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TOXICOLOGY
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina (Z.S.B., D.J.J., D.C.M.); and Department of Medicine, Vanderbilt University, Nashville, Tennessee (J.D.M.)
Received for publication
March 17, 2005
Accepted
April 13, 2005.
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Abstract |
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). Primaquine, the prototype 8-aminoquinoline tissue schizontocide, is effective against all four malarial species that infect humans and is the only drug approved for the radical cure of relapsing malaria. As resistance has developed from extensive use of blood schizontocides, such as chloroquine, the importance of primaquine in combating multiple drug resistance has increased, and it is now used in combination with several blood schizontocides for the treatment and prophylaxis of all forms of malaria, including the most lethal malarial species, Plasmodium falciparum (Shanks et al., 2001). Primaquine is also effective in the treatment of Pneumocystis carinii pneumonia, a common infection in AIDS patients (Toma et al., 1998). The clinical utility of primaquine, however, is restricted by its hemotoxicity, particularly in patients who are genetically deficient in erythrocytic glucose-6-phosphate dehydrogenase activity (Beutler, 1990).
The hemolytic activity of primaquine has long been known to be due to an intraerythrocytic oxidative stress that is mediated by redox-active metabolites rather than by the parent drug itself. The oxidatively damaged erythrocytes are thought to be recognized by splenic macrophages as the equivalent of senescent red cells, resulting in their selective removal from the circulation (Rifkind, 1966). 5-Hydroxyprimaquine (5-HPQ) is a putative human metabolite of primaquine that forms a redox pair with its quinoneimine form; continuous cycling of this redox pair is thought to generate reactive oxygen species (ROS) within erythrocytes (Vasquez-Vivar and Augusto, 1992). We have shown that when 5-HPQ is incubated with rat red cells in vitro, damage occurs within the cells such that they are removed rapidly from the circulation of isologous rats as compared with untreated controls (Bowman et al., 2004). Although it is clear that ROS attack on the cytosolic surface of the membrane leads to a signal for removal of damaged erythrocytes from the circulation, neither the nature of the crucial target(s), whether lipid or protein, nor the mechanism of transfer of the signal across the membrane is known.
The mechanism(s) by which senescent or damaged (but intact) erythrocytes are selected for splenic sequestration are not known. Two current hypotheses are 1) that red cell damage is analogous to the apoptotic response observed in other (nucleated) cell types, resulting in changes to the normal asymmetric distribution of phospholipids (Mandal et al., 2002); and 2) that protein oxidation interferes with normal protein-protein interactions among the cytoskeletal, integral membrane, and cell surface proteins that confer the recognition of "self" (Oldenborg et al., 2000; Bruce et al., 2003). The present study examines the role of the postulated truncated apoptotic pathway in the hemolytic activity of 5-HPQ, specifically in regard to potential roles for membrane lipid peroxidation and loss of phospholipid (i.e., phosphatidylserine) asymmetry. We also address the alternate hypothesis, that alterations to skeletal membrane proteins in 5-HPQ-damaged red cells provide a crucial link between internal oxidative stress and external recognition and removal of erythrocytes from the circulation.
The data indicate that under in vitro incubation conditions that provoke premature splenic sequestration of red cells in vivo, 5-HPQ generates intracellular ROS but does not induce lipid peroxidation or cause loss of phosphatidylserine asymmetry in the plasma membrane of the red cell. In contrast, profound alterations occur to certain proteins of the cytoskeleton in 5-HPQ-treated erythrocytes, and these alterations are due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. Further studies are warranted to determine the specific signal for erythrocyte phagocytosis; however, the present data are consistent with the concept that cytoskeletal protein damage, rather than alterations in the lipid bilayer, underlies the process of splenic uptake of red cells damaged during the course of primaquine therapy.
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Materials and Methods |
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). Bovine factor V, bovine factor Xa, and bovine prothombin were obtained from Enzyme Research Laboratories (South Bend, IN). Chromogenic substrate S-2238 was purchased from Chromogenix (DiaPharma Group, Inc., Westchester, OH). AlexaFluor 647-conjugated annexin V and 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) were obtained from Molecular Probes, Inc. (Eugene, OR). Calcium ionophore A23187
[GenBank]
(calcimycin), N-ethylmaleimide (NEM), cumene hydroperoxide (CH), and rabbit anti-rat hemoglobin antibodies were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of the highest grade commercially available. Erythrocytes and Incubation Conditions. Male Sprague-Dawley rats (75–100 g) were obtained from Harlan Laboratories (Indianapolis, IN) and maintained on food and water ad libitum. Animals were acclimated for 1 week to a 12-h light/dark cycle before their use. Blood was collected from the descending aorta of anesthetized rats into heparinized tubes and washed three times with Hanks' balanced salt solution with calcium (HBSS; Invitrogen, Carlsbad, CA) to remove the plasma and buffy coat. The cells were resuspended to a 40% hematocrit in HBSS and used on the same day they were collected. Erythrocytes were washed three times with HBSS and resuspended to a 40% hematocrit.
