A basis of echinocytosis and stomatocytosis in the disc-sphere transformations of the erythrocyte

A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3, the anion exchange protein, controls the shape. In essence, the mechanism hypothesizes that the membrane skeleton is used to generate different shapes and the alternate influx and efflux of anions mediated by Band 3, which recruit Band 3 to an inward-facing and an outward-facing conformation, contract and relax the skeleton by folding and unfolding spectrin. Spectrin is bound to Band 3 by the intermediary of ankyrin. The mechanism is shown to be consistent with rapid shape deformations of the erythrocyte in blood circulation. We have examined whether the mechanism could provide a basis of echinocytosis and stomatocytosis in disc-sphere transformations of the erythrocyte induced by a wide variety of agents. These agents were classified into four groups: lipids of the bilayer, Donnan equilibrium modifiers, Band 3 anion transport inhibitors and integral membrane protein modifiers. Evidence is presented that the lipids play a secondary function in the control of the erythrocyte shape, as indicated by the mechanism. Two possible functions of the lipids are suggested with respect to the mechanism. Without exception, echinocytogenic and stomatocytogenic Donnan equilibrium modifiers decrease and increase the equilibrium ratio of chloride (Cl-(i)/Cl-(o)), respectively, as predicted by the mechanism. Echinocytosis produced by competitive anion transport inhibitors slowly transported inward by Band 3 and by affinity labels of Band 3 is compatible with the mechanism. Evidence is presented which indicates that echinocytosis and stomatocytosis induced by amphiphilic drugs and detergents occur by inhibition of the Band 3 anion transport. Finally, echinocytosis and stomatocytosis induced by non-covalent and covalent modifiers of integral membrane proteins such as agglutinins and digestive enzymes are consistent with the mechanism.     

Mice Deficient in the Putative Phospholipid Flippase ATP11C Exhibit Altered Erythrocyte Shape,Anemia, and Reduced Erythrocyte Life Span

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.     

Thioridazine induces erythrocyte stomatocytosis due to interactions with negatively charged lipids

Despite the fact that thioridazine is used clinically as a neuroleptic drug, little is known about the molecular mechanisms underlying its biological effects, in particular about its interactions with membranes. In the present work we investigate the influence of thioridazine on model and cell membranes, using calorimetry, DPH fluorescence polarization measurements, studies of haemolysis and scanning electron microscopy. The experiments show that thioridazine interacts with lipid bilayers and intercalates into bilayer structure. We found that erythrocyte stomatocytosis induced by the drug might be related to preferential interaction of thioridazine with charged lipids.     

Dibucaine-induced modification of sodium transport in toad skin and of model membrane structures

The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.     

Genomic Organization and 5′-Flanking DNA Sequence of the Murine Stomatin Gene (Epb72)

Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5′-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over ∼25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5′-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins.     

Red blood cell membrane defects

We present an overview of the currently known molecular basis of red cell membrane disorders. A detailed discussion of the structure of the red cell membrane and the pathophysiology and clinical aspects of its disorders is reported. Generally speaking, hereditary spherocytosis (HS) results from a loss of erythrocyte surface area. The mutations of most cases of HS are located in the following genes: ANK1, SPTB, SLC4A1, EPB42 and SPTA1, which encode for ankyrin, spectrin beta-chain, the anion exchanger 1 (band 3), protein 4.2 and spectrin alpha-chain, respectively. Hereditary elliptocytosis (HE) reflects a diminished elasticity of the skeleton. Its aggravated form, hereditary pyropoikilocytosis (HPP), implies that the skeleton undergoes further destabilization. The mutations responsible for HE and HPP, lie in the SPTA1 and SPTB gene, and in the EPB41 gene encoding protein 4.1. Allele alpha LELY is a common polymorphic allele, which plays the role of an aggravating factor when it occurs in trans of an elliptocytogenic allele of the SPTA1 gene. Southeast Asian ovalocytosis derives from a change in band 3. The genetic disorders of membrane permeability to monovalent cations required a positional cloning approach. In this respect, channelopathies represent a new frontier in the field. Dehydrated hereditary stomatocytosis (DHS) was shown to belong to a pleiotropic syndrome: DHS + fetal edema + pseudohyperkalemia, which maps 16q23-24. Splenectomy is strictly contraindicated in DHS and another disease of the same class, overhydrated hereditary stomatocytosis, because it increases the risk of thromboembolic accidents.     

