ca2+ accumulation and loss by aberrant endocytic vesicles in sickle erythrocytes

JOURNAL OF CELLIJLAR PHYSIOLOGY 152:l-9 (1992)

Ca2+ Accumulation and Loss by Aberrant Endocytic Vesicles in Sickle Erythrocytes

PATRICK WILLIAMSON,* ESTELA PUCHULU, JOHN 1. PENNISTON, MAXWELL P. WESTERMAN, AND ROBERT A. SCHLEGEL

Department of Biology, Amherst college, Amherst, Massdchuseth 01 002 (P.W, E.P.), Department of Brochemistry dnd Mo/ecu/dr Biology, M d y o Clrnrc, Roche5ter, Mrnnesota 5590 7 (I.T.P.), HematologyIOncology Drvrbron, Unrversrly ot Hea/th ScrencesiChrcago Medical School, M t Sinai Hosprtal, Chicago, Illrnors 60608 IM,P. W.); Department of

Molecular and Cell Biology, The Pennsylvania State Unrversrty, Unrversrty Park, Pennsylvania 7 6802 (R A S.1

Sickle cells contain internal vesicles which accumulate CaL+ As 5hown here, the membrane enclosing the vesicle5 Lontdins the plasma membrane Ca*+-ATPasc, or CaL+ pump, as judged by staining with an antibody directed against the protein Moreover, the number of cells containing such vesicles increases upon deoxygenation. These findings argue strongly that the vesicles arise by endocyto- 515 from the plasma membrane, and explain how they accumulate CaL+ When sickle cells are depleted of ATP, Ca'+ is lost trom the vesicles, as judged by the disappearance of staining with the Ca'+/membrane probe chlortetrdcycline (CTC), without a corresponding 1055 of antibody staining This loss of Ca2 ' can be inhibited by nitrendipine, a CaL+ channel blocker These results suggest that the vesicle membrane allows outward passage of Ca2+ by a nitrendipine-sensitive pathway, which can be overcome by the inward-directed activity of the Ca2+ pump of the vesicle membrane. If 50, the Cazf which vesicles contain i s in dynamic equilibrium with the cytoplasm of the sickle erythrocyte 8 1992 Wiley-Liss, Inc

The primary defect in sickle cell anemia is the substi- tution of valine for glutamic acid in the P-globin chain. This defect, which is directly responsible for reduced hemoglobin solubility and its precipitation when sickle red cells are deoxygenated, also induces a variety of secondary cellular abnormalities and membrane de- fects (Padilla et al., 1973; Hoover et al., 1979; Hebbel et al., 1980a,b, 1985; Allan et al., 1981, 1982; Lubin et al., 1981; Westerman and Allan, 1983; Westerman et al., 1984; Hebbel and Miller, 1984; Hebbel, 1985; Choe et al., 1985; Franck et al., 1985; Hebbel, 1985; Schwartz et al., 19851, including an abnormally high Ca2+ content (Eaton et al.,. 1973; Palek, 1973) which increases upon deoxygenation (Palek, 1976), suggesting that HbS polymerization may be involved in the mechanism responsible for elevating Ca2 '.

A critical observation in deciphering the mechanism by which Ca2+ levels are increased in sickle cells was made by Bookchin and coworkers (19811, who found that red cell K+ transport, which is stimulated by elevated cytoplasmic Ca'+ (Gardos, 1958), was not abnormal in sickle cells. They suggested that the extra Ca2+ in sickle cells is compartmentalized, a hypothesis supported by the finding (Bookchin et al., 1981) that treat- ing sickle cells with the Ca2+ ionophore A23187 in the absence of exogenous Ca2+ potentiated K + transport, presumably by mobilizing internal Ca2' . Subsequent studies have confirmed that cytoplasmic CaZ+ levels are not generally elevated in sickle cells (Murphy et al., 1987; Rhoda et al., 1990). Q 1992 WILEY-LISS, INC

