Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca²⁺-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Phospholipid scramblase activation pathways in lymphocytes.

In erythrocytes and platelets, activation of a nonspecific lipid flipsite termed the scramblase allows rapid, bidirectional transbilayer movement of all types of phospholipids. When applied to lymphoid cells, scramblase assays reveal a similar activity, with scrambling rates intermediate between those seen in platelets and erythrocytes. Scrambling activity initiated in lymphoid cells by elevation of intracellular Ca(2+) proceeds after a lag not noted in platelets or erythrocytes. The rates of transbilayer movement of phosphatidylserine and phosphatidylcholine analogues are similar whether the scramblase is activated by elevated internal Ca(2+) or by apoptosis. Elevation of internal Ca(2+) levels in apoptotic cells does not result in an additive increase in the rate of lipid movement. In lymphoid cells from a patient with Scott syndrome, scramblase cannot be activated by Ca(2+), but is induced normally during apoptosis. These findings suggest that Ca(2+) and apoptosis operate through different pathways to activate the same scramblase.     

Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.     

Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structure-activity relationships for betulinic acid analogues

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH(2)OH, CONH(2)) caused formation of echinocytes, whereas those lacking this ability (CH(3), CHO, COOCH(3)) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC(50) values in the range 7-28 microM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.     

Shape transformations induced by amphiphiles in erythrocytes

Shape alterations induced in human erythrocytes by cationic, anionic, zwitterionic and nonionic amphiphiles (C10-C16) at antihaemolytic concentrations (CAH50 and CAHmax) and at a slightly lytic concentration (2-10% haemolysis) were studied. Anionic (sodium alkyl sulphates) and zwitterionic amphiphiles (3-(alkyldimethylammonio)-1-propanesulfonates) proved to be potent echinocytogenic agents. Among the nonionic amphiphiles there were potent stomatocytogenicagents (octaethyleneglycol alkyl ethers, pentaethyleneglycol dodecyl ether), one potent echinocytogenic agent (dodecyl D-maltoside) and one weak echinocytogenic agent (decyl beta-D-glucopyranoside). Shape alterations induced by cationic amphiphiles (alkyltrimethylammonium bromides, cetylpyridinium chloride and dodecylamine hydrochloride) showed a strong time-dependence. These amphiphiles immediately induced strongly crenated erythrocytes which during incubation shifted to less crenated erythrocytes or to stomatocytes. All of the echinocytogenic amphiphiles induced echinocytes immediately, and there were only small alterations of the induced shape during incubation. Among the stomatocytogenic amphiphiles there were some that induced stomatocytes immediately or after a short lag time while others first passed the erythrocytes through echinocytic stages before stomatocytic shapes were attained. Erythrocytes treated with amphiphiles did not recover their normal discoid shape following repeated washing and reincubation for 1 h in amphiphile-free medium. Our study shows that shape alterations induced by amphiphiles in erythrocytes cannot be explained solely by assuming a selective intercalation of differently charged amphiphiles into the monolayers of the lipid bilayer as suggested in the bilayer couple hypothesis (Sheetz, M.P. and Singer, S.J. (1976) J. Cell Biol. 70, 247-251). We suggest that amphiphiles, when intercalated into the lipid bilayer, trigger a rapid formation of intrabilayer non-bilayer phases which protect the bilayer against a collapse and bring about a transbilayer redistribution of intercalated amphiphiles as well as of bilayer lipids.     

Influence of new hybrid antioxidants ichphans on erythrocyte morphology

Scanning electron microscopy was used to study the effects of the new generation of compounds ICHFANs, which have a combined antioxidant and acetylcholine esterase inhibitory effect on the surface architectonics of erythrocytes. The incorporation of each of the studied compounds with the positively charged quaternary ammonium in the erythrocyte membrane and their distribution in the itramembraneous space were accompanied by the formation of echinocytes, stomatocytes, and compensative effects on erythrocyte shape. The time-dependent morphological transformation of erythrocytes apparently is determined by changes in the distribution of the compounds between the outer and inner monolayers of the erythrocyte membrane. A difference in the morphological effects of compounds with different hydrophobic properties was revealed.     

