Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells

Free RadicalBiology& Medicine,Vol. 18, No. 4, pp. 655-667, 1995 Copyright © 1995ElsevierScienceLtd Printed in the USA.All rightsreserved 0891-5849/95 $9.50 + .00

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- ~ OriginalContribution INHIBITION BY ACTIVATED NEUTROPHILS OF THE Ca 2÷ PUMP ATPase OF INTACT RED BLOOD CELLS

TROY T. ROHN, THOMAS R. HINDS, and FRANK F. VINCENZI Department of Pharmacology, University of Washington, Seattle, WA, USA (Received 23 March 1994; Revised 6 June 1994; Re-revised and accepted 29 July 1994)

Abstract--Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentationof the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca~+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2÷ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2÷pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derivedsuperoxide,dismutation of superoxide, to H202, diffusion of H202 into RBCs, a Fenton type ~eaction between oxyhemoglobin, and H202 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2÷ pump ATPase. Keywords---Rheumatoid arthritis, Ca2+pump ATPase, Neutrophils, Protein fragmentation,Reactive oxygen species, Free radicals

INTRODUCTION

mally not found in most biological fluids. However, free iron has been detected in synovial fluid, ~2 which may lead to the production of hydroxyl radical in the presence of H202, via the Fenton reactionJ 3 Synovial fluid of rheumatoid patients contains products of lipid peroxidation that correlate with disease severity as measured clinically and in the laboratory. ~2 O n the basis of these observations, it was hypothesized that in inflammation, such as RA, release of ROS by PMNs may damage the plasma membranes of nearby parenchymal (and circulating) cells, thereby causing an accumulation of intracellular calcium (Ca0. Some inflammatory processes are mediated by increased Cai,t4 and there is a great deal of evidence that chronically increased Ca~ is a final c o m m o n pathway in cell injury and death. 15'16 Cells have evolved mechanisms for removal of Ca 2÷, including a plasma m e m b r a n e Ca 2÷ p u m p A T P a s e ) 7 Previous work has shown that free radicals may promote inhibition of the plasma m e m brane Ca 2÷ pump A T P a s e J s-2° W e recently reported that tert-butyl hydroperoxide inhibits the Ca 2÷ p u m p

Neutrophils, or polymorphonuclear leukocytes (PMNs), play a critical role in host defense. W h e n activated, PMNs produce a variety of reactive oxygen species (ROS) t'2 that have been shown to induce methemoglobin formation, 3 lipid peroxidation,4 and hemolysis5"6of intact RBCs as well as inhibition of both the Na+/K + pump ATPase ~ and the Ca 2+ pump ATPase of cardiac sarcoplasmic reticulum. g It has been proposed that in m a n y inflammatory diseases, such as rheumatoid arthritis (RA), the causative agents are ROS, including superoxide anion (O2"-), H202, and the very reactive hydroxyl radical (HO'). 2'9 In RA, synovial fluid of inflamed joints contains many PMNs and macrophages. These phagocytic cells release HOC1, 02"-, and H202.* Release of the latter two agents into a fluid with free iron promotes the production of hydroxyl radical and extensive tissue damage, l°'lt Free iron is nor-

Address correspondence to: Frank F. Vincenzi, Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. 655

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ATPase in intact RBCs as a result of oxidative stress and lipid peroxidation, which can be prevented by certain antioxidants or free radical scavengers including stobadine, 2~ and thiol-containing compounds such as dithiothreitol. 22 In this study, using activated neutrophils, we found inhibition of the Ca 2÷ pump ATPase and fragmentation of membrane proteins of intact RBCs. It is suggested that inhibition of the Ca 2÷ pump occurred as a result of protein fragmentation promoted by hydroxyl and/or ferryl type radicals. It is further suggested that inhibition of the plasma membrane Ca 2+ pump may represent one mechanism of cell injury promoted by activated neutrophils. MATERIALS AND METHODS

Reduced glutathione (GSH) was purchased from Boehringer Mannheim (Indianapolis, IN). Luciferin-luciferase was purchased from Coral Biomedical (San Diego, CA). Glutathione peroxidase (GSHpx) and A23187 were purchased from Calbiochem. Bovine superoxide dismutase (SOD), histopaque- 1077, 1,1,3,3 tetraethoxypropane, thiobarbituric acid, cytochrome-C Type HI (Cyt c) from horse heart, phenylmethylsulfonyl fluoride (PMSF), sodium azide, catalase, and phorbol 12-myristate 13-acetate (PMA) (stored at -20°C in either ethanol or dimethyl sulfoxide (DMSO), goat antimouse IgG (Fab specific) peroxidase conjugate, and nonfat dried milk were all purchased from Sigma (St. Louis, MO). Dextran T500 was purchased from Pharmacia (Uppsala, Sweden). Tetramethyl benzidine membrane peroxidase substrate (1 component) was purchased from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). Polyvinylidene difluoride (PVDF) membranes (0.2/~m) were purchased from Novel Experimental Technology (San Diego, CA). Monoclonai (mouse) anti plasma membrane Ca 2÷ pump ATPase antibody (IgG2a) (clone 5F10) was a gift from Dr. John Penniston. All other chemicals were analytical reagent grade.