Stock solutions of 5-HPQ in argon-purged water were prepared to deliver the appropriate concentration of 5-HPQ in 10 µl to erythrocyte suspensions (1–2 ml). The erythrocyte suspensions (40% hematocrit) were allowed to incubate for up to 2 h at 37°C and then washed once with HBSS prior to biochemical analyses.
Measurement of ROS Formation in Erythrocytes. Rat erythrocytes were suspended in isotonic phosphate-buffered saline (pH 7.4) supplemented with 10 mM D-glucose to a 10% hematocrit. DCFDA (600 µM), dissolved in dimethyl sulfoxide, was added to the erythrocyte suspension and allowed to incubate for 15 min at 37°C. Immediately after the addition of various concentrations of 5-HPQ, fluorescence was measured at 2-min intervals for 20 min (excitation of 488 nm, emission of 529 nm) on a Molecular Devices SprectraMAX Gemini XS Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA).
Preparation of Erythrocyte Membrane Ghosts. Red cell ghosts were prepared from vehicle- and 5-HPQ-treated red cells as described previously with modification (Grossman et al., 1992). Briefly, washed red cells were centrifuged, and the packed cells were lysed in 30 ml of ice-cold phosphate buffer (5 mM, pH 8.0). The membrane ghosts were pelleted by centrifugation at 20,000g for 10 min. The supernatant was removed by aspiration, and the ghosts were repeatedly washed with phosphate buffer until the control cells yielded white ghosts, typically three to four washes.
Determination of Lipid Peroxidation in Erythrocytes. Lipid peroxidation was assessed by measuring the content of F2-isoprostanes in red cell ghosts prepared anaerobically from vehicle- and 5-HPQ-treated red cells as described previously (Bolchoz et al., 2002b). Briefly, the lipids were extracted from membrane ghost suspensions (250 µl) with chloroform/methanol (2:1, v/v). The extracted lipids were then subjected to alkaline hydrolysis to release the esterified F2-isoprostanes. The work-up procedure for quantifying F2-isoprostanes by GC/MS is described in detail elsewhere (Morrow and Roberts, 1999). Treatment of red cell suspensions for 1 h at 37°C with the lipid-soluble peroxide cumene hydroperoxide (1 mM) was used as a positive control for lipid peroxidation (van den Berg et al., 1992).
NEM and Calcium Ionophore Treatment of Erythrocytes. As a positive control for translocation of phosphatidylserine from the inner to the outer leaflet of the lipid bilayer, rat erythrocytes were treated with NEM to inhibit aminophospholipid translocase, and calcium ionophore A23187
[GenBank]
was added to induce membrane lipid scrambling as described previously (Kuypers et al., 1996). Briefly, rat erythrocytes were washed three times with HBSS (with calcium) and reconstituted to 40% hematocrit with HBSS. The red cell suspension (1 ml) was allowed to incubate with 10 mM NEM for 30 min at 37°C and then washed twice with HBSS. Calcium ionophore A23187
[GenBank]
(4 µM) in dimethyl sulfoxide was then added and allowed to incubate for 1 h at 37°C. After incubation, the red cells were washed with 2.5 mM EDTA (1 ml) to remove the calcium and then washed twice more with calcium-free HBSS containing 1% bovine serum albumin to remove the ionophore. The cells were then resuspended in 1 ml of annexin binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) and analyzed for annexin V labeling and prothrombinase activity as described below.