Phospholipid scramblase: An update

The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.     

Cytoplasmic pH and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.     

ATP-dependent transport of phosphatidylserine analogues in human erythrocytes

The plasma membrane of most cells contains a number of lipid transporters that catalyze the ATP-dependent movement of phospholipids across the membrane and assist in the maintenance of lipid asymmetry. The most well-characterized of these transporters is the erythrocyte aminophospholipid flippase, which selectively transports phosphatidylserine (PS) from the outer to the inner monolayer. Previous work has demonstrated that PS and to a lesser extent phosphatidylethanolamine (PE) are substrates for the flippase and that other phospholipids move across the membrane only by passive flip-flop. The present study re-evaluates these results. The incorporation and transbilayer movement of a number of short-chain (dilauroyl) phospholipid analogues in human erythrocytes was measured by observing lipid-induced changes in cell morphology, and the effect of an ATPase inhibitor (vanadate) and a sulfyhdryl reagent (N-ethylmaleimide) was determined. Incubation of cells with these lipids causes the rapid formation of echinocytes, because of the accumulation of the lipid in the outer monolayer. While dilauroylphosphatidylcholine-treated cells retained this shape, cells treated with sn-1,2-DLP-l-S, sn-1,2-DLP-d-S, or N-methyl-DLPS rapidly changed morphology to stomatocytes, which is consistent with the transport and accumulation of the lipid in the inner monolayer. A similar, although slower, stomatocytic shape change was induced by sn-2,3-DLP-l-S. Other lipids that were tested (dilauroylphosphatidylhydroxypropionate, dilauroylphosphatidylhomoserine, DLPS-methyl ester, or sn-2,3-DLP-d-S) reverted to discocytes only. In all cases, pretreatment with vanadate or N-ethylmaleimide inhibited the conversion of echinocytes to discocytes or stomatocytes. This is the first report of a protein- and energy-dependent pathway for the inwardly directed transbilayer movement of lipids other than PS and PE in the erythrocyte membrane and suggests that the flippase has broader specificity for substrates or that other lipid transporters are present.     

N-terminal Proline-rich Domain Is Required for Scrambling Activity of Human Phospholipid Scramblases

Human phospholipid scramblase 1 (hPLSCR1), a type II integral class membrane protein, is known to mediate bidirectional scrambling of phospholipids in a Ca²⁺-dependent manner. hPLSCR2, a homolog of hPLSCR1 that lacks N-terminal proline-rich domain (PRD), did not show scramblase activity. We attribute this absence of scramblase activity of hPLSCR2 to the lack of N-terminal PRD. Hence to investigate the above hypothesis, we added the PRD of hPLSCR1 to hPLSCR2 (PRD-hPLSCR2) and checked whether scramblase activity was restored. Functional assays showed that the addition of PRD to hPLSCR2 restored scrambling activity, and deletion of PRD in hPLSCR1 (ΔPRD-hPLSCR1) resulted in a lack of activity. These results suggest that PRD is crucial for the function of the protein. The effects of the PRD deletion in hPLSCR1 and the addition of PRD to hPLSCR2 were characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1 and PRD-hPLSCR2 showed Ca²⁺-dependent aggregation and scrambling activity, whereas hPLSCR2 and ΔPRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca²⁺-dependent scrambling activity by aggregation of protein. Our results provide a possible mechanism for phospholipid scrambling mediated by PLSCRs and the importance of PRD in its function and cellular localization.     