Cell preparation Blood (35 ml) was obtained from healthy human subjects by venipuncture and collected in heparinized tubes. Neutrophils were prepared as previously described by Ficoll-Paque separation followed by dextran sedimentation. 23 Briefly, collected blood was centrifuged at 1,500 x g, and the resulting supernate aspirated, being careful not to remove the buffy coat. All centrifugation steps were performed at room temperature using a Sorvall table top GLC-2 centrifuge with a type HL-4 swinging bucket rotor. Cold 10 mM phosphate buffer, pH 7.4, containing 135 mM

NaC1 (PBS) was added to the suspension to a volume of 32 ml. Eight ml of this suspension was layered onto 4 ml of Ficoll-Paque (density 1.077 g/ml) in four capped polypropylene tubes. These tubes were then centrifuged at 1,500 x g for 35 min, and the resulting mononuclear leukocyte was band removed. The remaining PMNs and RBCs were resuspended in PBS, mixed thoroughly, and added to a tube containing 20 ml of 3% dextran in PBS. This tube was allowed to stand for 20 min, and the resulting supernate was carefully removed using a Pasteur pipette, collected into a 50 ml conical tube, and centrifuged at 1,500 x g for 10 min. Following this step, the supernate was removed and any remaining RBCs were lysed by hypotonic saline. Finally, the relatively pure suspension of PMNs was washed two more times in cold PBS. After the final wash, the pellet was resuspended in 200 #l of PBS supplemented with 5 mM glucose. Ten #l of this suspension was used to count PMNs using a Coulter counter with typical yields being 4 0 - 8 0 x l06 PMNs/ml. Autologous erythrocytes were washed three times in PBS at 2,000 x g for 5 min, and, subsequently, the packed cells ( 7 5 - 8 0 % hematocrit) were placed on ice. As with neutrophils, RBCs were also counted using a Coulter counter.

Preincubation Fifty microliters of packed RBCs (3.9 x 108 cells) were added to reaction vessels, containing PBS and PMNs, to a final volume of 1 ml. Except where noted, NaN3 was also added to a final concentration of 2 mM. When present, test drugs were added before PMNs. Incubations were initiated by the addition of PMA at a final concentration of 200 nM. Immediately after addition of PMA, the reaction vessels were placed in a Dubnoff shaking water bath at 37°C. At the conclusion of the preincubation period, samples were immediately centrifuged at 2,000 X g for 5 min. The supernate was removed by aspiration, and cells were rewashed in 2 ml of PBS and spun again. After removal of the supernate, the packed RBCs were stored on ice until assayed. It should be noted that in all reactions in which PMA was added it was apparent, upon completion of the preincubation period, that the neutrophils had aggregated. This allowed for easy separation of the clumped neutrophils from the RBCs, thus assuring that the packed cells consisted primarily of RBCs.

Assay of the RBCs

C a 2+

pump ATPase activity in intact

C a 2+ pump ATPase activity was determined as previously described. 24 Packed RBCs (20 /zl), preincu-

Neutrophil-induced inhibition of the Caz+ pump ATPase bated under various conditions, were added to 1.0 ml of buffer containing 20 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (HEPES) (pH 7.4), 140 m M KCI, 2 mM MgClz, 1 m M iodoacetic acid (IAA, freshly prepared), and 0.1 mM CaC12. After incubation for 2 min at 37°C, 10 #1 of A23187 in ethanol was added to the suspension to a final concentration of 3.8 #M at time zero. This results in a massive influx of Ca 2+ and "short-circuits" the Ca 2+ pump ATPase, which rapidly consumes ATP. 24 Incubation at 37°C was continued with removal of 20 #1 aliquots every 2 min for 10 min. Aliquots were immediately diluted in 1.0 ml of a lysing solution consisting of 0.5 mM MgSO4 in 10 mM Tris buffer (pH 7.75). Then, 15 #1 of the lysed aliquot of RBCs was added to 40 #1 of a solution containing luciferin-luciferase (Picozyme F) in 0.25 m M MgSO4 in 5 mM Tris buffer (pH 7.75) in a Packard luminometer (model 6112B). The ATP content was determined in duplicate at each time point. In the presence of A23187, ATP levels in RBCs (including RBCs exposed to activated neutrophils) quickly declined with pseudo first order kinetics. The data were fitted with a first-order equation from which the rate constant of the Ca z+ pump ATPase was estimated.

TBARS measurement Thiobarbituric acid-reactive substances (TBARS) were assayed as previously described. 2z Briefly, 800 #1 of preincubation solution was " s t o p p e d " by the addition of 400 #1 of 28% (w/v) of trichloracetic acid containing NaAsO2. Samples were mixed and centrifuged at 1,500 x g for 5 rain at 4°C in a Fisher microcentrifuge (model 235 B). Eight hundred microliters of the supernate from each of these samples was then combined with 200 #1 of 1% thiobarbituric acid (w/v) in 0.05 M N a O H for a final volume of 1.0 ml. B H T was also included in the reaction mixtures at a final concentration of 0.05% as previously described by Braughler et al. 2s Samples were boiled for 15 min in capped microcentrifuge tubes and cooled on ice, and absorbance at 535 nm was determined with quantification based upon a molar extinction coefficient of 1.54 x 105/M x cm obtained from standard curves generated using l, 1,3,3-tetraethoxypropane.

Determination of methemoglobin and oxyhemoglobin Methemoglobin and oxyhemoglobin were determined by the spectrophotometric method of Winterbourn with the absorbances of each sample determined at 560, 577, 630, and 700 nm. 26 All values were expressed per heme group.

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Superoxide generation Generation of superoxide was measured as the SOD-inhibitable reduction of Cyt c as described by Curnutte and Babior z7 with some modification. PMNs (1 X 106) were added along with Cyt c (0.75 mM) and catalase (1,000 units) into plastic microcentrifuge tubes. Catalase was included in order to prevent possible detrimental effects by H202 on neutrophil function. PBS was added to a final volume of 1 ml. Reactions were initiated by adding 200 nM PMA, then mixed and incubated at 37°C. At the conclusion of the incubation period, samples were immediately spun down at 4°C in a Fisher microcentrifuge (model 235 B). Samples were then placed on ice until assayed. The supernate of each sample was transferred to cuvettes, and the absorbance at 550 nm was determined. Samples were then transferred to new microcentrifuge tubes to which excess sodium dithionite was added to completely reduce the Cyt c. Tubes were mixed vigorously, and the absorbance of each sample was again determined at 550 nm. All readings were performed using a Gilford Spectrophotometer, model 2400. Quantification was expressed as moles of reduced Cyt c using the extinction coefficients of 8.4 x 103/M × cm for oxidized Cyt c, 2.95 × 104/]VI × cm for reduced Cyt c, and 2.11 x 104/M x cm for reducedoxidized Cyt c. 28 Thus: moles of Cyt c A _

,04)x 84 x 2.11 × 104

-" 1,000,

where A is the absorbance of the sample at 550 nm and B is the absorbance at 550 nm of the same sample treated with excess sodium dithionite.