Compartmentalization of Ca2+ in sickle cells was demonstrated directly by electron probe X-ray analysis of cryosections (Lew et al., 1985) and by staining with the Ca2+-sensitive membrane probe chlortetracycline (Rubin et al., 1986). Both methods reveal the presence of relatively large, internal Ca2+-containing vesicles not present in normal red cells. Moreover, the number of these vesicles increases when sickle cells are deoxygenated (Rubin et al., 1986). In parallel experiments, sickle cells were found to endocytose a soluble fluorescent marker, suggesting that the Ca"-containing vesicles might arise by endocytosis (Rubin et al., 1984; Williamson et al., 1990). If so, then accumulation of Ca2' by vesicles might be explained by retention within the vesicle membrane of the Ca2*-ATPase present in the plasma membrane, whose orientation would be such that CaZt would be pumped from the cytoplasm of the cell into the vesicle. Direct proof of this hypothesis requires demonstration that the vesicle membrane contains the CaZ+-ATPase of the plasma membrane.

Lew et al., (1985) demonstrated that broken membrane preparations of sickle cells manifested ATP-dependent Ca2+ uptake into an ionophore-sensitive com- partment. However, a similar activity was observed in

Received September 3,1991; accepted January 28,1992. * To whom reprint requestsicorrespondence should be addressed.

2 WILLIAMSON ET AL.

broken membranes from normal red cells, raising the question of whether this uptake arises from the large, sickle cell-specific internal vesicles. Moreover, it is not clear that the plasma membrane Ca"-ATPase would be internalized with the plasma membrane, since receptor-mediated endocytosis by reticulocytes is selective for a subset of the proteins of the plasma membrane (Choe et al., 1987). To clarify this issue, we have used antibody to the plasma membrane Ca2+-ATPase to demonstrate that this protein is present in the large, Ca2+-containing vesicles. We also provide evidence that the accumulated Ca2+ is mobile and can leave the vesicles by a nitrendipine-sensitive pathway in ATP- depleted cells.

MATERIALS AND METHODS Preparation of cells

Blood was obtained from patients with homozygous sickle cell anemia and from normal controls, in accor- dance with NIH guidelines governing the use of material from human subjects. Ten microliters of whole blood was washed twice in phosphate buffered saline (PHS) containing 135 mM NaC1, 3 mM KC1, 10 mM Na,HPO,, and 1.5 mM KH,PO, (final pH, 7.4) and resuspended in 1 ml of the buffer. Ten microliter aliquots of the suspension were placed on coverslips, either clean or cationized by brief exposure to 150 pgiml Alcian Blue solution, and the cells allowed to attach for 30 min in a humidified chamber. Attachment was generally more efficient with Alcian Blue coated coverslips, but experimental results using either type were similar.

Fixation and permeabilization Coverslips of erythrocytes were rinsed in PBS to re-

move unattached cells and then fixed with 0.5% acrolein (Polysciences) in PBS for 5 min at room temperature, followed by permeabilization as described below. Alternatively, coverslips of cells were fixed in 5% formaldehyde in PBS for 15 min at room temperature; cells fixed under these conditions lost hemoglobin during subsequent permeabilization with detergent. When deoxygenated cells were examined, cells on coverslips in a Parafilm-covered Coplin jar were deoxygenated by bubbling humidified N, through the PBS covering them for 1 h, and then fixed by injection of sufficient deoxygenated 30% formaldehyde to yield a final concentration of 5%. After fixation, coverslips were rinsed in PBS, incubated for 30 min in 0.13 M glycine to elim- inate unreacted fixative, washed again, and permeabilized for 5 rnin in 0.1% Triton X-100 (Pierce) in PBS, and then nonspecific binding sites were blocked by incubation in 1 mgiml bovine serum albumin (BSA) in PBS (PBSIBSA) for 1 h.