Ethanol-induced alterations in human erythrocyte shape and surface properties: Modulatory role of prostaglandin E1

Summary Exposure of human erythrocytes to ethanol (1 to 20% by vol) in Ca²⁺ and Mg²⁺-free phosphate-buffered saline, pH 7.4, transformed biconcave discs into spiculated echinocytes within 3 min at 25°C. The effects of ethanol were concentration- and time-dependent, but reversible by washing in the incubation buffer system within 60 min of initial exposure to ethanol. After prolonged ethanol exposure (180 min), washing of cells resulted in the formation of stomatocytes (cup-forms). Ethanol-induced echinocytosis was also accompanied by a 30% enhancement in the agglutinability of erythrocytes by ligands with high affinity for negative surface charge (poly-l-lysine and wheat germ agglutinin, 20 l/ml) without any alterations in surface charge topography. Concomitant exposure of erythrocytes to prostaglandin E₁ (100nm) selectively prevented the enhancement of ligand-mediated agglutinability, but did not modify cell shape. These data indicate that certain erythrocyte surface properties may not be directly influenced by cell shape and suggest a unique modulatory action of prostaglandin E₁ on shape-transformed cells.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Recovery of human erythrocytes from the echinocyte shape induced by added choline-phospholipids is dependent on the acyl chain length

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocytes membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37 degrees C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8-C12 acyl chains and lysoPC with a C14-C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.     

NMR q-space analysis of canonical shapes of human erythrocytes: stomatocytes, discocytes, spherocytes and echinocytes

q-Space plots obtained experimentally using pulsed field-gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) spectroscopy from water diffusing in red blood cells (RBCs) of different canonical (distinct variant) morphologies have “signature” features. The experimental q-space plots from suspensions of stomatocytes, echinocytes and spherocytes generated chemically had no diffraction features; in contrast a sample of blood from a patient with hereditary spherocytosis showed diffraction minima. To understand the forms of q-space plots, mathematical/geometrical models of discocytes, stomatocytes, echinocytes and spherocytes were used as restricting boundaries in simulations of water diffusion with Monte Carlo random walks. These simulations indicated that diffusion-diffraction minima are expected for each of the cell shapes considered. The absence of diffusion-diffraction minima in stomatocytes generated by dithiothreitol treatment was surmised to be due to non-alignment of the cells with the magnetic field of the NMR spectrometer. Differential interference contrast microscopy images of the chemically generated spherocyte and echinocyte suspensions showed them to be heterogeneous in cell shape. Therefore, we concluded that the shape heterogeneity caused the loss of the diffusion-diffraction features, which were observed in the more homogeneous sample from a patient with hereditary spherocytosis, and in the simulations of homogeneous cell suspensions. This understanding of factors that affect q-space plots from RBC suspensions will assist morphological studies of other cell and tissue types.     

The shape of red blood cells as a function of membrane potential and temperature

Summary It is well known that a pH shift of the outside medium from 5 to 9 produces a shape transformation of washed human red blood cells from stomatocytes to echinocytes in isotonic salt solutions. In addition, a stomatocytogenic effect is demonstrated here due to solutions of low ionic strength (below 70mm). An analysis of the true cell state in these situations, proved by measurements of predicted volume changes, indicates a good correlation between transmembrane potential and cell shape. The fact that amphotericin B acts as echinocytogenic agent in low ionic strength medium at pH 7.4 but not at pH 5.1 underlines this explanation. Therefore, a transmembrane potential positive inside produces stomatocytes, slightly negative inside (below–10 mV), normocytes, and strongly negative, echinocytes. The temperature dependence of this process underlines the rigidity-pattern hypothesis of red blood cell shape (Glaser & Leitmannová, 1975, 1977).     

Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.     

The influence of chlorpromazine on the potential-induced shape change of human erythrocyte

The effect of chlorpromazine (CPZ) on the shape of human erythrocytes with different values of transmembrane potential (TMP) was investigated. The shape of red blood cells with negative values of the TMP remained unchanged after the formation of stomatocytes by chlorpromazine, while cells with positive TMP showed a characteristic time course of shape change during the incubation with CPZ. Experiments with vanadate show that this might be due to a difference in the activity of the phospholipid-translocase at different values of TMP.     

Membrane bilayer balance and erythrocyte shape: a quantitative assessment

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.