Expansion of phosphatidylcholine and phosphatidylserine/phosphatidylcholine monolayers by differently charged amphiphiles.

The degree and time-course of expansion of palmitoyloleoylphosphatidylcholine (PC) and bovine brain phosphatidylserine (PS)/PC (75:25, mol/mol) monolayers at 32 mN/m caused by differently charged amphiphiles (detergents) added to the sub-phase buffer (pH 7.4, 22 degrees C) were followed. Amphiphiles were added to the sub-phase at a concentration/monolayer area corresponding to the concentration/erythrocytes surface area where sphero-echinocytic or sphero-stomatocytic shapes are induced (0.46-14.6 microM). Nonionic, cationic and anionic amphiphiles expanded the PS/PC monolayer significantly more (1.7-4.2 times) than the PC monolayer. A zwitterionic amphiphile expanded both monolayers to a similar extent. The initial rate of monolayer-expansion was higher for all amphiphiles (1.7-20.4 times) in the PS/PC monolayer than in the PC monolayer. It is suggested that hydrophobic interactions govern the intercalation of amphiphiles into monolayers, and that monolayer packing, modulated by phospholipid head group interactions and alkyl chain saturation, strongly influence amphiphile intercalation. A possible relation between the monolayer-expanding effect of amphiphiles and their effect on erythrocyte shape is discussed.

Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions.

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37 C, 60 min) or glucose-starved (37 C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14-15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under non-starving conditions.

Oxyethylene chain-cation complexation: nonionic polyoxyethylene detergents attain a positive charge and demonstrate electrostatic head group interactions.

We report literature data indicating that the polyoxyethylene chain of polyoxyethylene detergents attracts cations via dipole-ion interactions thereby attaining a positive charge character. This implies that nonionic polyoxyethylene detergents like Triton X-100 and C12E8 may interact electrostatically with phospholipid head groups. We describe how a positive charge character of Triton X-100 and C12E8 can explain their hitherto mysterious stomatocytogenic shape altering effect in human erythrocytes.

Monitoring of MRP-like activity in human erythrocytes: inhibitory effect of isoflavones.

A method to fluorometrically monitor efflux of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was developed. Genistein, daidzein, sophoraisoflavone A, and licoisoflavone A induced 50% inhibition (IC(50)) of BCPCF efflux at 15-70 microM. The IC(50) value of the most efficient isoflavone, licoisoflavone A (15-25 microM), was comparable to that of indomethacin (approximately 10 microM) and markedly lower than for probenecid (100-200 microM), both known MRP1 inhibitors. Our results indicate that the human erythrocyte is a useful cell model in screening potential MRP inhibitors, that BCPCF is a good substrate for MRP, and that some isoflavones at low concentrations inhibit MRP-mediated efflux.

Torocyte shapes of red blood cell daughter vesicles.

The shape of the newly described torocyte red blood cell endovesicles induced by octaethyleneglycol dodecylether (C12E8) is characterized. A possible explanation for the origin and stability of the observed torocyte endovesicles is suggested. Three partly complementary mechanisms are outlined, all originating from the interaction of C12E8 molecules with the membrane. The first is a preferential intercalation of the C12E8 molecule into the inner membrane layer, resulting in a membrane invagination which may finally close, forming an inside-out endovesicle. The second is a preference of the C12E8-induced membrane inclusions (clusters) for small local curvature which would favour torocyte endovesicle shape with large regions of small or even negative membrane mean curvatures, the C12E8 membrane inclusion being defined as a complex composed of the embedded C12E8 molecule and some adjacent phospholipid molecules which are significantly distorted due to the presence of the embedded C12E8 molecule. The preference of the C12E8 inclusions for zero or negative local curvature may also lead to the nonhomogeneous lateral distribution of the C12E8 inclusions resulting in their accumulation in the membrane of torocyte endovesicles. The third possible mechanism is orientational ordering of the C12E8-induced inclusions in the regions of torocyte endovesicles with high local membrane curvature deviator.

Influence of band 3 protein absence and skeletal structures on amphiphile- and Ca(2+)-induced shape alterations in erythrocytes: a study with lamprey (Lampetra fluviatilis), trout (Onchorhynchus mykiss) and human erythrocytes.