Antibody staining Rabbit polyclonal serum or mouse monoclonal anti-

body (ascites fluid: clones JA3 (Borke et al., 1987) or 5F10 lBorke et al., 19911) directed against the human erythrocyte plasma membrane Ca"+-ATPase were used a t a dilution of 1:50 in PBSIBSA. Fixed and permeabi- lizcd cells were incubated with antibody for 15 min and washed twice in PBS containing 0.05% Tween 20 and once in PBSIBSA. PBSiBSA containing no antibody

was used as a negative control in all experiments. In some experiments, preimmune sera of the appropriate species were tested, with results similar to those using PBSiBSA as the negative control. Biotinylated donkey anti-rabbit IgG or goat anti-mouse IgG (Amersham) was used as the secondary antibody a t 1:50 dilution in PBSIBSA. After 15 min at room temperature, samples were washed as above and then either streptavidin- Texas Red (Amersham; 1:lOO dilution in PBSiBSA from a 1 mgIml stock), or streptavidin (Sigma; 1 : l O O dilution from a 1 mgiml stock) and biotinylated R-phycoerythrin (Molecular Probes; 1:lOO dilution from a 4.3 mgiml stock) were applied. After 15 rnin incubation at room temperature, coverslips were washed in PBSi BSA, ringed with vacuum grease, and mounted upside- down on slides in PBSIBSA. In some experiments, following antibody staining, coverslips were immersed in 10 ml of PBS to which was added 100 pl of a stock solution of Hoechst (Bisbenzimide H33258.3 HC1.3 H20, Calbiochem) a t 1 mgiml in DMSO. After 5 min at room temperature, coverslips were mounted in the same staining solution.

CTC staining Oxygenated samples. Cells from 100 pl of whole

blood were washed twice in PBS and fixed for 3 min in 1.4% glutaraldehyde added from a 70% stock (EM grade; Ladd). The cells were then washed 3 times in PBS and incubated in 100 pgiml of CTC (Sigma) in PBS (Rubin et al., 1986) on ice in the dark for 10 min before examination by microscopy.

Deoxygenated samples. Approximately 400 pl of washed packed erythrocytes were resuspended in 2 ml of PBS and deoxygenated under a humidified stream of N, for 60 min a t room temperature. Cells were then fixed with deoxygenated 70% glutaraldehyde injected to a final concentration of 1.4% and processed as above.

Cell counting To determine the frequency of cells containing vesi-

cles stainable with either antibody or CTC, cells containing no, 1 , 2 4 , or more than 4 vesicles were enumer- ated by counting 200-1,000 cells in randomly selected fields. The sum of the three positive categories is desig- nated in the tables as the fraction of cells labelled. Although this sum is the only value reported here, a higher frequency of cells which contained stainable vesicles invariably corresponded to a higher frequency of cells containing multiple vesicles (data not shown). Replicate measurement of frequency of cells containing vesicles in independently stained samples indicated that the uncertainty of frequencies was limited by the statistics of counting ( 2 3% from the given values in most of the cases shown here). The paired T-test of the RS1 data analysis package of the VAX 8550 computer was used to determine whether values were statisti- cally significantly different. In no case was the conclusion dependent (at a P < 0.05 level for significance) on whether the data were assumed to be normally distrib- uted.

ATP depletion of erythrocytes Erythrocytes were depleted of ATP accordin to Mid-

delkoop et al. (1988), either with or without C$+ in the

Ca2 ' -CONTAINING VESICLES IN SICKLE CELLS 3

Fig. 1. Staining of sickle and normal red cells with antibody to red cell plasma membrane Ca2'-ATPase. Undeplcted (A,R) or hemoglobin depleted (C,D) erythrocytes were labelled with antibody t o the Ca2 ' - ATPase, followed by biotinylated second antibody, streptavidin, and biotinylated R-phycoerythrin as described in Materials and Methods. Arrows in b indicate punctate staining observed in sickle, but not normal cells. A,C: Normal cells. B,D: Sickle cells. Bar = 10 pM.

buffer, with comparable results. CTC staining was performed in the depletion buffer except that CaCl,, MgC12, and streptomycin-penicillin were omitted.

