Department of Physiology, University of Tübingen, Tübingen, Germany
Submitted 1 March 2004 ; accepted in final form 15 September 2004
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ABSTRACT |
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cell volume; annexin; apoptosis; Gardos channel; calcium
Most recent experiments have disclosed that injured erythrocytes expose phosphatidylserine at their surface (4, 10, 11, 15, 35). Because macrophages are equipped with receptors specific for phosphatidylserine (20, 25), erythrocytes exposing phosphatidylserine at their surface will be rapidly recognized, engulfed, and degraded (6, 19) and thus are expected to be eliminated rapidly from circulating blood. Accordingly, the decreased life span of erythrocytes in the settings of sickle cell disease, thalassemia, and glucose-phosphate dehydrogenase deficiency has been shown to be paralleled by accelerated phosphatidylserine exposure (35).
Erythrocyte phosphatidylserine exposure is accomplished by a scramblase (16, 51) that is activated after an increase of cytosolic Ca2+ activity (4, 11, 15). Moreover, the scramblase is sensitized to Ca2+ by ceramide, which is formed after activation of a sphingomyelinase (34). Ca2+ activity may be increased by activation of a Ca2+-permeable cation conductance that is activated by osmotic shock, oxidative stress, and energy depletion (17, 18, 28, 33). Aside from increased scramblase activity, Ca2+ activates the Ca2+-sensitive K+ channels (7, 12), which similarly favors erythrocyte apoptosis (36). Pb+ ions have been demonstrated to directly activate Ca2+-sensitive Gardos K+ channels in human erythrocytes (44) and similar Ca2+-sensitive K+ channels in other cell types (13, 39). Therefore, the present experiments were performed to explore whether exposure to Pb+ ions triggers phosphatidylserine exposure and to elucidate the underlying mechanisms.
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MATERIALS AND METHODS |
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Solutions. Erythrocytes were drawn from healthy volunteers. They were used either without purification or after separation by centrifugation for 25 min at 2,000 g over Ficoll gradient (Biochrom, Berlin, Germany). Experiments with nonpurified or 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)-NaOH (pH 7.4), 5 glucose, and 1 CaCl2. Pb2+ ions were added to the NaCl-Ringer at final concentrations varying from 0.1 to 10 µM [10 mM Pb(NO3)2 stock solution; Sigma, Taufkirchen, Germany]. Clotrimazole (Sigma) was dissolved in dimethyl sulfoxide (DMSO; 80 mM stock solution) and used at a final concentration of 2 µM (0.02% DMSO). For Pb2+ stimulation, the final hematocrit was adjusted to 0.3%.
Fluorescence-activated cell sorting analysis. Fluorescence-activated cell sorting (FACS) analysis was performed as described previously (1). After incubation in the presence or absence of Pb2+, cells were washed in annexin-binding buffer containing (in mM) 125 NaCl, 10 HEPES-NaOH (pH 7.4), and 5 CaCl2. Erythrocytes were suspended in a solution composed of Annexin-V-Fluos (Boehringer Mannheim, Mannheim, Germany) and annexin buffer (1:50 dilution). After 10 min of incubation, samples were finally diluted 1:5 in annexin-binding buffer and measured using flow cytometric analysis (FACSCalibur; BD Biosciences, Heidelberg, Germany). Cells were analyzed by forward scatter, and annexin fluorescence intensity was measured in fluorescence channel FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
Light microscopy. The Pb2+ effect on annexin binding in erythrocytes was further studied using immunofluorescence as described previously (50). After 24-h exposure (37°C) to increasing concentrations of Pb2+ (0, 0.3, 1, and 3 µM in NaCl-Ringer solution), the erythrocytes were suspended for 20 min in annexin-binding buffer containing Annexin-V-Fluos (1:50 dilution), centrifuged, resuspended in NaCl-Ringer, and postincubated (37°C) for 10 min in a modified Ringer solution containing (in mM) 145 NaCl, 5 KCl, 5 glucose, 1.6 CaCl2, 0.8 MgCl2, and 5 HEPES-NaOH (pH 7.4) to wash the cells and to maintain an isosmotic bath condition (298 mosM). Finally, 10 µl of the cell suspension were analyzed under a fluorescence microscope (440/480-nm excitation and 535/50-nm emission wavelength, Q505LP beamsplitter, 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.
Patch-clamp recordings.