Determination of Phosphatidylserine Translocation in Erythrocytes. Erythrocyte labeling with annexin V was performed according to a modification of a flow cytometric technique (Kuypers et al., 1996). Following incubation with the vehicle, the positive control, or 5-HPQ (2 h at 37°C), aliquots (50 µl) of the suspensions were removed and washed twice with annexin binding buffer (1 ml). After the last wash, aliquots of the packed red cells (2 µl) were combined with annexin binding buffer (997 µl), and AlexaFluor 647-conjugated annexin V solution (1 µl) was added to the erythrocyte suspensions to bring the total volume to 1 ml. After a 30-min incubation in the dark at room temperature, the samples were washed and resuspended in annexin binding buffer (1 ml) and analyzed on a Becton Dickinson FACSCalibur analytical flow cytometer (BD Biosciences, San Jose, CA).
Phosphatidylserine translocation was also assessed by measuring the conversion of prothrombin to thrombin using a modification of a method described previously (Kuypers et al., 1996). Briefly, after incubation with the vehicle or 5-HPQ, erythrocyte suspensions were washed once with HBSS, and 1 µl of packed cells was added to 1 ml of Tris buffer (pH 7.4) at 37°C. Bovine factor V (0.33 U/ml) and bovine factor Xa (0.33 U/ml) were added, followed by 0.13 mg of prothrombinase. After 4 min, the reaction was quenched with 15 mM EDTA. The erythrocytes were pelleted by centrifugation, and 75 µlof the supernatant was added to 1 ml of chromogenic substrate S-2238 working solution. The increase in absorbance at 405 nm was determined over 1 min at 37°C in a Shimadzu UV-160 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan) equipped with software for enzyme kinetics. The amount of thrombin formed per unit of time was determined by comparison with thrombin standards.
Electrophoretic Analysis of Membrane Cytoskeletal Proteins. Red cell ghosts from control and 5-HPQ-treated erythrocytes were washed exhaustively to remove unbound hemoglobin (four washes). The ghost protein was then solubilized in NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA), with or without 40 mM dithiothreitol (DTT), and heated at 70°C for 10 min. Solubilized proteins (15 µg) were loaded and resolved on 4 to 12% NuPAGE Bis-Tris gels with MOPS Running Buffer (Invitrogen) at 200 V (constant) for 50 min. Resolved proteins were transferred to PVDF membranes for immunoblot analysis according to the Invitrogen protocol. For SDS-PAGE analysis, solubilized membrane ghosts were resolved on continuous gels under nonreducing conditions as described previously (McMillan et al., 1995), then stained with Gel Code Blue Stain Reagent (Pierce, Rockford, IL), and destained with water.
Rat red cell membrane proteins were identified according to molecular weight (Fairbanks et al., 1971) with the use of molecular weight protein standards (Invitrogen). Blotted proteins were blocked in Tris-buffered saline/Tween 20 (pH 7.5) containing 5% nonfat dry milk and incubated in Tris-buffered saline/Tween 20 containing 1% bovine serum albumin and primary antibody (rabbit anti-rat hemoglobin, 1:10,000, v/v). After washing and incubation with the peroxidase-conjugated secondary antibody (anti-rat IgG), the immunoblots were developed using enhanced chemiluminescence detection (Amersham Biosciences, Piscataway, NJ).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). To assess the formation of ROS in 5-HPQ-treated rat erythrocytes, erythrocyte suspensions were preincubated with DCFDA for 15 min followed by the addition of 5-HPQ. Fluorescence was then measured at 2-min intervals for 20 min using a fluorescence microplate reader. Preliminary experiments indicated that the concentration of red cells in the suspensions had to be reduced from a 40 to a 10% hematocrit to avoid significant quenching of the fluorescence signal by the cells. 5-HPQ concentrations were reduced accordingly (by 4-fold) to maintain the same 5-HPQ-to-red cell ratio that was used in the other experiments.
As shown in Fig. 1, incubation of erythrocytes with DCFDA in the absence of 5-HPQ resulted in a slight but measurable increase in fluorescence over a 20-min time period, consistent with the known low-level, steady production of ROS in normal red cells (Jandl et al., 1960). Inclusion of 5-HPQ (5–30 µM) in the erythrocyte suspension caused a marked and concentration-dependent increase in the generation of fluorescence, indicating enhanced production of ROS by 5-HPQ. This observation is consistent with previous observations, which showed that 5-HPQ has the ability to redox cycle and to cause oxidative stress in rat erythrocytes, as evidenced by glutathione oxidation and methemoglobin formation (Bowman et al., 2004). No fluorescence signal was detected in the absence of red cells or in the absence of DCFDA (data not shown).
