2008-7-4
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