Red blood cell membrane preparation Human RBC membranes were prepared as previously described z° with some modification. Membranes were prepared by hemolysis of 50 #I of washed, packed RBCs in 3 ml of imidazole (20 mM, pH 7.4) buffer with rapid mixing in centrifuge tubes. The hemolysate was centrifuged for 15 min at 17,000 x g, and the resultant supernate was removed by aspiration. This step was repeated two more times. At each centrifugation step, care was taken to remove the colored bottom that formed on the bottom of the membrane fraction. This button most likely consisted of methemoglobin (for those reactions run in the presence of

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activated neutrophils) and contaminating neutrophils. The membranes were finally washed with 3 ml (per original 50 #1 of packed RBCs) of 40 mM/40 mM histidine/imidazole buffer, pH 7.1. Membrane protein content was determined by the bicinchoninic acid method 29 using bovine serum albumin as a standard.

Catalase activity Catalase activity was determined spectrophotometrically by measuring the decomposition of H202 by catalase at 230 nm as described by Beutler. 3°

l

SDS-polyacrylamide gel electrophoresis (SDSPAGE) Sodium dodecyl sulfate-polyacrylamide slab gels were prepared according to the method of Laemmli 3~ using a Hoefer Scientific Instruments SE 250 mini-gel unit. A 10% polyacrylamide separating gel and a 4% polyacrylamide stacking gel were used for resolving proteins denatured with SDS. Gels were stained with Coomassie brilliant blue.

Western blot analysis Western blot analysis was performed as previously described 32 with some modification. SDS-PAGE was performed (each lane being loaded with 40 #g of total membrane protein) using a 4% stacking gel and a 7.5% separating gel. Following electrophoresis, the wet slab gels were placed on PVDF filter sheets, and the proteins were electroblotted (30 V, 6 0 - 2 0 0 mA) overnight at 4°C in a transfer buffer containing 8.6 g of glycine, 1.8 g of Tris base, 1 g of SDS, and 200 ml of methanol in a final volume of 1 liter. Blots were blocked with 3% wt/vol nonfat dry milk with 0.02% Tween 20. All washes were performed using PBS with 0.02% Tween 20. The first antibody, 5F10 monoclonal antibody to the plasma membrane Ca 2+ pump ATPase, was incubated for 1 h with agitation at room temperature. This antibody recognizes an epitope between amino acids 724-783 of the human erythrocyte Ca 2+ pump ATPase, located in the highly conserved hinge region on the intracellular loop between putative transmembrane domains M4 and M 5 . 33 Following four washes (5 min each) the second antibody was incubated with the blot for 1 h with agitation at room temperature. This antibody consisted of a horseradish-peroxidase conjugated goat antimouse IgG (Sigma). Following a washing step, peroxidase staining was developed using tetramethyl benizidine (TMB) Peroxidase substrate (component 1). Molecular weights of subsequent bands were ascertained using pre-stained SDS-PAGE stan-

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TIME (rain) Fig. 1. Typical raw data of the Ca 2+ pump ATPase assay in intact RBCs. Suspensions containing 3.0 × 108 RBCs and 4 x 106 PMNs without (11) or with 200 nM PMA ([23) were preincubated for 2 h at 37°C. Both suspensions also contained azide at a final concentration of 2 mM. Following preincubation, samples were washed 2x in cold PBS and the ATP content of the RBCs was assayed by luminometry. Data are shown as a semilogarithmic plot of the ATP content, normalized to time zero. In each case the data were fitted with an exponential, and the correlation coefficient was > 0.99. The slopes of the lines were taken as the pseudo first order rate constants (min ~) and, thus, as a measure of the activity of the Ca 2+ pump ATPase. 24

dards (broad range, Bio Rad). To determine protein transfer efficiency, unblocked blots were stained with Pelikan Fount India ink. 32

Statistical analyses To determine possible significant differences, analysis of variance was performed, followed by unpaired two-tailed Student t-tests between various data points. RESULTS

The effect of neutrophils on the of intact RBCs

C a 2+

pump ATPase

Exposure of normal RBCs to PMA-stimulated (activated) neutrophils resulted in inhibition of the Ca 2+ pump ATPase. Figure 1 shows typical raw data of an experiment with and without activation of the neutrophils. The loss of ATP was pseudo first order, and the

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Time (rain) Fig. 2. Time course of neutrophil-induced inhibition of the Ca2+ pump ATPase of intact RBCs. RBCs were preincubated with PMNs (4 × 10 6) and PMA (200 nM) in the absence ([~) or presence (11) of 2 mM azide for various times. The Ca2+ pump ATPase rate constant was then assayedas in Figure 1. Data represent two separate experiments and are expressed as the rate constant of the Caz+ pump ATPase as a function of time. Data represent the mean of three experiments, _+SEM.

solid lines represent the best fit equations for the data as previously described. 24 The disappearance of ATP was slower in RBCs preincubated with activated neutrophils. The rate constant for loss of ATP in RBCs preincubated with 4 x 106 PMNs, 200 nM PMA, and 2 mM azide was lower than that of control RBCs (absence of PMA). The respective rate constants were 0.144 rain-' and 0.282 min -1. Thus, there was a 50% inhibition of the Ca 2+ pump ATPase using a ratio of 97:1 RBCs to PMNs. There was no effect of PMA alone on the loss of ATP, and Ca 2+ pump ATPase rate constants for RBCs and PMNs were not significantly different from rate constants determined in samples with RBCs alone (data not shown). Inhibition of the RBC Ca 2÷ pump ATPase occurred only when PMA was added to the preincubation mixture containing neutrophils. Thus, the data were indicative of inhibition of the Ca 2÷ pump as a consequence of activation of the neutrophils. Concentrations of PMA higher than 200 nM did not result in greater inhibition of the Ca 2+ pump ATPase (data not shown). Inhibition of the Ca 2+ pump ATPase in intact RBCs was directly related to both the time of incubation and the neutrophil concentration. Figure 2 shows the time-

