Synergistic Effects of Oxidation and Deformation on ... - Blood Journal

Synergistic Effects of Oxidation and Deformation on Erythrocyte Monovalent Cation Leak By Paul A. Ney, Mary M. Christopher, and Robert P. Hebbel The normal red blood cell (RBC) membrane is remarkable for its durability (eg. preservation of permeability barrier function) despite its need t o remain deformable for the benefit of microvascular blood flow. Yet, it may be hypothesized that the membrane‘s tolerance of deformation might be compromisedunder certain pathologic conditions. We studied this by subjecting normal RBC in viscous suspending medium (20%dextran) t o elliptical deformation induced by application of shear stress under physiologic conditions (290 mOsm/L. 37°C. pH 7.40) in the presence of ouabain and furosemide. Measurement of resulting net passive K efflux (“K leak”) demonstrated that shearinduced RBC deformation causes K leak in a dosedependent fashion at shear stresses far below the hemolytic threshold, an effect shown to be due to deformation per se. To model the specific hypothesis that oxidatively perturbed RBC membranes would be abnormally susceptible t o this potentially adverse effect of deformation, we treated normal RBC with the lipid peroxidant t-butylhydroperoxide. Under conditions inducing only minimal K leak

due to either oxidation alone or deformation alone, deformation of peroxidant-pretreated RBC showed a markedly enhanced K leak ( P < .OOl). This highly synergistic oxidation-plus-deformation leak pathway is less active at low pH. is neither chloride-dependent nor calcium-dependent, and allows K efflux t o be balanced by Na influx so there is no change in total monovalent cation content or cell density. Moreover, it is fully reversible since deformationinduced K leak terminates on cessation of shear stress (even for oxidant-treated RBC). Control experiments showed that our results are not explained simply by hemolysis. RBC vesiculation, or development of prelytic pores. We conclude that oxidation and deformation individually promote passive leak of monovalent cation through RBC membranes and that a markedly synergistic effect is exerted when the two stresses are combined. We hypothesize that these findings may help explain the abnormal monovalent cation leak stimulated by deoxygenation of sickle RBC. 0 1990 by The American Society of Hematology.

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whether this is the case. However, actual sickling-induced deformation would involve a membrane that is abnormal and has been autoxidatively perturbed.’ Thus, the present studies were designed to address the hypothesis that minimal oxidative membrane damage might make the RBC membrane abnormally susceptible to any potentially adverse effects of deformation. To examine this, we used a Couette viscometer to induce elliptical deformation of normal RBC and RBC pretreated with a peroxidant, t-butylhydroperoxide (tBOOH).

H E NORMAL ERYTHROCYTE (RBC) membrane regulates cellular cation homeostasis by providing both a permeability barrier and the proper milieu for various ion transport pathways, even while remaining tolerant of physiologic degrees of cell deformation demanded by RBC circulation. However, pathologic conditions may place extraordinary demands on this balance between durability and deformability. In particular, the occurrence of sickling during deoxygenation of sickle RBC is of interest, since this is a case in which pathologic RBC deformation is associated with an abnormal flux of monovalent cation across the membrane.’-5Although this reversible leak pathway is still defined only by its phenomenology rather than by structural data, the necessary proximate event does appear to be mechanical membrane deformation per se, since exaggerated K efflux does not occur during deoxygenation if sickling itself is prevented! On the other hand, the presumed sufficiency of nonlytic deformation for inducing K or N a fluxes through normal RBC membranes has not been documented experimentally, so one goal of the present studies was to determine

From the Department of Medicine. University of Minnesota Medical School, Minneapolis. Submitted June 13,1989; accepted November 2,1989. Supported by National Institutes of Health Grants HL37528 and HL30160. Previously presented, in part, in abstract form: Blood 70:66a. I987 (suppl). Address reprint requests to Robert P. Hebbel, MD. Box 480 UMHC. University of Minnesota Hospital, Harvard Si at Easi River Rd, Minneapolis. MN 55455. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7505-OOI6$3.00/0 1192