Amphiphiles which induce either spiculated (echinocytic) or invaginated (stomatocytic) shapes in human erythrocytes, and ionophore A23187 plus Ca(2+), were studied for their capacity to induce shape alterations, vesiculation and hemolysis in the morphologically and structurally different lamprey and trout erythrocytes. Both qualitative and quantitative differences were found. Amphiphiles induced no gross morphological changes in the non-axisymmetric stomatocyte-like lamprey erythrocyte or in the flat ellipsoidal trout erythrocyte, besides a rounding up at higher amphiphile concentrations. No shapes with large broad spicula were seen. Nevertheless, some of the 'echinocytogenic' amphiphiles induced plasma membrane protrusions in lamprey and trout erythrocytes, from where exovesicles were shed. In trout erythrocytes, occurrence of corrugations at the cell rim preceded protrusion formation. Other 'echinocytogenic' amphiphiles induced invaginations in lamprey erythrocytes. The 'stomatocytogenic' amphiphiles induced invaginations in both lamprey and trout erythrocytes. Surprisingly, in trout erythrocytes, some protrusions also occurred. Some of the amphiphiles hemolyzed lamprey, trout and human erythrocytes at a significantly different concentration/membrane area. Ionophore A23187 plus Ca(2+) induced membrane protrusions and sphering in human and trout erythrocytes; however, the lamprey erythrocyte remained unperturbed. The shape alterations in lamprey erythrocytes, we suggest, are characterized by weak membrane skeleton-lipid bilayer interactions, due to band 3 protein and ankyrin deficiency. In trout erythrocyte, the marginal band of microtubules appears to strongly influence cell shape. Furthermore, the presence of intermediate filaments and nuclei, additionally affecting the cell membrane shear elasticity, apparently influences cell shape changes in lamprey and trout erythrocytes. The different types of shape alterations induced by certain amphiphiles in the cell types indicates that their plasma membrane phospholipid composition differs.

Gemini (dimeric) surfactant perturbation of the human erythrocyte.

We studied the ability of di-cationic gemini surfactantsdi (amphiphiles), i.e. 1,4-butanediammonium-N,N-dialkyl-N,N,N',N'-tetramethyl bromides (Di-Cm-di-QAS (s = 4), where m = 8, 11, 13, 16 and s = the number of alkyl groups in the spacer) to induce shape alteration, vesiculation, haemolysis and phosphatidylserine exposure in human erythrocytes, and to protect erythrocytes against hypotonic haemolysis. At high sublytic concentrations the Di-Cm-di-QAS (s = 4) amphiphiles rapidly induced echinocytic (spiculated) shapes and a release of exovesicles, mainly in the form of tubes, from the cell surface. Following 60 min incubation erythrocytes were sphero-echinocytic and a few cells with invaginations/endovesicles were observed. No phosphatidylserine exposure was detected. The haemolytic potency increased with an increase of the alkyl chain length. At sublytic concentrations the Di-Cm-di-QAS (s = 4) amphiphiles protected erythrocytes against hypotonic haemolysis. It is suggested that the Di-Cm-di-QAS (s = 4) amphiphiles perturb the membrane in a similar way as single-chain cationic amphiphiles, but that they do not easily translocate to the inner membrane leaflet.

Torocyte membrane endovesicles induced by octaethyleneglycol dodecylether in human erythrocytes.

Endovesicles induced in human erythrocytes by octaethyleneglycol dodecylether (C12E8) were studied by confocal laser scanning microscopy, using fluorescein isothiocyanate dextran as a nonspecific fluid marker. The endovesicles appeared to consist mainly of a ring-formed toroidal part joined with a central flat membrane segment. The torocyte contour length was several microm. There was usually one torocyte endovesicle per cell. The endovesicles seemed to be located near the cell surface. In sections of C12E8-treated erythrocytes transmission electron microscopy revealed the frequent occurrence of flat membrane structures with a bulby periphery, which apparently are cross sections of torocyte endovesicles. The possible physical mechanisms leading to the observed torocyte endovesicle shape are discussed. The torocyte endovesicles seem to be formed in a process in which an initially stomatocytic invagination loses volume while maintaining a large surface area. Because intercalation of C12E8 in the erythrocyte membrane induces inward membrane bending (stomatocytosis) we assume that C12E8 is preferentially located in the inner lipid layer of the erythrocyte membrane, i.e., in the outer lipid layer of the endovesicle membrane. It is suggested that local disturbances of the lipid molecules in the vicinity of the C12E8 molecules in the outer lipid layer of the endovesicle membrane form membrane inclusions with the effective shape of an inverted truncated cone. If the interaction between the inclusion and the membrane is weak, the membrane of such an endovesicle can be characterized by its negative spontaneous curvature, which may lead to a torocyte endovesicle shape with a small relative volume. Effects of a possible strong interaction between the C12E8-induced membrane inclusions and the membrane on the stability of the torocyte endovesicles are also indicated.

Liposomes composed of a double-chain cationic amphiphile (vectamidine) induce their own encapsulation into human erythrocytes.