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). The 2-h incubation time was chosen because this time period was used in an earlier study to document the hemolytic activity of 5-HPQ (Bowman et al., 2004), and it was important to determine the state of erythrocyte lipids and proteins under the same incubation conditions that were used to measure hemolytic activity. The red cells were then washed and lysed in argon-purged hypotonic buffer for the preparation of red cell ghosts, as described previously (Grossman et al., 1992). Esterified isoprostanes were extracted from the ghosts and hydrolyzed, and the total F2-isoprostane content in the samples was quantified by GC/MS (Morrow and Roberts, 1999). As shown in Fig. 2, F2-isoprostane content in 5-HPQ-treated incubates was not different from that of control incubates, regardless of the concentration of 5-HPQ. In contrast, treatment of red cells with the positive control cumene hydroperoxide (CH; 1 mM) resulted in a 3-fold increase in F2-isoprostane formation. Treatment with this concentration of CH was the lowest that produced a significant elevation in F2-isprostane formation that was not associated with significant frank lysis of the erythrocytes; concentrations of CH greater than 1 mM caused progressive and complete red cell lysis (data not shown). Effect of 5-HPQ on Red Cell Membrane Phospholipid Asymmetry. To determine whether exposure to 5-HPQ resulted in a loss of phosphatidylserine asymmetry, the transmembrane location of phosphatidylserine was assessed by two methods: enhancement of prothrombinase activity and annexin V labeling of intact erythrocytes.
Erythrocyte suspensions (40%) were incubated with hemolytic concentrations of 5-HPQ (10–70 µM) for 2 h. At the end of the incubation, the red cells were washed, resuspended in HBSS, and analyzed for prothrombinase activity, which is dependent on cell surface exposure to phosphatidylserine (Bevers et al., 1982). For a positive control, erythrocytes were treated with 10 mM NEM for 30 min, followed by treatment with 4 µM calcium ionophore A23187
[GenBank]
in the presence of calcium for 1 h. As shown in Fig. 3A, thrombin formation in 5-HPQ-treated erythrocytes was not significantly different from the control. In contrast, the positive control, NEM plus ionophore and calcium, induced an 8-fold increase in prothrombinase activity compared with the control.
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The presence of phosphatidylserine in the outer leaflet of the lipid bilayer was confirmed by direct labeling of erythrocytes with fluorescently conjugated annexin V, which binds to acidic phospholipids, particularly phosphatidylserine (Kuypers et al., 1996). As shown in Fig. 3B, annexin-positive erythrocytes from 5-HPQ-suspensions constituted less than 2% of the red cells, which was similar to the vehicle-treated control. In contrast, treatment of red cells with NEM plus ionophore and calcium resulted in labeling of about 45% of the erythrocytes.
Effect of 5-HPQ on Rat Erythrocyte Membrane Skeletal Proteins. To determine whether 5-HPQ-induced hemolytic activity was associated with alterations to the membrane cytoskeleton, rat erythrocyte ghost proteins from control and 5-HPQ-treated erythrocytes were separated by SDS-PAGE and either stained with Gel Code Blue or transferred to PVDF membranes and immunostained with antibodies to rat hemoglobin. As shown in Fig. 4A, 5-HPQ caused concentration-dependent changes to the normal electrophoretic pattern of erythrocyte skeletal proteins compared with that of the vehicle-treated control. 5-HPQ treatment induced the appearance of new protein bands at 16, 32, and 64 kDa, which is consistent with formation of membrane-bound hemoglobin monomers, dimers, and trimers, respectively (Grossman et al., 1992). In addition, there was a concentration-dependent loss of resolution of protein bands 1 and 2 (
and 
spectrins), splitting of band 2.1 (ankyrin), and loss of band 4.2. On the other hand, band 4.1 and band 5 (actin) appeared not to be affected significantly by treatment with 5-HPQ.
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Discussion |
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Previous studies showed that the phenolic metabolite 5-HPQ has the capacity to redox cycle and generate ROS, suggesting that it could contribute to the hemotoxicity of the parent drug (Vasquez-Vivar and Augusto, 1992). We recently demonstrated the hemolytic potential of 5-HPQ by showing that it is a direct-acting hemolytic agent in rats (Bowman et al., 2004). In these studies, 51Cr-tagged erythrocytes were treated with 5-HPQ in vitro then re-administered to rats. The treated cells were rapidly removed from the circulation as compared with saline-treated controls. The concentration dependence of this response was sharp, with a minimum response at 25 µM, an EC50 at 40 µM, and a maximal response at 75 µM. Additionally, hemolytic activity was strongly correlated with glutathione depletion and formation of glutathione-protein mixed disulfides. These observations were similar to the pattern of oxidative stress-induced damage observed previously in red cells exposed to other directacting, pro-oxidant hemolytic agents, such as dapsone hydroxylamine (Bradshaw et al., 1997). However, the precise intracellular molecular targets of oxidative damage have not been identified nor has the mechanism whereby internal oxidative damage is translated to external cell-surface marker(s) that enable splenic macrophages to recognize and remove damaged red cells.