659

dependent inhibition of the Ca 2+ pump ATPase in RBCs by 4 × 106 PMNs. Inhibition did not increase after 2 h whether azide, a potent inhibitor of catalase, was present or not. Azide increased the degree of inhibition of the Ca 2+ pump ATPase. We determined (data not shown) that 2 mM azide gave the maximal increase in inhibition. At the end of a 2-h preincubation period, the average rate constant for ATP loss in the absence of azide was 0.211 min -1 and in the presence of azide (2 mM) was 0.174 min -~ (Fig. 2). Thus, there was an approximately 18% increase in the degree of inhibition of the Ca 2+ pump ATPase in samples preincubated in the presence of azide. For all other experiments, azide was included at a final concentration of 2 mM. In separate experiments, 2 mM azide completely inhibited catalase activity measured in RBC hemolysates (data not shown). In the presence of 2 mM azide and 200 nM PMA, inhibition of the Ca 2+ pump ATPase was dependent on PMN concentration (Fig. 3). Detectable inhibition (--10%) occurred at a ratio of 1560:1 RBCs to PMNs, and maximal inhibition of the Ca 2+ pump ATPase (approximately 50% inhibition) was observed at 97:1. Direct cell to cell contact was apparently not required for inhibition of the Ca 2÷ pump ATPase either in the presence or absence of azide. Cell suspensions were allowed to incubate in a homogenous manner with occasional mixing throughout the preincubation. No initial centrifugation of the samples to establish cell to cell contact was utilized as previously reported by Weiss. 3'5 In summary, inhibition of the Ca 2+ pump ATPase occurred only when PMA was added to the preincubation medium. Neither neutrophils nor PMA alone caused inhibition. Ca 2+ pump ATPase rate constants were typically slightly higher in the presence of PMNs and RBCs than in the presence of RBCs alone (data not shown). Incubation of RBCs with activated neutrophils resulted in both a concentration and time-dependent inhibition of the Ca 2+ pump ATPase, with maximal inhibition occurring after 2 h in the presence of 4 × 106 PMNs and 2 mM azide. Therefore, concentrations of 200 nM PMA, 2 mM azide, and 4 x 106 PMNs were used in the standard preincubation reaction for all further experiments. It should be noted that this standard preincubation reaction resulted in negligible hemolysis of RBCs (less than 0.05%, data not shown).

Time course of neutrophil-induced inhibition of the Ca 2÷ pump ATPase, superoxide production, lipid peroxidation, and oxidation of hemoglobin Addition of PMA to neutrophils in the absence of RBCs resulted in the immediate production of superox-

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and inhibition of the Ca 2+ pump ATPase occurred more gradually, reaching a maximum only after 2 h.

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Mechanism of inhibition of the Ca 2+ pump ATPase in intact RBCs

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PMNs (x 10^6) Fig. 3. Effect of PMN concentration on inhibition of the Ca 2+ pump ATPase of intact RBCs. Various concentrations of neutrophils were preincubated with 50 #1 of packed RBCs (3.9 × 10s), azide (2 mM), and P M A (200 nM) in a final volume of 1 ml at 37°C for 2 h. Following preincubation, Ca 2+ pump ATPase activity was determined as in Figure 1. Data represent the mean of three experiments, ___SEM.

ide as evidenced by the reduction of Cyt c (data not shown). In addition, incubation of RBCs with activated neutrophils also resulted in a decrease of oxyhemoglobin from 16.2 mmole/1 RBC to 3.6 mmole/1 RBC in 60 min and remained constant for up to 3 h. At the same time there was an increase in methemoglobin from 0 to 5.9 mmole/l RBC in 60 min, which also remained constant for up to 3 h (data not shown). Superoxide production by the activated neutrophils was measured by the reduction of cytochrome C, (Cyt c) as described in the methods. Reduced Cyt c increased in a hyperbolic fashion reaching a plateau of about 70 nmol by 60 min. The reduced Cyt c remained constant for an additional 60 min. As previously reported by Claster et al., 4 we were able to demonstrate the formation of TBARS in intact RBCs following preincubation with activated neutrophils. The formation of TBARS increased in a hyperbolic fashion. At 60 min about 6 /zmol/1 RBC were formed, and TBARS reached a plateau of about 10 pmol/1 RBC at 120 min and remained constant for 60 min more. Thus, it appeared that upon incubation of RBCs with activated neutrophil, superoxide increased, followed by the loss of oxyhemoglobin and increased methemoglobin. In contrast, the formation of TBARS