Vectamidine is a liposome-forming double-chain cationic amphiphile. The present work was aimed to microscopically study the interactions of Vectamidine liposomes with the human erythrocyte plasma membrane. Vectamidine rapidly induced stomatocytic shapes. Attachment of Vectamidine liposomes to the erythrocyte induced a strong local invagination of the membrane. This frequently resulted in a complete encapsulation of the liposome. Liposomes composed of phosphatidylcholine (neutral) or phosphatidylserine/phosphatidylcholine (anionic) did not perturb the erythrocyte shape. Our results indicate that besides an attraction of Vectamidine liposomes to the plasma membrane, there is a preference of Vectamidine for the inner bilayer leaflet. We suggest that cationic amphiphiles may transfer from membrane-attached liposomes to the plasma membrane and then translocate to the inner bilayer leaflet where they induce a strong local inward bending of the plasma membrane resulting in an encapsulation of the liposome.

The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.

Membrane skeleton detachment in spherical and cylindrical microexovesicles.

We observed that amphiphile-induced microexovesicles may be spherical or cylindrical, depending on the species of the added amphiphile. The spherical microexovesicle corresponds to an extreme local difference between the two monolayer areas of the membrane segment with a fixed area, while the cylindrical microexovesicle corresponds to an extreme local area difference if the area of the budding segment is increased due to lateral influx of anisotropic membrane constituents. Protein analysis showed that both types of vesicles are highly depleted in the membrane skeleton. It is suggested that a partial detachment of the skeleton in the budding region is favoured due to accumulated skeleton shear deformations in this region.

Amphiphile-induced phosphatidylserine exposure in human erythrocytes.

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time- and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37 degrees C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.

A possible physical mechanism of red blood cell vesiculation obtained by incubation at high pH.

The membrane of human red blood cells is essentially composed of two parts, the lipid bilayer and the membrane skeleton that interacts with the lipid bilayer. The normal resting shape of the red blood cells at physiological pH 7.4 is the discocyte. However, at alkaline pH approximately equal to 11 the shape of red blood cells is composed of a spherical parent cell and large spherical daughter vesicles. The daughter vesicles may be free or connected to the parent cell by a narrow neck. In this paper we show that the shapes of red blood cells at pH approximately equal to 11 correspond to some of the calculated shapes of a closed lipid bilayer having an extreme area difference between the outer and the inner monolayer. Therefore, it is suggested that the observed shapes of the red blood cells at pH approximately equal to 11 are a consequence of the abolishment of the skeleton bilayer interactions at this pH.

Membrane skeleton and red blood cell vesiculation at low pH.

Shapes of red blood cells at low pH were studied theoretically. It is assumed that the equilibrium shape of the red blood cell corresponds to the minimum of its membrane elastic energy which consists of the bending energy and relative stretching energy of the bilayer, the stretching energy of the skeleton and the interaction energy between the skeleton and the bilayer. It is shown that the aggregation of the skeleton at low pH can cause the red blood cell shape transformation from the stomatocytic shape to the cell shape composed of a spherical parent cell having the bilayer completely underlaid with the skeleton and spherical daughter vesicles without the skeleton.

Vesiculation induced by amphiphiles and ionophore A23187 in porcine platelets: a transmission electron microscopic study.

Amphiphiles, known to induce exo- and endovesiculation in human erythrocytes, were studied by means of transmission electron microscopy (TEM) for their ability to induce shedding of vesicles (microparticles) from the porcine platelet plasma membrane. While echinocytogenic amphiphiles induced shedding of vesicles to the extracellular medium (exovesiculation), stomatocytogenic amphiphiles did not induce endovesiculation. The rapid (< 1 min) formation of many thin spicules in platelets upon treatment with echinocytogenic amphiphiles, indicates that spicule-formation is caused by a primary interaction of the amphiphile with the plasma membrane. Agonist- (Ca(2+)-ionophore A23187, thrombin and collagen) induced shape changes, however, seem to involve contractile cytoskeletal processes since treated cells attained heavily irregular shapes with broad pseudopods. Our study indicates that the mechanisms involved in amphiphile- and agonist-induced exovesiculation differ. Amphiphile-induced exovesicles are mainly electron-dense spherical structures (phi 150-200 nm) which originate from the formed spicules. Ionophore A23187-induced exovesicles are large (phi 200-800 nm) predominantly electron-lucent structures which are mainly shed from the cell body and seem to originate from extrusions of the canalicular system. Our study shows that there are several similarities but also obvious differences in the response of platelets and erythrocytes to amphiphile-treatment.