Since mature erythrocytes lack organelles, lipids, and proteins of the plasma membrane are the most logical targets of intracellular ROS. In regard to lipids, it is known that they are prime targets for ROS-initiated lipid peroxidation, which is thought to result in membrane blebbing and cell lysis. Additionally, oxidative stress was shown to alter the asymmetric distribution of phospholipids within the bilayer (Jain, 1984). A functional role for this asymmetry was demonstrated when it was shown that phosphatidylserine externalization resulted in increased susceptibility of erythrocytes to phagocytosis (Zwaal and Schroit, 1997).
More recently, studies on the normal removal of senescent erythrocytes have suggested that they undergo a process analogous to apoptosis. Human erythrocytes treated with t-butylhydroperoxide were shown to activate caspase 3 and induce phosphatidylserine externalization (Mandal et al., 2002). Additional studies showed that peroxidation of phosphatidylserine in cells undergoing apoptosis causes a reduced affinity of aminophospholipid translocase for the oxidized phospholipid, resulting in the accumulation of phosphatidylserine in the outer leaflet (Tyurina et al., 2000). Other investigators have found no association between oxidative stress and loss of erythrocyte phospholipid asymmetry (de Jong et al., 1997).
An alternate hypothesis is that erythrocyte proteins are critical targets of intracellular ROS. Previous studies showed that hemoglobin and cytoskeletal proteins of the membrane cytoskeleton are altered in response to prooxidant hemolytic agents, which is reflected by the generation of reactive forms of hemoglobin, such as ferrylhemoglobin (Bolchoz et al., 2002a) and hemoglobin thiyl radicals (Bradshaw et al., 1995), and by alterations to the mobility of skeletal proteins on nonreducing SDS-PAGE gels (Grossman et al., 1992; McMillan et al., 1995, 2001). Evidence for the importance of normal protein-protein interactions among cytoskeletal, integral membrane, and external cell surface proteins is illustrated by recent studies showing that these interactions are necessary to confer the recognition of self to circulating erythrocytes (Oldenborg et al., 2000; Bruce et al., 2002).
The present studies were undertaken to determine the effect of the primaquine metabolite 5-HPQ on the generation of ROS in erythrocytes and on the consequences of ROS generation to erythrocyte membrane lipids and proteins. Given the association between 5-HPQ-induced oxidative stress and its hemolytic activity, we analyzed rat erythrocyte lipids for peroxidation and loss of phosphatidylserine asymmetry, and skeletal proteins for electrophoretic alterations and hemoglobin binding under experimental conditions associated with generation of intracellular ROS.
DCFDA is a fluorescence-based probe developed to detect intracellular production of ROS. DCFDA diffuses passively into cells where it is deacetylated to form 2',7'-dichlorodihydrofluorescein. 2',7'-Dichlorodihydrofluorescein is oxidized to a fluorescent product, 2',7'-dichlorofluorescein, by ROS, thus providing a general assessment of intracellular oxidative stress (Hempel et al., 1999; Halliwell and Whiteman, 2004). The data presented in Fig. 1 show a concentration-dependent increase in DCFDA oxidation in rat erythrocytes treated with 5-HPQ, indicating that ROS formation is associated with the hemolytic response in both a concentration- and time-dependent manner. DCFDA oxidation continued for at least 20 min, which is of interest because 5-HPQ has an extremely short half-life (<1 min) in erythrocyte suspensions (Bowman et al., 2004). Although the reason for this relatively long generation of ROS relative to 5-HPQ stability is not understood, it suggests that some unknown reactive intermediate with a longer half-life is generated in erythrocytes under hemolytic conditions.