MATERIALS AND METHODS

RBC preparation. Normal RBC from fresh heparinized blood were washed three times in buffer A (10 mmol/L HEPES, 10 mmol/L glucose, 2 mmol/L CaCI,, 2 mmol/L MgCI,, 0.1 mmol/L ouabain, 1 mmol/L furosemide, NaCl to 290 mOsm/L, pH 7.4) with removal of buffy coat. RBC were then suspended to hematocrit 10%in buffer A containing 20% dextran (average molecular weight 40 Kd; Sigma Chemical Co, St Louis, MO) and having osmolarity of 290 mOsm/L (measured using a Wescor 5100B Vapor Pressure Osmometer; Wescor, Logan, UT) and viscosity of 10 cp at 37OC. RBC in this medium are morphologically normal (by phase contrast microscopy) and were documented to have normal total monovalent cation content (Na + K = 110.2 f 2.2 mEq/L RBC; n = 4) and MCV (88 1 fl; n = 4) and MCHC (34.1 t 0.9 g/dL; n = 7). Moreover, one additional experiment provided functional evidence that this medium supports normal RBC hydration by documenting that it allows normal RBC deformability during ektacytometry (data not shown). Because of the osmotic contribution of dextran to this medium, the resulting increased chloride ratio (nearly unity) predicts near identity of cytoplasmic and buffer pH. Measurement of the former on freeze-thawed RBC pellets confirmed that RBC internal pH averaged only 0.03 f 0.05 unit lower than buffer pH. The benign nature of this suspending medium was further documented by finding that rates of K leak from normal RBC (under static conditions) in buffer A with and without the dextran were identical (data not shown). Deformation-induced net potassium eBux (“K leak”). Fourteen milliliters of RBC suspension were pipetted into the gap (0.63 mm) of a Couette viscometer (built locally). Temperature of the suspenBlood, Vol 75, No 5 (March 1). 1990: pp 1192-1 198

OXIDATION-PLUSDEFORMATION LEAK PATHWAY

sion was maintained at 37OC by perfusion of the stationary metal inner cylinder using a high-velocity circulating water bath, and the desired shear rate was obtained by adjusting the rotational speed of the plexiglass outer cylinder. In parallel, 11 mL of RBC suspension were incubated at 37OC under static (unsheared) conditions. At zero time and various intervals, I-mL aliquots of each RBC suspension (in triplicate) were added to 2 mL of ice-cold 10 mmol/L Trisbuffered MgCI, (98 mmol/L), pH 7.4. One milliliter of this supernatant was added to 1 mL of 6 mmol/L LiCI, and the K concentration was determined by flame photometer (Radiometer model FLM3; Louisville, KY). Based on microhematocrit of the RBC suspension, results were converted to mEq K leak per liter RBC. Sensitivity of this technique allowed reliable detection of K leak amounting to 0.02 mEq K/L RBC/h. In every experiment, K leak was corrected for hemolysis. To do this, we derived percent hemolysis from the concentrations of hemoglobin in the sample supernatant and in a lysate of the RBC suspension, both prepared using 0.1% Triton X-100.Based on the intracellular K content (at zero-time), the percent lysis was converted into the corresponding mEq K and subtracted to yield the K leaks reported here. The hemoglobin concentrations were determined spectrophotometrically at 412 nm, the A'""" for both lysate and supernatant hemoglobin from both control and tBOOH-treated RBC. Likewise, for experiments on phenazine methosulfate (PMS)treated methemoglobin containing cells (see below), readings were taken at the documented Xma"for those specific cells. REC cation contents and REC density. Intracellular K contents were determined by washing samples three times in ice-cold 10 mmol/L Tris-buffered MgCI, (98 mmol/L), pH 7.4. After 0.2 mL of packed RBC were lysed by addition to 3.8 mL of diluent (5.3% trichloroacetic acid and 3.2 mmol/L LiCI), supernatant K concentrations were determined by flame photometry. Total intracellular Na and K contents of RBC were determined by atomic absorption spectroscopy (Varian SpectrAA-10, Mulgrave, Australia) using similar extracts (without LiCI) diluted as necessary for optimal detection. RBC density distribution was documented using microcapillary differential flotation with phthalate ester mixtures having specific gravity increments varying by 0.001*; sheared and static RBC were examined side by side during the same centrifugation run. RECmanipulations. For experiments requiring oxidant pretreatment, fresh RBC were first washed three times in buffer B (10 mmol/L HEPES, 10 mmol/L glucose, 4 mmol/L KCI, NaCl to 290 mOsm/L, pH 7.4). Next, the RBC were suspended to hematocrit 10% in buffer B containing the desired concentration of tBOOH and incubated at 37OC for 30 minutes, after which 30 pL of 0.1 mol/L butylated hydroxytoluene (BHT) in ethanol was added (final concentration 0.1 mmol/L). Control RBC were handled identically in all respects except for exposure to tBOOH. After washing RBC three times in buffer A, deformation-induced K leak was measured as described above. Generation of thiobarbituric acid-reactive substances (TBARS) by tBOOH-treated RBC was measured after addition of BHT and with correction for non-TBARS chromagen, as described? In some experiments, after exposure to tBOOH, RBC were washed twice and suspended to hematocrit 10% buffer B containing 10 to 100 pmol/L 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) and incubated at 37OC for 20 minutes. Then the RBC were washed three times with buffer A, and deformation-induced K leak was measured as above. For some experiments we deliberately altered RBC deformability. To decrease it, we exposed RBC at hematocrit 10%(for 30 minutes) to buffer B containing l%acetaldehyde at room temperature'' or 2 mmol/L diamide at 37OC. Conversely, to increase RBC deformability we increased the ratio of external to internal RBC viscosity" by raising the concentration of dextran in buffer A, final osmolarity was still maintained at 290

