Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers

Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK_a of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 °C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.     

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.     

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.     

Phosphatidylserine exposure and aminophospholipid translocase activity in Rh-deficient erythrocytes

Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated for seven unrelated cases of Rh_null erythrocytes. Endogenous PS exposure was measured by prothrombinase activity. Out of six cases studied, two Rh_null samples exhibited abnormal aminophospholipid exposure, as suggested by the measurement of a lower Km of factor Xa for prothrombin. Aminophospholipid translocase activity was measured through the transbilayer redistribution of spin-labelled analogues of phospholipids. Provided that incubation conditions allow the maintainance of intracellular ATP level, no difference was observed between Rh_null and control erythrocytes, clearly indicating that the aminophospholipid translocase and Rh polypeptides are different molecular species.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Incorporation and translocation of aminophospholipids in human erythrocytes

Cell morphology changes are used to examine the interaction of exogenous phosphatidylserine and phosphatidylethanolamine with human erythrocytes. Short-chain saturated lipids transfer from liposomes to cells, inducing shape changes that are indicative of their incorporation into, and in some cases translocation across, the cell membrane bilayer. Dioleoylphosphatidylserine and low concentrations of dilauroyl- and dimyristoylphosphatidylserine induce stomatocytosis. At higher concentrations, dilauroylphosphatidylserine and dimyristoylphosphatidylserine induce a biphasic shape change: the cells crenate initially but rapidly revert to a discocytic and eventually stomatocytic shape. The extent of these shape changes is dose dependent and increases with increasing hydrophilicity of the phospholipid. Cells treated with dilauroylphosphatidylethanolamine and bovine brain lysophosphatidylserine exhibit a similar biphasic shape change but revert to discocytes rather than stomatocytes. These shape changes are not a result of vesicle--cell fusion nor can they be accounted for by cholesterol depletion. The reversion from crenated to stomatocytic forms is dependent on intracellular ATP and Mg2+ concentrations and the state of protein sulfhydryl groups. The present results are consistent with the existence of a Mg2+- and ATP-dependent protein in erythrocytes that selectively translocates aminophospholipids to the membrane inner monolayer engendering aminophospholipid asymmetry.     

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.     

Identification of a potential artifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells

The suitability of electron microscope autoradiography for studying the uptake and intracellular localization of lipid vesicles (liposomes) containing radiolabeled saturated phospholipids has been examined. Data are presented showing that preparation of specimens for electron microscope autoradiography by conventional methods is accompanied by significant translocation and intercellular redistribution of radiolabeled saturated lipids, causing spurious labeling patterns. Intercellular redistribution of radiolabeled lipid was demonstrated by mixing glutaraldehyde-fixed mouse L1210 cells that had been incubated with sonicated lipid vesicles containing [³H]dipalmitoyl phosphatidylcholine with an indicator cell population (fixed avian erythrocytes) which had not been exposed to vesicles and showing that after electron microscope processing radiolabeled grains were present in both cell types. The same redistribution artifact also probably affects the intracellular localization of radiolabeled lipids. This artifact is discussed in relation to previous work in which autoradiographic methods have been used for ultrastructural localization of saturated phospholipids in cells and tissues.     

Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers

Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK(a) of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 degrees C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.     

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.     

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

Effect of oxygen concentration on trasverse water proton relaxation times in erythrocytes homozygous and heterozygous for hemoglonin S.

The transverse water proton relaxation times (T₂) of erythrocytes homozygous and heterozygous for hemoglobin S have been measured as a function of oxyhemoglobin concentration at 37 °C. An immediate decrease in T₂ is observed in S/S erythrocytes as the amount of oxyhemoglobin is decreased and the maximum change is observed at 50% deoxyhemoglobin S. In heterozygous erythrocytes, the T₂ remains unchanged until a critical level of deoxyhemoglobin is attained. The critical level of deoxyhemoglobin is a function of the percentage of hemoglobin S in the heterozygous erythrocytes. A Hill plot of the data obtained from S/S erythrocytes gives an n value of around 2.4. These results suggest that the measurement of T₂ is sensitive to the very early stages of the polymerization process. This suggestion is supported by calculations; our T₂ measurements are sensitive to a range of correlation times expected for hemoglobin monomers at one extreme and linear polymers of seven hemoglobin molecules at the other extreme.     

A dilatometric investigation of the effects of general anaesthetics,alcohols and hydrostatic pressure on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The effect of general anaesthetics, alcohols and hydrostatic pressure on the thermal transition in dipalmitoyl phosphatidylcholine multilayer liposomes has been measured using dilatometry. The volume increasse at the transition (ΔV_t) is 0.0350 ± 0.0003 ml/g. the transition temperature (T_t) 41.84 ± 0.09°C and the width of the transition 1.025 ± 0.18°C. ΔH calculated by the Clapeyron-Clausius equation is 8.4 kcal/mol. The n-alcohols C₃C₅ reduced the transition temperature without affecting the transition width which was however, increased by n-hexanol. Trichloroethylene, the fluorescent probe N-phenyl-1-naphthyl-amine, and methoxyflurane all increased the transition width (reduced the cooperativity of the transition) with a simultaneous depression of T_t. Methoxyflurane caused a two-stage transition expansion. Diethyl ether's effect has similarities with both the C₃ and C₆ alcohols. Generally ΔV_t was unaffected by the agents.Pressure increased T_t by 0.0238°C/atm linearly over the range 1–300 atm in both treated and untreated liposomes, and therefore cannot be said to antagonize anaesthetics. In both treated and untreated liposomes ΔV_t and the width of the transition were unaffected by pressure. Pressure thus reverses the effects of anaesthetics on T_t but not their spread of the transition width.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

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.     

Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes

The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.     

Impact of thermohaemolysis-associated membrane alteration on the passive ion permeability and life-span of erythrocytes

Suspending erythrocytes in medium containing sucrose prevented heat-induced lysis at its early stage. This allowed determination of the thermohaemolysis-related ion permeability by measuring the initial rate of the stipulated shrinkage of erythrocytes. Thus, correspondingly, the coefficient P of the ion permeability was calculated for heated human erythrocytes using ouabain-pretreated cells in 37–45°C range and intact cells in 50–58°C range. The values obtained for P obeyed a straight line Arrhenius plot over the entire 37–58°C range suggesting that the ion permeability was activated by a single mechanism earlier identified as relevant to thermohaemolysis. At the 37–58°C range, the activation energy of the P was 250±15 kJ/mol which was markedly different from the value of 56 kJ/mol known for the 10–37°C range. For erythrocytes from five mammals, similar temperature dependencies of the P were obtained over 45–60°C range. For erythrocytes from all species, excluding horse, the P, extrapolated at 37°C, had a value comparable with the known coefficient of the passive, ouabain-insensitive cation permeability at 37°C. For ouabain-treated human erythrocytes at 37°C, the period of thermohaemolysis-related shrinkage in sucrose containing media was found to be about six times shorter than the life-span of intact cells which substantiated the role of the active transport in balancing the thermohaemolysis-related diffusion of ions at 37°C. Consequently, the thermal resistance of erythrocytes, which was earlier related to their sphingomyelin content, was now found also to be in good correlation with their life-span in the circulation of 11 mammals.