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

The behavior of the human erythrocyte as an imperfect osmometer: a hypothesis

The human erythrocyte does not behave as a perfect osmometer that is its volume does not change as predicted with the change of the tonicity of the medium, as if there was a fraction of the cell water not participating in the osmotic exchange. A mechanism of control of the erythrocyte shape has been previously proposed in which Band 3 (AE1), the protein anion exchanger of Cl(-) and HCO(3)(-), plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation and inward-facing conformation (Band 3(o)/Band 3(i)) contract and relax the membrane skeleton, thus favoring echinocytosis and stomatocytosis, respectively. The equilibrium Band 3(o)/Band 3(i) ratio is determined by the Donnan equilibrium ratio of anions and protons, increasing with it (r=Cl(i)(-)/Cl(o)(-)=HCO 3(i)(-)/HCO 3(o)(-)=H(o)(+)/H(i)(+)). The Donnan ratio is influenced by the erythrocyte transport and metabolic activities. The volume change of the human erythrocyte alters the skeleton conformation as it is accompanied by a change of the membrane curvature. Thus, the mechanism could be a hypothesis for explaining the behavior of the human erythrocyte as an imperfect osmometer since the Donnan ratio controls the Band 3(o)/Band 3(i) ratio which controls the volume by a control of the degree of contraction or relaxation of the skeleton. Predictions made by the hypothesis on the Ponder's coefficient R' values in the presence of sucrose or Band 3 substrates slowly transported as well as on the participation of Band 3 in the osmotic hemolysis appear to be corroborated by previous observations. If the hypothesis was valid, it would follow that there is a pressure gradient across the erythrocyte membrane. The equilibrium volume is antagonistically determined by the Donnan ratio per se and Band 3. Band 3, rather than the ratio of surface-to-volume, primarily controls the osmotic hemolysis.     

Membrane raft actin deficiency and altered Ca-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to < 10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

Membrane effects of trifluoperazine, dibucaine and praziquantel on human erythrocytes

Trifluoperazine (TFP) is a potent antipsychotic agent, dibucaine (DBC) is a local anaesthetic and praziquantel (PZQ) is a highly effective agent against schistosomiasis. The present work was conducted to (i) investigate the cytotoxic effects of TFP, DBC and PZQ on human erythrocyte membranes; and (ii) compare the alterations induced by the cationic drugs (TFP and DBC) with those induced by the uncharged compound (PZQ), in an attempt to have a better insight on the pathways of each drug-membrane interaction. The erythrocyte morphological alterations induced by sublytic concentrations of TFP, DBC and PZQ were evaluated by scanning electron microscopy and expressed quantitatively by the morphological index. Haemolysis and release of membrane lipids (phospholipids and cholesterol) produced by selected concentrations of TFP, DBC and PZQ, were compared with those resulting from the corresponding triple concentrations of each drug. Our results showed that the uncharged molecule of PZQ induces the same morphological alterations (stomatocytosis) as the cationic drugs TFP and DBC. Haemolysis was shown to vary with the drug used and to be concentration-dependent, with values approximately 10-fold more elevated for TFP and DBC than for PZQ, which revealed a maximum of 6% haemolysis for the highest concentration tested. Different concentration-response curves were obtained for lipid elution, although the profiles of cholesterol and phospholipids released were similar for all drugs. Nevertheless, at a fixed rate of 50% haemolysis, TFP induced a approximately 2-fold increment in the elution of cholesterol when compared with that produced by DBC (P<0. 05). The different effects induced by TFP, DBC and PZQ on erythrocyte morphology, haemolysis and lipid exfoliation are related to the physical and chemical characteristics of each compound. These results suggest that distinct cell membrane interaction pathways lead to drug-specific mechanisms of cytotoxicity.     

Membrane proteins in hereditary spherocytosis, elliptocytosis and stomatocytosis

Cell membrane proteins of patients with hereditary spherocytosis, elliptocytosis and stomatocytosis were analyzed by SDS acrylamide gel electrophoresis. From 7 patients with hereditary spherocytosis only one can be supposed to have a reduction of a band with a molecular weight of about 80,000 daltons. 3 members of a family with hereditary elliptocytosis and 1 patient with hereditary stomatocytosis did not differ from the donors. The question of the nature of membrane defect in haemolytic anaemias could not be answered by the results obtained.     

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.     

Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues

Human stomatin (band 7.2b) is a 31-kDa erythrocyte membrane protein of unknown function but implicated in the control of ion channel permeability, mechanoreception, and lipid domain organization. Although absent in erythrocytes from patients with hereditary stomatocytosis, stomatin is not linked to this disorder. A second stomatin homologue, termed SLP-1, has been identified in nonerythroid tissues, and other stomatin related proteins are found in Drosophila, Caenorhabditis elegans, and plants. We now report the cloning and characterization of a new and unusual stomatin homologue, human SLP-2 (stomatin-like protein 2). SLP-2 is encoded by an approximately 1.5-kilobase mRNA (GenBank(TM) accession no. AF190167). The gene for human SLP-2, HUSLP2, is present on chromosome 9p13. Its derived amino acid sequence predicts a 38,537-kDa protein that is overall approximately 20% similar to human stomatin. Northern and Western blots for SLP-1 and SLP-2 reveal a wide but incompletely overlapping tissue distribution. Unlike SLP-1, SLP-2 is also present in mature human erythrocytes ( approximately 4,000 +/- 5,600 (+/- 2 S.D.) copies/cell). SLP-2 lacks a characteristic NH(2)-terminal hydrophobic domain found in other stomatin homologues and (unlike stomatin) is fully extractable from erythrocyte membranes by NaOH, pH 11. SLP-2 partitions into both Triton X-100-soluble and -insoluble pools in erythrocyte ghost membranes or when expressed in cultured COS cells and migrates anomalously on SDS-polyacrylamide gel electrophoresis analysis with apparent mobilities of approximately 45,500, 44,600, and 34,300 M(r). The smallest of these protein bands is believed to represent the product of alternative translation initiated at AUGs beginning with nt 217 or 391, although this point has not been rigorously proven. Collectively, these findings identify a novel and unusual member of the stomatin gene superfamily that interacts with the peripheral erythrocyte cytoskeleton and presumably other integral membrane proteins but not directly with the membrane bilayer. We hypothesize that SLP-2 may link stomatin or other integral membrane proteins to the peripheral cytoskeleton and thereby play a role in regulating ion channel conductances or the organization of sphingolipid and cholesterol-rich lipid rafts.     

A hypothesis of the disc-sphere transformation of the erythrocytes between glass surfaces and of related observations

Erythrocytes suspended at a low hematocrit in a non-buffered isotonic saline change from biconcave discs to spheres between glass surfaces of a slide and of a coverslip with the echinocyte as an intermediate. A pH increase is a major factor responsible for this disc-sphere transformation or glass effect. It is also observed between surfaces made of various polymers and of mica provided that the distance between them is controlled (0.1 mm). The glass effect is antagonized by serum, plasma, serum albumin, ammonium salts and CO2. It is not observed above a 1-2% hematocrit, but is enhanced by gamma-globulins. The sites of reappearance of the spicules are the same and the order of their disappearance is the inverse of the order of their reappearance during the repetitive cycle of the disc-sphere transformation and reversal when a small glass rod is alternatively approached near a site on the erythrocyte surface and withdrawn. A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3 (AE1), the anion exchange protein, plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation (Band 3o) and inward-facing conformation (Band 3i) contract and relax the membrane skeleton, promoting the echinocytosis and stomatocytosis, respectively. The Band 3o/Band 3i equilibrium ratio is determined by the Donnan equilibrium ratio of Cl-, HCO3- and H+ (r=Cl(i)-/Cl(o)-=HCO3i-/HCO3o-=Ho+/Hi+), increasing with it. The mechanism could explain by a change of the Donnan ratio the above observations with the assumptions that polymers are permeable to CO2 and that an unstirred layer slows the propagation of the change occurring at the site of approach of the glass rod to peripheral sites. The presence of HCO3- in serum or plasma may be the basis for the absence of the glass effect in these fluids.     

The basis of echinocytosis of the erythrocyte by glucose depletion

Echinocytosis of erythrocytes by glucose depletion is attributed to adenosine triphosphate depletion, but its process still remains unknown. A mechanism of control of the erythrocyte shape has been previously proposed in which the anion exchanger Band 3, linked to flexible membrane skeleton, has a pivotal role. Recruitments of its inward facing (Band 3(i) ) and outward facing (Band 3(o) ) conformations contract and relax the membrane skeleton, thus promoting echinocytosis and stomatocytosis, respectively. The Band 3(o) /Band 3(i) equilibrium ratio increases with the increase of the Donnan equilibrium ratio, and preferential inward and outward transport by Band 3 of substrates slowly transported are echinocytogenic and stomatocytogenic, respectively. The mechanism suggests the following process. The major organic phosphate 2,3-bisphosphoglycerate is catabolized to lactate to form inorganic phosphate, 3-phosphoglycerate, and adenosine triphosphate. The last two products can be reversibly transformed into 1,3-bisphosphoglycerate and adenosine diphosphate by the glycolytic enzyme phosphoglycerate kinase, thus allowing 2,3-bisphosphoglycerate formation by 2,3-bisphosphoglycerate synthase/phosphatase. The catabolic and cyclic processes initially oppose echinocytosis by increasing the Donnan ratio and outward transport of slowly transported inorganic phosphate by Band 3 (its basic form is transported with a hydrogen ion). Echinocytosis occurs when inward transport of this product becomes predominant. This process can rationalize direct and indirect observations.     