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mOsm/L. The effect of external calcium on K leak was assesed by substituting 1 mmol/L EGTA for calcium in buffer A. In other experiments, all chloride-containing compounds in buffer A were replaced with the bromide or nitrate salts to examine the effect of anion substitution. Postshear REC integrity. RBC integrity after oxidant pretreatment and shear exposure was examined in several ways, Evidence for RBC vesiculation was sought by adding 2 mL of postshear RBC to 36 mL of buffer B, centrifuging at 1,000 x g for 5 minutes to remove most of the RBC, centrifuging the supernatant at 30,000 x g for 20 minutes, and examining the pellet for vesicles using phase microscopy." Potential spherocyte formation during shear was assessed by measuring the osmotic fragility of postshear RBC. Evidence for a prelytic pore was sought by measurement of colloid osmotic lysis in the presence of various sized protectants." Twenty microliters of packed postshear RBC were directly added, without intervening washes, to each of six tubes containing 2 mL of diluent. Four tubes contained 12.5 mmol/L sodium phosphate, 4 mmol/L KCI, NaCl to 290 mOsm/L, pH 7.4 and 30 mOsm/L of one sugar (mannitol, sucrose, raffinose, or dextran). One tube contained 2 mL of the same buffer with no sugar, and one contained 2 mL of distilled water. These were gently agitated for 24 hours at room temperature. Percent protection was calculated from the hemoglobin concentration in the supernatants, with 100% protection defined by the tube with 30 mOsm/L dextran and 0% by the tube with distilled water. Failure of a given substance to osmotically protect the postshear RBC would be evidence for the presence of a pore large enough to allow that sugar to pass. To provide a positive control, actual prelytic pores were induced in a second group of RBC by incubation at 37OC for 35 minutes in buffer B containing 2 mmol/L tBOOH and 5 mmol/L sodium azide.'' These RBC were suspended in buffer A with dextran (but not sheared), and colloid osmotic lysis was measured as above. RESULTS