To assess whether lipid peroxidation was associated with 5-HPQ-induced hemolytic injury, the formation of F2-isoprostanes was quantified in erythrocyte ghosts by GC/MS. Treatment with a range of concentrations of 5-HPQ, from subhemolytic (10 µM) to maximally hemolytic (70 µM), did not reveal any increase in F2-isoprostanes content in 5-HPQ-treated erythrocytes compared with the control (Fig. 2). In agreement with the lipid peroxidation data, analysis of phosphatidylserine externalization (Fig. 3) supported the concept that erythrocyte membrane lipids are not targets of 5-HPQ-generated ROS and suggests that phosphatidylserine exposure on the external cell surface does not occur and thus is not a signal for uptake of 5-HPQ-treated erythrocytes into the spleen.
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) and divicine (McMillan et al., 2001). The process by which hemoglobin becomes bound to the membrane cytoskeleton is not well understood but is thought to arise from ROS-induced oxidation of hemoglobin free-sulfhydryl groups to thiyl free radicals (Bradshaw et al., 1995), dissociation of the normal tetramer, and recombination of oxidized monomers via disulfide bond formation into polymeric forms that can be reduced back to monomers by treatment with DTT (Grossman et al., 1992).
The disappearance of band 4.2 was perhaps the most intriguing effect. Band 4.2 has been shown to be a member of the band 3 complex, which includes band 3, band 2.1 (ankyrin), and CD47 (Bruce et al., 2003). Band 3 is an anion transporter protein that is anchored to the underlying cytoskeleton through its interaction with ankyrin. The extracellular domain of CD47 acts as a ligand for inhibitory signal regulatory protein on splenic macrophages (Oldenborg et al., 2001). As noted above, this interaction is thought to confer self-recognition to normal erythrocytes, preventing their phagocytic removal (Oldenborg et al., 2000). These findings are of relevance to the hemolytic response because other studies have shown that band 4.2 forms a vertical association between CD47 and ankyrin, thereby mediating cell-surface expression of CD47 and its membrane skeleton attachment in human erythrocytes (Dahl et al., 2004). Although the fates of band 4.2 and ankyrin are not clear from these experiments, immunoblotting with anti-rat hemoglobin showed that significant amounts of hemoglobin are bound to multiple proteins of the band 3 complex (Fig. 4B). Treatment of erythrocyte ghosts with the disulfide-reducing agent DTT indicated that covalent binding of hemoglobin to these proteins occurred through the formation of intermolecular disulfide bonds (Fig. 5).
Our working hypothesis for 5-HPQ-induced hemolytic anemia is shown in Fig. 6. This scheme illustrates the preferential attack of ROS on hemoglobin as a consequence of 5-HPQ redox cycling. As noted above, attack of ROS on hemoglobin may result in the formation of hemoglobin thiyl radicals. Thiyl radicals are highly reactive, but the globin chain lacks sufficient lipophilicity to penetrate into the membrane to initiate lipid peroxidation. Thiyl radicals instead react with free sulfhydryl-groups on skeletal proteins to form disulfide-linked adducts (Jollow and McMillan, 2001). Additional studies are necessary to determine whether thiyl radicals or other reactive species can be detected in response to treatment with 5-HPQ. Additional studies are also warranted to determine the functional importance of hemoglobin adduct formation; however, it is plausible that binding of hemoglobin to the band 3 complex would lead to disturbances in cytoskeletal protein-protein interactions that are transmitted to the external surface of the erythrocyte, providing a signal for macrophage recognition.
In summary, we have shown that a hemolytic metabolite of primaquine, 5-HPQ, is not associated with lipid peroxidation or phosphatidylserine translocation despite the generation of ROS. Rather, the data are consistent with the concept that cytoskeletal protein damage, in the form of disulfide-linked hemoglobin adducts, underlies the removal of damaged erythrocytes. Additional studies are warranted to elucidate the signal that underlies the loss of self-recognition.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HPQ, 5-hydroxyprimaquine; ROS, reactive oxygen species; DCFDA, 2',7'-dichlorodihydrofluoresein diacetate; NEM, N-ethyl maleimide; CH, cumene hydroperoxide; HBSS, Hank's buffered saline solution; GC/MS, gas chromatography-mass spectroscopy; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis.
Address correspondence to: Dr. David C. McMillan, Dept. of Cell and Molecular Pharmacology, Med. Univ. South Carolina, 173 Ashley Ave., Charleston, SC 29425. E-mail: mcmilldc{at}musc.edu
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), 5 µM(
), 15 µM (
), and 30 µM (
) 5-HPQ. Fluorescence emission intensity was monitored on a fluorescence microplate reader (excitation, 488 nm; emission, 529 nm) at 2-min intervals. The values are means ± S.D. (n = 3).