RESULTS When sickle cells are stained with the Ca2+-sensitive

membrane probe CTC (Caswell, 1979; Wolniak et al., 1980), relatively bright punctate fluorescence is observed from structures inside the cells (Rubin et al., 1986). The edges of the vesicles in sickle cells are often resolvable in the light microscope (data not shown), confirming electron microscopic evidence (Lew et al., 1985) that the vesicles are large (> ca 0.5 pm). Al- though such vesicles are observed in much higher frequencies than in normal cells, they are nevertheless present in only a fraction (1040%) of sickle cells and are relatively few in number (1-5 per positive cell). Because of this low frequency, we have used light microscope methods to examine them further, because these methods permit survey of many cells.

If the vesicles which CTC detects contain the red cell plasma membrane Ca2+-ATPase, staining with antibody directed against the protein should produce similar punctate fluorescence. To determine if this is the case, sickle cells were fixed with acrolein, permeabilized with detergent, and the labelled with polyclonal antibody directed against the Ca2+-ATPase (Niggli et al., 1979; Verma et al., 1982) followed by detection with secondary antibody. When the secondary antibody was conjugated with fluorescein or Texas Red, only the plasma membrane was fluorescent (data not shown). In order to increase the sensitivity of the assay, the secondary antibody was conjugated with biotin and then located by sequential application of streptavidin and biotin-conjugated phycobiliproteins. As shown in Figure l B , sickle cells, stained with these reagents, exhibited instances of clearly punctate fluorescence in addition to the faintly granular plasma membrane fluorescence. The punctate fluorescence was not observed when normal red cells were stained in similar fashion

4 WILLIAMSON ET AL.

TABLE 1. Frequency of cells with punctate staining following labelling with antibody to Ca2+-ATPase or with CTC'

% of cells labelled Normal Sickle

FixativeAabel cells cells

None/CTC 3.0 48 Acrolein/antibody 2.6 5 Formaldehyde/antibody 6.4 44

'Red cells from d normal control and a slckle cell patient were labelled with CTC or with antibody to the Caz+ ATPase as described in Matenals and Methods, using either acrolein fixahon to meld cells from which hemoglobm was not depleted or formaldehyde fixation tomeld hemoglobin depletedcells Cellsln which atleast one instance of punctate fluorescence was observed were counted and expressed a8 a percent of total cells

(Fig. 1A). The frequency of cells displaying this pattern of staining, however, was lower than the frequency of cells with punctate fluorescence following staining with CTC (Table 1).

The difference in frequency of cells with punctate staining as detected by CTC versus antibody might be artifactual. Experiments with permeabilized and non- permeabilized cells indicated that the antibody is directed primarily against determinants on the cytoplasmic side of the membrane (data not shown). As a result, i t is possible that the phycobiliprotein fluorophore is quenched by hemoglobin while it remains present. To test this possibility, cells were fixed with formaldehyde rather than acrolein; during permeabilization of formaldehyde-fixed red cells, most of the hemoglobin is lost while normal cellular morphology is retained. When cells prepared in this manner were stained for Ca2+- ATPase, punctate fluorescence was again observed in sickle cells (Fig. 1D) and not in normal red cells (Fig. 1C). However, the frequency of cells with observable punctate staining was increased almost to the level measured with CTC (Table 1). The slightly lower frequency seen with the antibody was observed in every case in which such comparisons were made and is dis- cussed in more detail below. The objects visualized by antibody, like those seen with CTC, were present in many but not all cells, with individual cells containing only one or a few of them.