Deformation-inducedK leak. In these experiments, RBC at 37OC in viscous suspending medium were elliptically deformed by application of shear stress in a Couette viscometer. Since these studies were done in the presence of ouabain and furosemide (to inhibit Na-K-ATPase and Na/K cotransport, respectively), the resulting appearance of K in RBC supernates reflects net passive K efflux ("Kleak"). We find that shear stress induces K leak in a dose-dependent fashion, with a threshold of about 110 dynes/cm* (Fig IA). The effect is still quite small at 220 dynes/cm2, the shear stress used in all subsequent experiments. This K leak measurement is reproducible, as evidenced by four consecutive runs using RBC from the same donor (Fig IB). It should be noted that for all results presented here, K leak has been corrected for hemolysis, even though this was always trivial relative to the amount of K leak. Specifically, RBC lysis due to shearing at 220 dynes/cm2 never exceeded 0.5% (even for oxidanttreated cells described below), and this never contributed more than 4% of total supernate K. To confirm that shear-induced K leak is due to RBC deformation per se, we showed that it depends on RBC deformability (ie, not just on applied shear stress) which, in turn, depends partly on the ratio of external to internal viscosity." We increased this ratio by using higher concentrations of dextran in the suspending medium and held shear stress constant by proportionately decreasing the shear rate. Increasing normal RBC deformability in this manner en-

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NEY, CHRISTOPHER, AND HEBBEL

Fig 1. Deformation induces net K leak from normal RBC. As described in Materials and Methods. K leak was measured at 37°C as cells were elliptically deformed in a Couette viscometer ("shear") or during parallel static incubation ("static"). Panels A, C, end D depict single representative experiments (1 of 5 , l of 2. and 1 of 4, respectively). Panel (A) shows the dose response for K leak from normal RBC during deformation as a function of applied shear stress. Panel (6) shows the reproducibility of K leak for four consecutive paired runs (static and shear [220 dyneslcm']) using RBC from the same donor. Points are indicated as mean f SD. Panel (C) shows that K leak actually depends on RBC deformation per se. Here degree of RBC deformation was increased by raising external viscosity while shear stress was kept constant at 220 dyneslcm' by proportionately lowering shear rate. Panel (D) shows that applied shear stress (220 dynes/cm') causes no enhancement of K leak if RBC are unable t o deform by virtue of prior treatment with diamide (2 mmol/L).

hances shear-induced K leak (Fig IC). Conversely, we decreased RBC deformability by pretreatment with diamide, a cytoskeletal crosslinking agent which stiffens RBC membra ne^.'^ Despite a higher baseline K leak, the poorly deformable diamide-treated cells show no shear-induced effect (Fig 1D). Thus, K leak from RBC subjected to shear stress is truly dependent on RBC deformation per se. Further support for this conclusion is derived from experiments using acetaldehyde and phenazine methosulfate (see Fig 5 , to be described below). Importantly, induction of K leak during RBC deformation is completely reversible so that the rate of K leak promptly returns to the static baseline on cessation of deformation (Fig 2A). Oxidation-induced K leak. The lipid peroxidant tBOOH induces K leak from normal RBC in a dose-dependent fashion, with a threshold around 1 mmol/L tBOOH (Fig 3). Likewise, this perturbant causes RBC to generate peroxidation byproducts (TBARS) in a dose-dependent fashion: 0.6 mmol/L tBOOH yields 0.3 k 0.2 nmol/mL RBC, 0.8

Fig 2. Deformation-induced K leak is reversible. Net K leak is shown as a function of time for this representative experiment (1 of 5).Panels compare K leak from RBC during static incubation (0) with that from a parallel aliquot examinedfor 2 hours during shear (220 dynes/cm') after which shear was stopped (0). Panel (A) shows normal control RBC, and panel (6) shows RBC pretreated with tBOOH (0.8 mmollL). In both cases. rate of net K leek is promptly restored t o that of the static control on cessation of deformation.

mmol/L yields 3.7 + 2.2 nmol, and 1.0 mmol/L yields 14.7 +. 1.5 nmol (mean k SD for n = 5 ) . tBOOH (at 1 mmol/L) has no appreciable effect on membrane proteins as judged by gel electrophoresis and thiol-disulfide exchange ~hromatography'~;nor does it significantly diminish the number of membrane thiols titratable with DTNB (data not shown). As shown in Fig 3, the concentration of tBOOH used in all subsequent experiments (0.8 mmol/L) has a minimal effect on K leak in the absence of deformation. Synergistic eflect of oxidation and deformation on K leak. Compared with the minimal increase in K leak registered by normal RBC exposed to either deformation alone or oxidant treatment alone, RBC subjected to equivalent deformation after equivalent oxidant pretreatment show greatly enhanced K leak (Fig 4). This effect of oxidation-plus-deformation on K leak is significantly greater than the sum of the two effects alone (P< .001), and it is abrogated if RBC aretreated with the antioxidant BHT before exposure to tBOOH (data not shown) rather than after (as in our standard conditions). Hence, there is marked synergism between effects of oxidative membrane damage and deformation on RBC K leak.