The role of neutrophil-derived ROS in hemoglobin oxidation and eventual inhibition of the Ca 2+ pump ATPase was examined. Because either superoxide or H 2 0 2 may react with oxyhemoglobin to form methemoglobin, 34'35 the potential role of these species was investigated by the addition of either a GSH/GSHpx system, SOD, or both, to preincubation mixtures. We chose to use GSH/GSHpx because azide, present in all experiments, is a potent inhibitor of catalase, but not GSH/GSHpx. 36 GSHpx in the presence of GSH is capable of reducing H202 t o H 2 0 . 37 Figure 4 presents results of an experiment in which RBCs were preincubated with activated neutrophils in the presence of various agents for 2 h. Panel A shows the C a 2+ pump ATPase rate constants obtained from such an experiment. As shown, typical inhibition of the C a 2+ pump ATPase activity (to less than 50% of control) was observed when RBCs were incubated with neutrophils to which PMA had been added (column labeled "activated"). Although SOD (270 units) had no effect on this inhibition, GSH (10 mM)/GSHpx (5 units) did provide modest protection. The rate constants for the Ca 2+ pump ATPase in RBCs exposed to activated neutrophils were 0.148 -4- 0.004 min -L and 0.195 _+ 0.007 min 1, respectively, in cells without and with GSIMGSHpx. Further addition of SOD did not result in additional protection of the Ca 2+ pump ATPase (Fig. 4A). A similar pattern emerged for the lipid peroxidation, as measured by the production of TBARS. As shown in Fig. 4B, there was a seven-fold increase in TBARS in RBCs when PMA was added (as compared to controls, RBCs, and PMNs only). In this experiment, TBARS increased in RBCs from a control value of 1.76 + 0.83 to 12.6 _+ 2.35 #mol/l in the presence of activated neutrophils. Addition of SOD had no effect on the level of TBARSs. In contrast to SOD, GSH/ GSHpx was very effective in preventing the formation of TBARS: TBARS production was limited to 5.02 _ 0.53 #mol/l RBC in the presence of 10 mM GSH and 5 units of GSHpx. However, neither GSI-I/GSHpx nor GSH/GSHpx plus SOD was completely effective in inhibiting lipid peroxidation (Fig. 4B). In addition to partial protection of the C a 2+ pump ATPase and prevention of lipid peroxidation in intact RBCs, the GSIM GSHpx system was also effective in preventing the oxidation of hemoglobin. In the presence of activated neutrophils, GSH/GSHpx prevented the loss of oxyhemoglobin and the production of methemoglobin.

Neutrophil-induced inhibition of the Ca 2+ pump ATPase

661

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Fig. 4. Protection by GSHpx, but not SOD from neutrophil-induced lipid peroxidation and inhibition of the Ca 2÷ pump ATPase of intact RBCs. In panels A and B, the columns labeled "control" represent RBCs preincubated with PMNs (4 × 106), and the columns labeled "activated" represent RBCs preincubated with PMNs (4 x 106), 2 mM azide, and PMA (200 nM). Other columns, as labeled, represent additional inclusions in the preincubation, such as superoxide dismutase (SOD, 270 units), glutathione peroxidase (GSHpx, 5 units), or SOD (270 units) plus GSHpx (5 units). Samples with GSHpx also contained 10 mM GSH. Samples were preincubated for 2 h. Subsequently, in both experiments, Ca 2+ pump ATPase activity (A) or TBARS (B) were determined. Data represent the mean of three experiments, ±SEM (A), and a separate set of three experiments, ±SD (B). Values statistically different from one another, p < 0.01, are denoted*. NS, p > 0.05.

Greater than half-maximal protection was caused by 1 unit of GSHpx (data not shown). The potential role of hemoglobin in mediating neutrophil-induced inhibition of the Ca 2+ pump ATPase was examined by pretreating RBCs with carbon monoxide (CO). In these experiments, the Ca 2÷ pump ATPase rate constant of RBCs alone was 0.326 _+ .011 min -1. In the presence of activated neutrophils, this value decreased to 0.202 ___ .008 min -1 (n = 2). Under the same conditions when CO-treated RBCs were incubated with activated neutrophils, the Ca 2÷ pump ATPase rate constant was 0.256 ± .002 (n = 2). Thus, in the presence of activated neutrophils, the Ca 2+ pump ATPase rate constant was 17% higher in CO-treated RBCs than in untreated RBCs (p < 0.02).

SDS-PAGE analysis of RBC membranes isolated from intact RBCs preincubated under various conditions The possibility that neutrophil-derived ROS may result in cross linking and/or fragmentation of RBC membrane proteins was first examined by SDS-PAGE. Results of SDS-PAGE are shown in Figure 5, with lane 1 representing isolated RBC membrane proteins from intact RBCs preincubated alone; lane 2, RBC membrane proteins from intact RBCs preincubated with PMNs, and lane 3, RBC membrane proteins from intact RBCs incubated with PMNs in the presence of PMA. For all three conditions, the RBC membranes were examined for the presence of methemoglobin us-

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bulk of methemoglobin. Indeed, methemoglobin could be observed during the electrophoresis as a brown band just behind the dye front. The next major difference was a broad, diffuse band running at about 32 kDa. This might represent crosslinked methemoglobin subunits, though there was no observable color associated with this band prior to staining. It is also possible that this band represents fragmentation products of other membrane proteins. Other additional bands in lane 3 appear, in general, to be diffuse and not as sharp as their counterparts in the control lanes (1 and 2). Heterogeneity of the membrane proteins in these bands could be due to fragmentation of the constituent proteins by the free radical species generated during the preincubation process. It is also possible that, similar to methemoglobin, other nonhemoglobin cytosolic proteins bound to the RBC membranes. These additional proteins could be responsible for some of the increased background staining compared to the controls.

kDa 200--* 116 66 45

1

2

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Fig. 5. SDS-PAGE of RBC membranes isolated from intact RBCs preincubated under various conditions. Preincubation reactions included RBCs (3 X 108 cells), without or with PMNs (4 x 10 6 cells), or PMA (200 k~M). In all cases azide was added at a final concentration of 2 mM. Following a 2-h preincubation period, membranes were prepared from washed RBCs (see Methods), and were subjected to SDS-PAGE. Lane 1, RBC membrane proteins (15 #g) following preincubation in the presence of intact RBCs only; lane 2, RBC membrane proteins (15 #g) following preincubation of intact RBCs and PMNs; lane 3, RBC membrane proteins (15 #g nonmethemoglobin protein) following preincubation of intact RBCs, PMNs, and PMA.