To test whether the antibody was absorbing nonspe- cifically, sickle cells were stained with nonimmune serum. As shown in Figure 2, nonspecific labelling does not occur with rabbit antibodies under the conditions used. The specificity of the antibody was further confirmed by replacing the polyclonal antibody with two different monoclonal antibodies against the Ca2'- ATPase (Borke et al., 1987, 1991); cells with punctate fluorescence were observed at identical frequencies, although the fluorescence levels were considerable lower (data not shown).

When sickle cells are examined by darkfield microscopy, internal objects, and particularly precipitated hemoglobin, can be visualized in large numbers in essen- tially all sickle cells (Schneider et al., 1972). The relationship between these objects and those identified by the anti-Ca2+-ATPase antibody was determined by comparing fluorescence images with darkfield images. As shown in Figure 3, such a comparison revealed that the majority of the Ca2+-ATPase-containing objects correspond to a structure visible in darkfield, while the converse is not true: there are generally many more

objects visualizable by darkfield microscopy in sickle cells than are observed with antibody (or CTC).

Even at the higher level of detection obtained by removing hemoglobin, the frequency of cells containing vesicles stained with CTC was always higher than the frequency of cells from the same patient stained in parallel with anti-Ca2+-ATPase (Table 21, suggesting that not all of the objects revealed by CTC derive from the plasma membrane. We observed that CTC-stained objects occasionally colocalized with Howell-Jolly bodies (Larrimer et al., 1975) visualized by staining with the DNA-specific probe Hoechst 33258 (Cesarone et al., 1979) (data not shown). To test whether these remnants of the nucleus contained the Ca2+-ATPase, cells were double-stained with antibody and Hoechst 33258 and localization of the two stains compared. As shown by the sample field presented in Figure 4, the two probes do not identify the same objects, implying that Ca2+- ATPase-containing objects do not contain DNA, and DNA-containing objects do not contain the Ca2+- ATPase. This result suggests that Howell-Jolly bodies may represent the subpopulation of vesicles stained by CTC but not by anti-Ca"-ATPase. To test this possibility, samples of blood from different patients were stained separately with each of the three probes and the number of cells containing each type of vesicle enumer- ated. As shown in Table 2, the number of cells containing vesicles labelled with CTC is significantly different than the number of cells labelled with anti-Ca2+- ATPase (P < 0.05), but not significantly different from the sum of cells labelled with Hoechst 33258 and cells labeled with anti-Ca2+-ATPase (P > 0.4). This result suggests that the population of vesicles stained with CTC contains two and only two different subpopulations: Howell-Jolly bodies and vesicles containing the plasma membrane Ca2+ -ATPase. Evidence that CTC stains HowellJolly bodies was also obtained from examination of patients who had undergone surgical splenectomy. In these cells, CTC-stained inclusions were occasionally observable; in this case, however, all such inclusions could also be stained with Hoechst (data not shown).

Earlier studies indicated that the number of cells containing CTC-stainable vesicles increases upon deoxygenation (Rubin et al., 1986). The results presented above raise the question of whether deoxygenation is increasing the number of vesicles containing the Ca2+- ATPase. To investigate this possibility, oxygenated and deoxygenated samples of sickle cells were stained with antibody or with CTC, and the number of cells containing vesicles was compared. As seen in Table 3, deoxygenation resulted in a significant increase in both the number of cells containing vesicles stained with CTC (P < 0.01) and the number stained with antibody (P < 0.03), whereas a comparison of the size of the increase for the two probes revealed no significant difference (P > 0.5). As would be predicted from these results, separate analysis showed that the number of Howell- Jolly bodies in sickle cells remains constant upon deoxygenation (data not shown). These results suggest that the deoxygenation-induced increase in the number of Ca2+-ATPase-containing vesicles accounts for the increase in the number of vesicles containing Ca2+, as visualized with CTC.