Patch-clamp experiments have been performed in whole cell voltage-clamp mode as described elsewhere (17, 27) using patch-clamp pipettes made of borosilicate glass (150 TF-10; Clark Medical Instruments) with resistances ranging from 8 to 12 M. Currents were recorded and low-pass filtered at 3 kHz using an EPC-9 amplifier (Heka, Lambrecht, Germany), Pulse software (Heka), and an ITC-16 interface (Instrutech, Port Washington, NY). 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, and the potentials were corrected for liquid junction potentials as estimated according to the method of Barry and Lynch (3). Whole cell currents were evoked using 10 voltage pulses (400 ms each), from 30 mV holding potential to voltages between 100 and +80 mV, in increments of 20 mV. The inward currents, defined as flow of positive charge from the pipette into the cell, were negative currents and are depicted as downward deflections of the original current traces. The original current traces are depicted after 1,000-Hz low-pass filtering. Whole cell currents were averaged in the 350- to 375-ms time window of each 400-ms square pulse, and these data were used to calculate the means ± SE of different cells.
The pipette solution used in whole cell and on-cell mode contained (in mM) 80 K+-gluconate, 60 KCl, 10 HEPES-NaOH (pH 7.2), 1 MgATP, 1 MgCl2, 3 EGTA, and 2 CaCl2 ( 0.3 µM free Ca2+ concentration). NaCl and KCl bath solutions contained (in mM) 115 NaCl, 20 HEPES-NaOH (pH 7.4), 10 MgCl2, 5 CaCl2, 140 KCl, 10 HEPES-NaOH (pH 7.4), 1 CaCl2, and 1 MgCl2, respectively. The high concentrations of Mg2+ and Ca2+ in the NaCl bath solution are suggested to increase the probability of obtaining high-resistance seals. In addition, the NaCl bath solution was used in the pipette in further cell-attached recordings. Pb2+ was applied to the bath solution [superfusion of 110 µM Pb(NO3)2] during continuous whole cell and cell-attached recording.
Measurement of intracellular free Ca2+ concentration. Intracellular Ca2+ measurements were performed as described previously (1). Erythrocytes were loaded with fluo-3 AM (Calbiochem, Bad Soden, Germany) by addition of fluo-3 AM stock solution (2 mM diluted in DMSO) to 1 ml of erythrocyte suspension (0.16% hematocrit in Ringer solution; 4 µM fluo-3 AM final concentration). The cells were incubated at 37°C for 15 min under protection from light. An additional 2-µl aliquot of fluo-3 AM was added, and then the mixture was incubated for 25 min. Fluo-3-loaded erythrocytes were centrifuged at 1,000 g for 5 min at 22°C and then washed twice with Ringer solution containing 0.5% bovine serum albumin (Sigma) and once with Ringer solution. For flow cytometry, fluo-3-loaded erythrocytes were resuspended in 1 ml of Ringer solution (0.16% hematocrit) containing 1 µM Pb(NO3)2 and 1 µM Ca2+ ionophore ionomycin (Sigma) incubated at 37°C for 30 min and 5 min, respectively. For negative control, cells were incubated for 30 min at 37°C with vehicle alone. Subsequently, Ca2+-dependent fluorescence intensity was measured in fluorescence channel FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. To test for an interaction between Pb2+ and fluo-3, the fluorescence dye (4 µM fluo-3) was incubated with increasing concentrations of Pb2+ (and for positive control Ca2+; 102 to 109 M total divalent cations) and fluorescence intensity was measured using photometry. In these experiments, Pb2+ did not induce changes in fluo-3 fluorescence intensity (data not shown), while Ca2+ did.
Statistics. Data are expressed as arithmetic means ± SE, and statistical analysis was performed using paired or unpaired t-test or ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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In cell-attached mode, Pb2+ (10 µM) increased the macroscopic inward and outward currents when recorded with a K+-containing pipette solution (Fig. 4, D and E) but only outward currents when recorded with a Na+-containing pipette solution (Fig. 4, F and G). This further indicated K+ selectivity for the Pb2+-stimulated current. The Pb2+ (10 µM)-stimulated whole cell current fraction in KCl bath solution amounted to 15 ± 4 and +10 ± 3 pA (n = 8) at 100 and +80 mV, respectively (differences between open and solid symbols in Fig. 4C). In the cell-attached mode, the Pb2+ (10 µM)-stimulated outward and inward current fractions (when recorded with K+ in the pipette) were +8.6 ± 2.2 and 9.0 ± 2.2 pA (n = 4) at +100 and 100 mV, respectively (differences between open and solid bars in Fig. 4E).