ing the method of Winterbourn 26 as previously described. Lanes 1 and 2 contained RBC membrane proteins (15/zg each) in which no detectable methemoglobin was found. In contrast, membrane proteins in lane 3 contained 0.73 mg methemoglobin/mg total protein. Therefore, to compensate for the methemoglobin bound to RBC membranes, lane 3 was loaded with 15 #g of nonmethemoglobin protein. The amount of spectrin (bands I and II) was more or less equal in all three lanes, indicating minimal changes of this protein due to neutrophil activation (Fig. 5). Other than some minor variation in certain other bands there was little difference between lanes 1 and 2. By inference, quiescent PMNs had little effect on RBC membrane proteins. The small differences could be due to proteins of residual PMN membranes that copurified with the RBC membranes during preparation. By contrast, lane 3 showed considerable changes in the banding pattern as compared to the two controls. Near the dye front, there was a significant amount of protein staining which corresponded to the

Western blot analysis of the C a 2+ pump ATPase from intact RBCs preincubated in the presence of activated neutrophils Western blotting (Fig. 6A) was used because the C a 2+ pump ATPase is present in insufficient quantities

to visualize by typical SDS-PAGE. In Figure 6A, the treatments are identical to those described in Figure 5, with lane 1 representing membrane proteins from intact RBCs incubated alone; lane 2, membrane proteins from intact RBCs incubated with PMNs, and lane 3, membrane proteins from intact RBCs incubated with PMNs in the presence of PMA. Each lane was loaded with 40 #g of total nonmethemoglobin membrane protein. In Figure 6A, lanes 1 and 2 show a predominant band running at about 140 kDa. This band represents the Ca 2+ pump ATPase. All three lanes also displayed a more diffuse band appearing at approximately 180190 kDa. This band represents natural aggregation products of the pump that are normally recognized by the 5F10 monoclonal antibody. 33 There were two additional bands present in the sample containing RBCs and quiescent PMNs (lane 2). These additional bands appeared at about 81 and 68 kDa respectively, and most likely represent natural proteolytic products of the C a 2+ pump ATPase. The reason these two bands are more pronounced in lane 2 is unknown, but may represent the contribution of the C a 2+ pump ATPase from PMNs that copurified along with RBC membranes. As previously mentioned, activation of PMNs by PMA resulted in the eventual aggregation of the PMNs, allowing for easy separation from RBCs following the preincubation step. Therefore, we were able to remove most, if not all, of the clumped PMNs from

Neutrophil-induced inhibition of the Caz+ pump ATPase

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1

2

3

2

3

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Fig. 6. Western blot analysis of the Ca2+ pump ATPase from intact RBCs preincubated under various conditions. Preincubation reactions included RBCs, without or with PMNs, or PMA as in Figure 5. Azide was added to each reaction at a final concentration of 2 mM. Following a 2-h preincubation period, membranes were prepared from washed RBCs, and subjected to SDS-PAGE as in Figure 5 with each lane containing 40 #g of membrane protein. (A) Western blot; lane 1, RBC membrane proteins following preincubation of intact RBCs only; lane 2, RBC membrane proteins following preincubation of intact RBCs and PMNs; lane 3, RBC membrane proteins following preincubation of intact RBCs, PMNs, and PMA. (B) Identical set of lanes as in (A), blotted onto the same PVDF membrane. In this case, lanes were stained for total membrane protein using India ink. For description of various arrows, see text.

R B C s before membrane preparation in the sample represented in lane 3, but not lane 2. As shown in Figure 6A, the 81 k D a and 68 kDa bands were not present in lane 3. The presence of these two bands in lane 2 m a y indicate that the Ca 2+ p u m p ATPase from P M N s is subjected to more (auto?) proteolysis; however, further work must be done to clarify this question. In contrast to lanes 1 and 2, lane 3 showed consider-

663

able differences in the banding pattem o f the C a 2+ pump ATPase. Most evident was the decrease in intensity o f the band running at 140 kDa. This is probably not a reflection o f unequal protein loading or of inadequate transfer o f proteins onto P V D F membranes of this lane, because when identical sets o f lanes were stained with India ink (Fig. 6B), it appeared that all 3 lanes contained an approximately equal amount of protein. Importantly, Figure 6 A also shows that in lane 3 there were two additional bands running at about 8.2 and 6.8 k D a that were not present in lanes 1 and 2 (small open arrows). These additional bands represent some of the fragmented products o f the Ca 2+ p u m p ATPase from the R B C as caused by the free radical species produced upon activation of coincubated PMNs. Further evidence o f protein fragmentation is depicted in Fig. 6B. A band, most likely representing actin (indicated by the small triangle), is lower in intensity in lane 3 than in lanes 1 and 2. The lower intensity of this band could be attributed to fragmentation o f this protein. In addition, the bands running at 8.2 and 6.8 kDa (small open arrows) are more intense in lane 3 than in lanes 1 and 2. These bands probably represent multiple fragmented proteins. Supporting the hypothesis that generalized protein cross-linking did not occur to the Ca 2+ p u m p ATPase (Fig. 6A) or o f the other R B C membrane proteins (Fig. 6B), is the absence of high molecular weight material at the interface o f the stacking gel and the separating gel (large closed arrows at the top of each blot). It should be noted that differences in the protein banding pattern between Figure 5 and Figure 6B m a y be attributed to the fact that membrane proteins blotted onto P V D F membranes (Fig. 6B) were run on a 7.5% separating gel, which allowed for better separation o f proteins than membrane proteins seen in Figure 5, where a 10% separating gel was used. Using a 7.5% separating gel allowed for more efficient transfer o f membrane proteins onto P V D F filters. DISCUSSION In this study we found that PMA-stimulated (activated) neutrophils inhibited the Ca 2+ p u m p ATPase o f the intact R B C by mechanisms apparently dependent, at least in large part, upon neutrophil-derived superoxide and H202. Inhibition o f the Ca 2+ p u m p ATPase and oxidation of hemoglobin did not appear to depend on direct cell to cell contact. This result is in contrast to that o f Weiss who reported a requirement for c e l l cell contact to observe neutrophil-mediated hemoglobin oxidation) In our model system, centrifugation of samples to concentrate the cells into a pellet was not