The presence of Ca2+-ATPase in Ca2+-containing vesicles presents the possibility that Ca2+ is accumu-

Caz ' -CONTAINING VESICLES IN SICKLE CELLS 5

Fig. 2. Staining of sickle cells with non-immune rabbit antibody. Hemoglobin-depleted sickle cells were incubated with non-immune rabbit serum, followed by the detection system as described in Fig. Id. A Phase contrast image. B: Fluorescence image. Bar = 10 pM.

Fig. 3. Comparative patterns of CaZ+-ATPasexontaining vesicles and inclusions identifiable by darkfield microscopy in sickle cells. Hemoglobin-depleted sickle cells were labelled for CaZ+-ATPase, and then viewed using standard darkfield (A) or fluorescence (B) microscopy. Bright spots in the darkfield images correspond to scattering

centers. The darkfield pattern obtained is similar to that observed in unfixed cells by the same technique, although the scattering intensity is considerably higher in the hemoglobin-depleted cells shown here. Bar = 10 pM.

lated by this enzyme, oriented so that Ca2+ is pumped into the vesicles. To test whether ATP-dependent pumping is required to maintain the Ca2+ in vesicles, cells were depleted of ATP using the protocols of Mid- delkoop et al. (1988), where ATP levels decrease to values below 0.01 mg/ml of packed cells, or less than 1% of normal levels, after 24 h of incubation. As shown in Table 4, this treatment significantly reduced the number of cells containing CTC-stainable vesicles (P < 0.01). The number of cells with CTC-stained objects which remained was not significantly different (P > 0.7) than the number of cells in the same population with objects stained with Hoechst (Table 6, below), suggesting that the remnant population of cells stained by CTC consists of cells containing Howell-Jolly bodies; separate analysis showed that the numbers of these objects are not changed by ATP depletion (data not

TABLE 2. Comparison of CTC, anti-Cay+-ATPase, and Hoechst labelling of sickle cells from several patients'

% of cells labelled Patient CTC Antibody Hoechst

H.C. 38.3 37.4 2.1 Sa.C. 49.2 40.2 8.3 Se.C. 46.4 39.2 6.7 L.S. 48.8 43.4 1.7

'Aliquots of blood from four patients were labelledwith CTC, or double-labelled with anti-Ca2+-ATPase and Hoechst. Cells displaying punctate fluorescence were tabulated for each label.

shown). Finally, when ATP-depleted cells were deoxygenated, the number of cells containing CTC-stained vesicles was not significantly increased (P > 0.3) ((Table 4), as expected if only Howell-Jolly bodies were labelled (see above).

6 WTLLIAMSON ET AL.

Fig. 4. Comparison of anti-Ca'+-ATPase labelling with Hoechst 33258 labelling of Howell-Jolly bodies. Hemoglobin-depleted sickle erythrocytes were double labelled with antibody (A) and Hoechst 33258 (B) as described in Materials and Methods. The Hoechst labelling pattern is heavily overexposed, allowing the low level non-specific membrane staining which this probe displays t,o be revealed, so

that corresponding images can he located in the two fields. Non-specific membrane staining by the Hoechst dye is the source of the faint punctate labelling seen scattered in B. Howell-Jolly bodies stain with bright fluorescence, as seen in the cells near the center of the field. Bar = 10 pM.

TABLE 3. Effect of deoxygenation on Ca2+-ATPase and CTC labelling'

TABLE 5. Comparison of anti-Cazt-ATPase and CTC labelling of fresh and ATP depleted cells1

% of cells labelled Probe Fresh ATP-deoleted Antibody CTC

Patient 0 2 N P 0 2 Nz CTC Antibody

48.2 47.9

20.8 49.7

S.L. 41.3 57.7 48.8 64.7 Sa.C. 40.2 55.1 49.2 59.9 Se.C. 39.2 54.8 46.4 53.2 H.C. 37.5 53.6 38.3 60.1

'A sample of sickle cells was divided into two aliquots and one depleted of ATP. Both were then labelled with either CTC or anti-Ca2+-ATPase and the frequency of cells displaying punctate fluorescence tabulatd.