To explore the functional significance of K+ channel activation for the Pb2+-induced phosphatidylserine exposure and cell shrinkage, cells were incubated in an elevated (125 mM) extracellular K+ concentration to dissipate the K+ gradients and thus to impede K+ exit after opening of the K+ channels. In addition, the Ca2+-sensitive K+ channel blocker clotrimazole (2 µM) was applied to cells suspended in NaCl solution. Increase of extracellular K+ concentration to 125 mM and to a lesser extent clotrimazole both significantly inhibited the increase of annexin binding after exposure to Pb2+ (1 µM; Fig. 5, A and B).
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DISCUSSION |
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The effect of Pb+ ions was paralleled by erythrocyte shrinkage, another typical feature of apoptotic cell death (32). The cell shrinkage apparently was secondary to cellular K+ loss, because it was reversed by an increase in extracellular K+ concentration, which dissipated the chemical driving force for K+ exit. Moreover, the Pb2+-induced cell shrinkage was blunted by clotrimazole, an inhibitor of the Ca2+-sensitive Gardos K+ channels (7, 12). In theory, hyperpolarization of erythrocyte membrane potential after Gardos K+ channel activation might be a direct determinant of phosphatidylserine exposure. However, similarly to clotrimazole, anion channel inhibitors blunt the ionomycin-stimulated phosphatidylserine exposure in human erythrocytes (34). Because anion channel inhibitors increase Gardos K+ channel-mediated hyperpolarization but decrease cell shrinkage, hyperpolarization does not seem to directly stimulate the phosphatidylserine exposure.
Pb2+ may activate K+ channels by increasing cytosolic Ca2+ activity. On the basis of fluo-3 fluorescence measurements, Pb2+ increased cytosolic Ca2+ activity, an effect that may be secondary to Pb2+-induced cell shrinkage (27), activation of a Ca2+ channel not visible in patch-clamp experiments, or inhibition of Ca2+-ATPase (5, 24, 37). Ca2+ in turn is suggested to further activate Ca2+-sensitive Gardos K+ channels that amplify the Pb2+ signal in intact cells but not during whole cell recording. Beyond its effect on cytosolic Ca2+ activity, Pb2+ may activate Ca2+-sensitive K+ channels even without an increase in intracellular free Ca2+ concentration. Previous patch-clamp (23, 43) and tracer flux experiments (44) demonstrated that Pb2+ activates Ca2+-sensitive K+ channels in human erythrocytes in the absence of free Ca2+, and it has been concluded that Pb+ ions activate Gardos K+ channels by more direct interaction. In particular, Pb2+ has been shown to act at the intracellular face of the K+ channel protein (13), and band 3 has been proposed to transport Pb2+ as Pb(CO3)2 or ternary Pb(CO3)2-anion complex across the erythrocyte membrane by anion exchange (46). Cytosolic Pb2+ activates Gardos K+ channels at subnanomolar threshold concentrations in ATP-depleted, hemoglobin- and Pi-free resealed ghosts (44). Binding to hemoglobin (as well as the formation of phosphate complexes) has been estimated to decrease the cytosolic free Pb2+ concentration by a factor of 6,000 (45). According to this estimate, the threshold concentration for Gardos K+ channel activation should increase to submicromolar concentrations in intact erythrocytes. The present study did not reveal Pb2+-induced cell shrinkage and phosphatidylserine exposure at Pb2+ concentrations less than 0.3 µM. Activation of K+ channels after relatively short exposure during patch-clamp experiments required an extracellular Pb2+ concentration of 10 µM.
Increasing extracellular K+ concentration and inhibitors of K+ channels not only reversed the Pb2+-induced cell shrinkage but also significantly interfered with Pb2+-induced phosphatidylserine exposure. As reported recently (4, 11, 15), activation of the erythrocyte K+ channels and subsequent cell shrinkage participate in the triggering of scramblase activation. The sensitivity of erythrocytes to cellular K+ loss is similar to that observed in other cell types. Cellular loss of K+ has been demonstrated to parallel and support apoptosis of a variety of nucleated cells (8, 9, 21, 29, 30, 38, 41).
Further mechanisms involved in the stimulation of erythrocyte apoptosis after Pb+ ion exposure may include decreased cytosolic ATP concentration, because energy depletion was previously shown to trigger erythrocyte apoptosis and Pb+ ion exposure is known to decrease erythrocyte ATP concentration (2, 22).
In conclusion, exposure to Pb+ ions at concentrations relevant for toxicity activates K+ channels, leading to K+ loss and erythrocyte shrinkage and favoring phosphatidylserine exposure at the surface of the cell membrane. The affected erythrocytes are prone to being cleared from circulating blood, which presumably contributes to the decrease in erythrocyte life span and the development of anemia after Pb+ intoxication.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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