664

T.T. RoHN et

employed. Both RBCs and neutrophils were in suspension throughout the preincubation time. Activity of the Ca 2+ pump ATPase was measured using an assay in intact RBCs. We have shown that, under the conditions of the assay, the rate constant for ATP loss in intact RBCs is a measure of the Ca 2+ pump ATPase activity. 24 The advantages of using this assay include that it can be carried out on very small volume of RBCs (e.g., 10 /A) and that it allows for minimal handling of the RBCs. In addition, the assay may be a better physiological model than assays using RBC ghosts or isolated membranes because the integrity of the cell remains intact. For these and other reasons, assay of the Ca 2÷ pump ATPase in intact RBCs was well suited for this study. Using this assay we typically preincubated 50 #1 of packed RBCs (3.9 × 108 cells) together with 4 × l06 PMNs. Inhibition of the Ca 2+ pump ATPase in intact RBCs was dependent upon both the neutrophil concentration and time of incubation. With a ratio of 97:1 RBCs:PMNs, we observed approximately 50% inhibition of the Ca 2+ pump ATPase with 2 h of coincubation. At 1560:1, detectable inhibition ( - 1 0 % ) of the Ca 2+ pump ATPase was also observed. Normal ratios for RBCs:PMNs in the circulation are roughly 2,000:1. 38 Thus, the observed inhibition at a 1560:1 may reflect in vivo potentialities for neutrophil-induced damage of RBCs and, possibly, other cells. These data may bear on certain pathophysiological conditions where the ratio of RBCs to PMNs is lower, for example, at a site of inflammation. 39 There is evidence that the environment in the RA patient is hostile to RBCs. Mild hemolytic anemia is associated with RA, and RBCs from normals exhibit a shortened life span when administered to patients with RA. 4° We demonstrated that GSH/GSHpx limited the degree of inhibition of the Ca 2÷ pump ATPase in intact RBCs (Fig. 4). By contrast, SOD failed to protect the Ca 2+ pump ATPase. Because SOD dismutes superoxide to peroxide, these results suggest a direct role for H202, but probably not superoxide, in causing inhibition of the Ca 2÷ pump ATPase and other changes in intact RBCs. Our data support the interpretation that as superoxide is generated by activated neutrophils it spontaneously dismutes to H202. The H202 thus formed then diffuses into the RBC where it interacts with the iron moiety on hemoglobin, oxidizing it to methemoglobin. In a study by Weiss, the addition of either SOD or catalase alone partially prevented the oxidation of hemoglobin, while a combination of both enzymes provided the best protection. 3 The results of our study do not support a role for superoxide in oxidizing hemoglobin. We cannot explain why our results differ from those of Weiss. Because we did not centri-

al.

fuge RBCs and PMNs into a pellet for direct cell to cell contact, as did Weiss, 3 there may have been a shift toward H202 as the major mediator of damage in our system. In any event, reactive products released by the PMNs diffused to the RBCs and promoted inhibition of the Ca 2÷ pump ATPase without cell-to-cell contact. It should be noted that inclusion of azide in our system would inhibit myeloperoxidase of neutrophils making it unlikely that hypochlorous acid 41 was a causative agent in inhibition of the Ca 2÷ Pump ATPase. Activation of neutrophils may also lead to the release of proteolytic enzymes, such as elastase, 2 which could mediate some of the neutrophil-induced damage in the intact RBCs. However, this did not appear to be the case in the system we employed, because when PMSF (2 mM) was added to the preincubation medium, there was no protection of the enzyme (data not shown). Our data support the interpretation that hemoglobin within the RBC may act as a Fenton reagent 42 in the presence of H202, thereby causing production of hydroxyl radical and/or ferryl radical. GSH/GSHpx prevented the oxidation of hemoglobin and formation of methemoglobin (data not shown). GSH with GSHpx, can reduce both hydrogen peroxide and organic hydrogen peroxides. 43 Second, inhibition of the Ca 2+ pump ATPase in RBCs by activated neutrophils was enhanced in the presence of azide, a potent inhibitor of catalase. RBCs contain substantial amounts of catalase, 44 which catalyzes the breakdown of H202 into O2 and H20. 45 We chose to inhibit catalase instead of GSHpx even though GSHpx has a higher affinity for H202 than does catalase. K m values reported for HzO2 vary, but are generally in the low #M range for GSHpx 46 and are generally in the mM range for catalase. 47 However, RBCs exhibit a 60-fold greater specific activity of catalase than GSHpx. 48 Thus, as H202 is increased in RBCs, for example, by activated PMNs as in our system, catalase becomes relatively more important. Recent evidence suggests a role for catalase in protection against H202-mediated lipid peroxidation at physiologic levels, 49 a role previously assigned predominantly to G S H p x 9 It has been suggested that erythrocyte catalase might function as a " s i n k " for exogenous H202. 44 Our data support the conclusion that azide inhibited intracellular RBC catalase, allowing a greater proportion of H202 to react with hemoglobin, thus producing greater quantities of hydroxyl radical and greater inhibition of the Ca 2+ pump ATPase. Pretreatment of RBCs with CO limited the degree of Ca 2+ pump ATPase inhibition by activated neutrophils. It is suggested that the CO-hemoglobin complex is not effective as a generator of hydroxyl/ferryl radicals since CO occupies the sixth coordination position of iron. Taken together, these data support the hypothesis