'Aliquots of sickle cells were oxygenated or deoxygenated, labelled with eitber CTC or anti-CaZ+-ATPase, and cells displaying punctate fluorescence were tabulated.

TABLE 4. Effect of ATP depletion on CTC labelling'

% of cells labelled Fresh ATP depleted

Patient 0 2 N2 0 2 N2

Sa.C. 49.2 59.9 16.6 17.2 Se.C. 46.4 53.2 13.8 13.9 H.C. 38.3 60.1 12 22.1 L.S. 48.2 - 20.8 -

'Samples of sickle cells were divided into two aliquots, and one depleted of ATP as described in Materials and Methods. These aliquota were subdivided and one of each deoxygenated. All four samples were stainedwith CTC as describedin Materials and Methods and cells displaying punctate fluorescence tabulated.

The reduction in CTC-stainable inclusions following ATP depletion could be the consequence of reduced levels of Ca2+ inside Ca"-ATPase-containing vesicles which persist, or it could be the consequence of the disappearance of the vesicles themselves. These two alternatives are easily distinguished by staining for Ca2+-ATPase. In four experiments with samples from two different patients, ATP-depleted cells were found to still contain Ca2+ -ATPase positive vesicles; a represen- tative experiment is shown in Table 5. This result implies that ATP depletion of sickle cells compromises the

abilit of vesicles to maintain the high internal levels of Ca'+ required for CTC to detect them.

These results in turn suggest that if Ca"-ATPase activity is compromised, previously accumulated Ca2+ dissipates. As shown in Figure 5 for one patient, and summarized for several in Table 6, when the Ca2+ channel blocker nitrendipine (Janis et al., 1987) was included in the incubation medium during ATP depletion and staining, the number of cells containing vcsi- cles stained with CTC was not significantly reduced (P > 0.71, in marked contrast to the result obtained with ATP depletion alone. The fluorescence was not produced by nitrendipine itself, since vesicles were not visible in the absence of CTC.

DISCUSSION This study directly demonstrates that the internal

vesicles in sickle cells which contain high levels of Ca2+ also contain the red cell plasma membrane Ca2+- ATPase. This finding, together with the observation that additional cells containing these vesicles appear upon deoxygenation, strongly suggests that the vesicles arise by endocytosis. It is important to emphasize, however, that this endocytosis is not likely to be the same as the clathrin-mediated endocytosis characteristic of normal immature red cells, even though the number of immature cells is increased in the circulation of sickle patients. Although receptor-mediated endocytosis does

Ca2+-CONTAINING VESICLES IN SICKLE CELLS 7

Fig. 5. Effect of nitrendipine on CTC labelling of ATP-depleted sickle cells. Sickle cells were depleted of ATP either in the absence (A,B) or presence (C,D) of M nitrendipine, then labelled with CTC as described in Mat,erials and Methods. Samples treated with nitrendipine were washed and stained using buffers which also contained M nitrendipine. Bar = 10 pM.

TABLE 6. Effect of nitrendipine on reduction in CTC labelling in ATP-depleted cells'

ATP-depleted Patient Fresh - Nitrendiuine + Nitrendiuine

E.P. 28.7 10.3 (11.81 25.5 R.R. 42.9 17.1 (17.4j 43.5 R.H. 46 28.0 (24.8) 47.1

'Samples of sickle cellswere divided into threealiquots, two of which were depleted of ATP either in the presenceor absence of M nitrendipine. All samples were then labelled with CTC: samples treated with nitrendipine were washed and stained using buffers which contained nitrendipine. Numbers in parentheses are cells stained by Hoechst 332.58 in the same blood samples, included for reference.

occur in sickle cells, the vesicles which contain Ca2+ differ from clathrin-coated vesicles in that they are much larger, are generated more slowly and in fewer numbers per cell, do not concentrate transferrin or the transferrin receptor, and are not preferentially found in immature cells (Williamson et al., 1990).