Fig. 3. Peroxidetive stress induces K leak. RBC were pretreated with the indicated tBOOH concentration for 30 minutes, after which net K leak was measured during static incubation at 37°C. Data are shown for six experiments (mean SD).

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The exaggerated oxidation-plus-deformationK leak remains fully reversible (Fig 28). We also sought evidence for induction of this synergistic leak pathway in normal RBC treated with PMS, which stimulates intracellular generation of superoxidei6 and induces lipid peroxidation," oxidation of membrane protein thiols,17 and very poor RBC deformability as judged by micropipette aspiration and ektacytometry." Notably, this stiffness prevents expression of the oxidation-dependentsyn-

ergistic leak pathway unless dithiothreitol (DTT) is used to restore deformability by reversing the thiol oxidation induced by PMS (Fig 5A). DTT has no effect on the synergistic K leak induced by tBOOH (data not shown). However, deliberately diminishing the deformability of tBOOHtreated RBC using 1% acetaldehyde, a nonoxidative membrane stiffening agent," inhibits expression of the synergistic leak response to deformation (Fig 5B). Nature of the oxidation-plus-deformation K leak. Additional experiments further characterized the leak pathway induced by oxidation-plus-deformation.The rate of K leak is significantly diminished at lower external pH (Fig 6A). Substitution of other anions such as bromide (Fig 6B) or nitrate (data not shown) for chloride in these experiments has no effect on magnitude of the synergistic K leak. Removal of ouabain (Fig 6C) or furosemide (data not shown) from our standard suspending medium has a minimal effect on the synergistic K leak. Substitution of EGTA for calcium in the suspending medium has no influence on K leak (Fig 6D). The synergistic K leak is not diminished by 10 Fmol/L DIDS (data not shown), but it is diminished at 25 pmol/L (or higher) DIDS (Fig 6E). However, this is due to a higher baseline of K leak, making results obtained using this agent suspect and of questionable significance. Since it is total intracellular monovalent cation content that ultimately determines RBC hydration, we used several types of measurements to detect or reflect this. For example, K efflux can be balanced by equivalent Na influx during activation of the synergistic leak pathway so that total intracellular Na + K content for tBOOH-treated RBC subjected to deformation does not significantly change. In the specific example depicted in Fig 6F, the change in total Na+K is typical (an average change of +0.7 + 1.2 mEq Na + K/L RBC for seven experiments) and, being within experimental error, is of no significance. Consistent with this, activation of this leak pathway leads to no significant

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Fig 6. Characteristics of the synergistic leak pathway. All panels show results for tB00H-treated RBC and compare those subjected t o deformation (open symbols) and those from the parallel static incubation (closed symbols). As described in detail in the text, panels illustrate the synergistic leak pathway's dependence on external pH (panel A), independence of external anion (panel 6). persistence even if ouabain is not included (panel C). end lack of requirement for external calcium (panel D). Panel (E) shows the effect of 25 MmollL DlDS (see text). Panel IF) shows balancing of K efflux by Na influx, with no significant change in total Na K content. Panel (GIillustrates RBC density distribution on phthalate gradients, as identified by separate centrifugation runs covering upper and lower density ranges (circles and squares). Indicated specific gravity is that measured at room temperature, and curve positions have not been corrected for heating of the esters during centrifugation. Panel (HI shows that shearing does not alter osmotic fragility. All panels show single. but representative, experiments (1of 3 experiments in panel A; 1 of 5 in 6; 1 of 6 in C; 1 of 8 in D; 1 of 4 in E; 1 of 7 in F; 1 of 3 in G; 1 of 2 in H).