Neutrophil-induced inhibition of the Ca2+ pump ATPase that the critical event for inhibition of the Ca 2+ pump ATPase was a Fenton reaction between H202 and hemoglobin producing either hydroxyl radical or a ferryl radical. 48 As previously stated, we found that the addition of GSH/GSHpx provided only partial protection of the Ca 2+ pump ATPase, while SOD provided no protection at all. Other authors using activated neutrophils found that the addition of either SOD and catalase alone, or in combination, is only moderately successful in preventing neutrophil-mediated lipid peroxidation, 4 methemoglobin formation, 3 or K + leak in RBCs. 5t The failure to protect from neutrophil-mediated damage may be due to an inability to scavenge all superoxide and H202 or to other unknown mechanisms. Our data are suggestive of a reaction between H202 and the iron moiety on hemoglobin producing hydroxyl radical. However, because there was no direct demonstration of the hydroxyl radical and its inactivation of the Ca 2+ pump ATPase, the proposed mechanism must be regarded as plausible rather than demonstrated. It is possible that instead of a reaction of Fe 2+ and H202 producing hydroxyl radical, a ferryl radical was formed instead. Formation of a ferryl radical, in which the iron has a valency of +4, has been suggested to occur instead of hydroxyl radical. 48 It should be noted that either of these two radicals are extremely reactive, and if they are not produced near the membrane, they would likely react with other cytosolic molecules. It is also possible that the methemoglobin and hemichrome produced in our system associated with the RBC membrane and contributed to the inhibition of the Ca 2+ pump ATPase. Generation of hydroxyl radical or some other ROS by H202 and hemoglobin also resulted in lipid peroxidation in RBC membranes. TBARS were assayed by the reaction of malondialdehyde (MDA), with thiobarbituric acid. M D A is a secondary component of lipid peroxidation capable of cross-linking membrane proteins containing amino groups. 52 Therefore, one potential mechanism for neutrophil-mediated inhibition of the Ca 2+ pump ATPase is through cross-linking 53's4 of the proteins. However, our data are suggestive that the production of TBARS had little or nothing to do with inhibition of the Ca 2+ pump ATPase in intact RBCs. First, although a G S H / G S H p x system substantially inhibited the formation of TBARS, protection of the Ca 2+ pump ATPase was modest at best. Second, although lipid peroxides promote protein cross-linking, we were unable to detect cross-linking of membrane proteins by SDS-PAGE or western blot analysis. Finally, there were observable differences between the production of TBARS and inhibition of the Ca 2+ pump ATPase. Most of the inhibition of the Ca 2+ pump ATPase occurred

665

at the end of 1 h even though TBARS had only increased by 50% during the same time (data not shown). These data can be interpreted to suggest that inhibition of the Ca 2+ pump ATPase, as caused by activated PMNs, occurred by some other additional mechanism. Thus, a direct reaction of the Ca 2+ pump ATPase with either hydroxyl radical or a ferryl radical may be responsible for its inhibition. Free radicals, such as hydroxyl radical, are capable of directly modifying amino acids such as tryptophan, tyrosine, histidine, and cysteine. 55 Hydroxyl radical reactivity may result in the fragmentation, increased proteolytic susceptibility, 56 and modification of secondary and tertiary structure s7 of affected proteins. Membrane proteins have been suggested as primary targets for free radicals causing cytolysis. 58 The results presented in Figures 5 and 6 support the interpretation that significant alteration of at least some membrane proteins, and possible fragmentation of the Ca 2÷ pump ATPase, was associated with neutrophil-mediated inhibition of the Ca 2+ pump ATPase. In this regard, a more complete picture of the extent of protein fragmentation of the Ca 2+ pump ATPase might have been accomplished if a polyclonal antibody to the pump had been employed. Only the specific epitope clipped from the Ca 2÷ pump ATPase will be recognized by the monoclonal antibody and visualized on a western blot although there were certainly other regions of the enzyme that were fragmented. As stated previously, we were unable to detect gross protein cross-linking of RBC membrane proteins by SDS-PAGE, or of the Ca 2÷ pump ATPase by western blot analysis. We have reported that certain ROS can lead to the cross-linking of proteins in isolated RBC membranes. 2° However, the data presented here support a role for protein fragmentation of membrane proteins, including the Ca 2+ pump ATPase. Thus, exposure of cells to ROS may produce different types of membrane protein damage depending upon the particular free radical species involved, the intactness of the cell membranes, antioxidant enzymes, and so forth. In conclusion, we have shown that PMA-stimulated neutrophils promoted oxidation of hemoglobin and inhibition of the Ca 2+ pump ATPase of intact RBCs. Inhibition of the Ca 2+ pump ATPase may occur as a result of protein fragmentation as caused by either hydroxyl radical or a ferryl radical generated by a reaction with PMN-derived H202 and hemoglobin within RBCs. The RBC may be viewed as a model of itself or other cells. Thus, we suggest that inhibition of the Ca 2+ pump ATPase by neutrophil-generated ROS may lead to the accumulation of intracellular Ca 2+ to toxic levels in a variety of cell types. Decreased Ca 2÷ pump ATPase and increased intracellular Ca 2+ may be re-

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lated to premature RBC "aging ''s9 and the anemia associated with chronic inflammatory disease, n° Neutrophils may cause similar changes in parenchymal cells. Thus, increased intracellular Ca 2+ may cause or contribute to the cell injury and death seen in numerous inflammatory diseases including RA. Similar, but more acutely developing injuries are associated with organ damage in ischemia/reperfusion and other conditions in which PMNs have been associated with cell damage. 6°,61

Acknowledgements - - Supported in part by NIH Training Grant

GM-07750. The authors thank Peter van der Ven for providing numerous blood samples. They also thank John Penniston for providing the monoclonal antibody to the Ca2+ pump ATPase.

REFERENCES

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Cai--intracellular calcium CO--carbon monoxide Cyt c--cytochrome c (type III) DMSO--dimethyl sulfoxide GSH--reduced glutathione GSHpx--glutathione peroxidase PBS--phosphate buffered saline PMA--phorbol 12-myristate 13-acetate PMNs--polymorphonuclear leukocytes RBCs--red blood cells ROS--reactive oxygen species SOD--superoxide dismutase TBARS--thiobarbituric acid-reactive substances