Probably because this endocytic process is abnormal and is not selective for the proteins which i t incorpo-

rates, the vesicles which it forms contain the plasma membrane Ca2+-ATPase. Upon internalization, the Ca2+-ATPase would be oriented such that Ca2 ' is transported into the lumen of the vesicles. Since the enzyme can maintain a 104-fold concentration gradient between the inside and the outside of the cell, i t should be able t o concentrate Ca2+ at least to millimolar levels inside vesicles. Ca2+ a t such a concentration is sufficient to account for the ability of CTC, whose affinity constant for the ion is less than millimolar, to allow visualization of Ca"-containing vesicles. In fact, the elemental analysis by Lew et al. (1985) suggests that the estimate of millimolar Ca2+ concentrations is probably low by at least 1-2 orders of magnitude. How long vesicles must persist to accumulate such very high levels of Ca2+ is not yet known.

The high levels of Ca" found in sickle cells could have potentially serious consequences were the Ca2+ free in the cytoplasm and in contact with the plasma membrane. However, the fact that the excess Ca2+ is actually sequestered within membrane-bounded vesicles implies that its pathological potential is critically

WILLIAMSON ET AL 8

dependent on whether Ca2+ remains sequestered or whether there are conditions under which it can esca e the vesicle lumen, increasing free cytoplasmic Ca concentration. Vesicles might be disrupted and lose their Ca2+ through mechanical damage occurring during deoxygenation-induced sickling, or by osmotic lysis as a consequence of accumulation of ions in the small volume of the lumen. As yet there is no evidence that vesicles are ever broken by either of these mechanisms. However, the disappearance of Ca2 ' from vesicles when the ATP supply of the cell is depleted demonstrates that sequestered Ca'+ can leak into the cytoplasm under conditions where the cells is neither lysed nor deoxygenated.

The possibility that leakage occurs through a Ca'+ channel is supported by the finding that the Ca2+ channel blocker, nitrendipine, can prevent the loss. Any conclusion that vesicles contain an actual Ca2+ leak channel is at present only tentative, since the Ca2' permeability of red cells is low and red cells have generally been considered t o lack them (Janis et al., 1987, although see Engelmann and Duhm, 1989). Regardless of the mechanism by which nitrendipine acts, however, it is interesting to note that Ohnishi and coworkers (1986) have shown that the drug inhibits the formation of irreversibly sickled cells (ISCs) in vitro during cycles of oxygenation and deoxygenation, raising the possibility that Caz+ leakage from vesicles during these cycles may play a role in ISC formation. Moreover, sickle cell patients treated with the nitrendipine-related drug, nifedipine, have decreased indirect bilirubin and plasma hemoglobin levels (Rodgers et al., 19881, suggesting the Ca2' leakage may contribute to the hemol- ysis characteristic of the disease. Further evidence for these possibilities awaits additional experimentation.

From the data presented here, a model emerges in which leakage of Ca2+ from vesicles occurs as a con- tinuing process balanced by the action of the Ca2+- ATPase; inactivation of the enzyme results in dissipa- tion of the Ca" gradient established by the ATPase. Perturbation of the dynamics of these Ca2+ fluxes through the vesicle membrane could lead to fluctua- tions in cytoplasmic CaZt concentrations at the micro- molar level or higher, sufficient to induce many of the membrane abnormalities characteristic of sickle cells (see Williamson et al., 1990, for review). Whether phys- iological conditions exist under which such fluctua- tions occur will also require addition experimentation to resolve.

ACKNOWLEDGMENTS This work was supported by grant HL37477 from the

National Institutes of Health and performed during the tenure of R.A.S. as an Established Investigator of the American Heart Association.

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%+

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ca2+ accumulation and loss by aberrant endocytic vesicles in sickle erythrocytes

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