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differences in MCHC (static greater than shear by an average difference of 0.3 f 0.8 g/dL for eight experiments) or in RBC density distribution on phthalate gradients (Fig 6G) or in osmotic fragility (Fig 6H). Control experiments. To provide further evidence for a leak pathway reversibly induced by deformation, we excluded the Occurrence of other phenomena potentially associated with deformation. In the supernatant of RBC exposed to oxidation plus 2 hours of deformation, the ratio of K to hemoglobin increases an average 27-fold over the ratio in intact or in totally lysed RBC. This specificity of the leak for K over hemoglobin rules out overt hemolysis as an explanation for deformation-induced K leak. We excluded RBC vesiculation as an explanation by documenting an absence of vesicles in the postshear supernate using phase contrast microscopy (data not shown) and an absence of reciprocal spherocyte formation amongst postshear RBC by measurement of osmotic fragility (Fig 6H). In addition, we sought to determine if deformationinduced K leak might be due to creation of prelytic pores. By definition, these defects initially would be smaller than hemoglobin but large enough to allow rapid loss of RBC K, and they would be lasting and lead ultimately to cell lysis. Their existence was excluded by measurement of the colloid osmotic lysis of postshear RBC in the presence of protectants

varying in size from NaCl (smallest) to raffinose (largest). This was donedirectly postshear, without intervening washes, so there was no opportunity to lose RBC by lysis during preparative washing. Even for oxidant-pretreated RBC, virtually 100% protection is provided by all protectants, including NaCl (Fig 7). In contrast, positive control RBCI3 illustrate how progressively smaller molecules offer less protection in the presence of actual prelytic pores (Fig 7). Thus, we find that the RBC membrane remains competent and free of pathologic or prelytic pores after cessation of deformation, even for oxidant-pretreated RBC. DISCUSSION

Using the experimental model of elliptical deformation of RBC suspended in viscous medium, we have documented that normal RBC leak potassium in response to deformation and demonstrated that RBC membranes minimally perturbed with the lipid peroxidant tBOOH have greatly enhanced susceptibility to this deformation-induced K leak. Although we have not yet performed formal ion selectivity studies, data reported here do indicate that activation of this leak pathway allows Na influx to balance K efflux. These results clearly are explained by deformation per se, since the leak pathway is shown to depend not only on applied shear stress but also upon RBC deformability. The shear stress

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Fig 7. Deformation-induced leak is not explained by development of prelytic pores. RBC were pretreated with tBOOH (0.8 mmol/L) and then subjected to shear-induced (220 dynes/cm2) deformation for 2 hours. Immediately thereafter, RBC were incubated for 24 hours in isotonic salt solution containing 30 mOsm/L of potential protectants of varying size. RBC thus subjected to our standard tBOOH exposure followed by deformation (closed symbols) have not developed prelytic pores, as evidenced by the fact that even incubation with NaCl does not lead to colloid osmotic lysis. Open symbols show an example of a positive control, development of prelytic pores in RBC subjected to high concentrations of tBOOH plus azide. After this extreme stress, NaCl provides virtually no protection from colloid osmotic lysis, mannitol provides some protection, and high-molecular-weight dextran provides complete protection.

used in these studies (220 dynes/cm2) is far below the established hemolytic shear stress threshold of 1,500 dynes/ cm2.19As described herein, various experiments have ruled out RBC vesiculation, frank RBC lysis, and development of prelytic pores as potential explanations for this phenomenon. Furthermore, the deformation-induced leak in normal and in tBOOH-treated RBC is completely reversible so that the leak pathway returns to its basal state on cessation of deforming stress. We believe it is likely that this synergistic oxidation-plus-deformation leak pathway develops uniformly throughout the population of mildly stressed normal RBC examined in these studies; but at present it is not possible to prove this point. The precise mechanism underlying exposure of this leak pathway has not yet been identified. We can be confident that it is not caused by the tank-treading (orbital passage of membrane around cytoplasm in the direction of cell elongation) that occurs under conditions used here. Because tanktreading frequency is linearly dependent on shear rate and independent of suspending medium viscosity,20the experiment shown in Fig 1C excludes this explanation. Consequently, we hypothesize that activation of the synergistic