Erythrocyte membrane defects in hemolytic anemias found through derivative thermal analysis of electric impedance

Hereditary hemolytic anemias originate mainly from defects in hemoglobin and plasma membrane proteins. Here, we propose a new method, thermal analysis of impedance, sensitive to membrane defects. It detects three processes in erythrocyte membrane; fall in membrane capacity at 49.5 degrees C and activation of passive PO(4)(2+) permeability at 37 degrees C and inorganic ions at 61.5 degrees C. The denaturation of spectrin is involved in the first process whilst the anion channel is involved in latter processes. Using this method three persons with xerocytosis were found whereby the fall in membrane capacity and spherization of erythrocytes were both postponed (53 degrees C) compared to control (49.5 degrees C). In contrast to control cells, strong activation of passive permeability for Cl(-) at 37 degrees C and sucrose at 61 degrees C were detected that were both eliminated by pre-inhibition of the anion channel with 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). In addition, erythrocytes from 15 patients with various forms of anemia were studied in intact state and after refreshment. The results were compared with the data of clinical laboratory and osmotic fragility test. The final conclusion is that this method detects membrane defects with altered spectrin and anion channel syndrome (hereditary xerocytosis, spherocytosis, poikilocytosis and pyropoikilocytosis, elliptocytosis and stomatocytosis) and, after refreshment, helps differentiate them from the anemia with hemoglobinopathy.     

Effect of bilirubin on toxicity induced by trifluoperazine, dibucaine and praziquantel to erythrocytes

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 micromol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37 degrees C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p<0.05), and in elution of membrane cholesterol (73%, p<0.01) and phospholipids (123%, p<0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (-36%, p<0.01), as well as that of phospholipids by TFP (-28%, p<0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.     

Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology

In recent studies, we showed that flunitrazepam (FNTZ) and other benzodiazepines interact with artificial phospholipid membranes locating at the polar head group region, inducing a membrane expansion, reducing the molecular packing and reorganising molecular dipoles. In the present paper we investigated the possibility that those phenomena could be transduced into changes in the curvature of membranes from natural origin. Hence we studied the effect of FNTZ on cellular morphology using human erythrocyte as a natural assay system. Shape changes of erythrocytes were evaluated by light microscopy and expressed as a morphological index (MI). FNTZ induced echinocytosis in a time-dependent manner with MI values significantly higher than those of control (without drug) or DMSO (vehicle) samples. Lidocaine, a local anesthetic known to induce stomatocytosis by incorporating in the inner monolayer, counterbalanced the concentration-dependent FNTZ crenating effects. FNTZ induced protective effects, compared with control and DMSO, against time-dependent hemolysis. Hypotonic-induced hemolysis, was also lowered by FNTZ in a concentration-dependent manner. Both antihemolytic effects suggested a drug-induced membrane expansion allowing a greater increase in cell volume before lysis. In such a complex system like a cell, curvature changes triggered by drug partitioning towards the plasma membrane, might be an indirect effect exerted through modifications of ionic-gradients or by affecting cytoskeleton-membrane linkage. In spite of that, the curvature changes can be interpreted as a mechanism suitable to relieve the tension generated initially by drug incorporation into the bilayer and may be the resultant of the dynamic interactions of many molecular fluxes leading to satisfy the spontaneous membrane curvature.     

Role of membrane lipid distribution in chlorpromazine-induced shape change of human erythrocytes

This is a study of the morphology and transbilayer lipid distribution of human erythrocytes treated with chlorpromazine (CPZ) over extended time courses. At 0°C, treatment of dilauroylphosphatidyl[1-¹⁴C]choline-labeled erythrocytes with 120 μM CPZ produced an immediate stomatocytic transformation (t_1/2<5 min) with no concurrent change in transbilayer distribution of radiolabeled lipid, as determined by bovine serum albumin extractability. At 37°C, CPZ treatment of cells produced two sequential morphological effects: immediate stomatocytosis (t_1/2<1 min) with no concurrent change in radiolabel transbilayer distribution, followed by gradual increase in stomatocytic extent over several hours, with concurrent redistribution of radiolabeled lipid to the inner monolayer. Cells pretreated with vanadate at 37°C exhibited a triphasic morphological response: CPZ produced immediate stomatocytosis, followed by a transient reversion to echinocytes lasting about 2 h, before returning to stomatocytic morphologies over the next several hours. The echinocytic reversion was accompanied by exposure of phosphatidylserine on the cell surface, as indicated by increased activation of exogenous prothrombinase. These findings suggest that while CPZ induces transbilayer lipid redistribution over extended time periods (which may mediate the complex morphological transformations observed), the early stomatocytic response elicited by addition of CPZ is not due to lipid reorganization.