oxidation-plus-deformation leak pathway involves application of areal tension and that leak reflects the membrane’s relative inability to withstand isotropic forces.2’ Presence of lipid hydroperoxides would perturb the hydrophobic core of the lipid bilayer by adding polarity, decreasing order, and causing packing defects, thereby possibly providing opportunities for abnormal permeation by monovalent ~ a t i o n . ~ ~ . ~ ’ However, other tBOOH-related possibilities include effects of various peroxidation byproducts (eg, malondialdehyde and other carbonyl compounds), oxygenated sterols, disturbances at lipid/protein interfaces, or even secondary adverse effects on protein per se. In theory, any of these oxidative defects could result in the synergistic leak pathway either by altering the sterochemical characteristics of the minimal leak pathway detectable during deformation of normal RBC or by altering membrane biophysical properties. It is anticipated that future studies will precisely define the nature of this abnormal leak pathway. It should be noted that prior investigations have examined effects of peroxidation alone on permeability of red cell membranes. In particular, Deuticke reported that tBOOH induces a K leak pathway 0.5 to 0.7 nm in diameter and having characteristics of an aqueous pore.” However, it is essential to note that those investigations were done using a much higher degree of peroxidative stress (2 mmol/L tBOOH plus azide) that induces pores that are unequivocally prelytic, while the leak pathway induced in our model of minimal oxidation clearly is not a prelytic pore. Likewise, the prelytic effects of other pharmacologic oxidants are of uncertain relevance to the reversible, synergistic leak pathway identified in the present studies. The present data may be relevant to monovalent cation leak induced by deoxygenation of sickle RBC. This phenomenon has long been assumed to reflect distortion of the membrane by spicules of polymerized hemoglobin during the sickling process, but only recently have experimental data demonstrated that such distortion is necessary for deoxygenation-induced potassium leak from sickle RBC.6 Our data document not only that deformation alone is sufficient to alter membrane permeability but also that deformation has an exaggerated effect on an otherwise abnormal membrane. The relevance of this stems from ample evidence of autoxidative damage to sickle RBC membranes.’ Notably, the amount of TBARS generated by tBOOH treatment in our experiments is equivalent to that generated spontaneously by sickle cells during one-half day of aerobic incubation in vitro.24 Thus, we have approximated a physiologic degree of peroxidative stress, albeit on a collapsed time scale and in an artificial manner. Therefore, we hypothesize that autoxidatively damaged sickle RBC membranes may be particularly susceptible to the potentially adverse effects of deformation (ie, induction of monovalent cation leak) in the same sense that we have modeled herein. However, it would be hazardous to assume that shearinduced elliptical deformation is necessarily an accurate model of sickling per se. Indeed, existing biophysical analyses do not allow direct and formal comparison of isotropic (or other) forces in the two cases, although stresses on the membrane are assumed to be even greater during sickling.

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Hence, it is worth noting that the leak pathway induced by oxidation-plus-deformation (as defined herein) is similar in magnitude, pH dependence, reversibility, and anion-independence to the leak of monovalent cation observed during sickling (as defined by the literature describing its phen~menology'.~.~.~~.~~). The synergistic leak pathway allows passive K efflux to be balanced by passive N a influx; data are conflicting for sickling but perhaps tend to support a balanced leak.2-5The synergistic leak pathway does not depend on entry of external calcium, as is the case for most (but perhaps not all) of the K leak occurring during sickling.26

Thus, despite inherent differences between elliptical deformation used here and actual sickling, both appear to expose leak pathways with similar features. It will be of interest to determine whether this synergistic leak pathway can be identified in sickling RBC per se and, if this is the case, whether it plays any role in sickle disease pathobiology. ACKNOWLEDGMENT

We thank Krishna Goyal for technical assistance, Carol Taubert for assistance in manuscript preparation, Dr Perry Blackshear for providing the viscometer, and Dr Narla Mohandas for helpful suggestions.

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