Interactions of surface-active alkyltrimethylammonium salts with the erythrocyte membrane.

The interactions of ahomologue series of surface-active alky Itrimethylammonium salts (C₁₀-C₂₀) with the rat erythrocyte membrane were studied. The surfactants were found to have a biphasic effect on the erythrocyte membrane. At low concentrations they protected or stabilized erythrocytes against hypotonic haemolysis, but at higher concentrations they caused rapid haemolysis. The stabilizing and lytic effect increased with an increase in length of the alkyl chain to maximum activity at about C₁₆. It is suggested that laminar-micellar transitions in the lipid bilayer of the membrane are responsible for the lytic activity of the surfactants. Micellar regions in the lipid bilayer abolish the ability of the membrane to prevent the free exchange of ions, and haemolysis of the cell results from a secondary osmotic effect. The stabilizing effect, on the other hand, is proposed to stem from an expansion of the membrane caused by a fluidizing effect of the surfactants on the lipid bilayer. Binding studies with the C₁₆ homologue revealed that at a concentration causing 50 per cent haemolysis in an isotonic solution there are about 780,000 molecules bound per μm² of the erythrocyte membrane. At a concentration giving 50 per cent protection against hypotonic haemolysis, the number of molecules bound per μm² of the erythrocyte membrane was estimated to be 190,000.     

Dark effect of 8-methoxypsoralen on human erythrocytes

Abstract— Furanocoumarin 8-methoxypsoralen (8-MOP) (1–100 μg mL^?1) in the dark showed a protective affect against hypotonic haemolysis of the erythrocyte membrane. However, the effect against heat-induced haemolysis was dependent on the concentration of 8-MOP; lower concentrations of 8-MOP showed an inhibiting effect, whereas higher concentrations caused acceleration of haemolysis. 8-MOP was not able to induce haemolysis in isotonic solution at 20 or 37 C. Reaction of erythrocytes with 8-MOP in the dark resulted in a shrinkage of the cells and alterations of their shapes. We conclude that modification of erythrocyte membrane by 8-MOP proceeds via reaction with membrane lipids and proteins. This indicates that the effect on the cell membrane plays an important role in the mechanism of the action of 8-MOP on the cells.     

The Action of Thioridazine and Promazine on Biological Membranes: Relationship between ATPase Inhibition and Membrane Stabilization

The effects of promazine and thioridazine on hypotonic haemolysis of human erythrocytes are compared with their effect on Na⁺-K⁺-ATPase of washed human erythrocyte ghosts. Promazine (5 × 10^-5 - 5 × 10^-4 M) and thioridazine (10^-5 - 10^-4 M) stabilize erythrocytes against hypotonic haemolysis, but have lytic effects at higher concentrations. Both drugs inhibit Na⁺-K⁺-ATPase of erythrocyte membranes. This inhibition is slight at drug concentrations which have a membrane-stabilizing action, but is complete and irreversible at lytic concentrations of the drugs. Promazine and thioridazine also inhibit Na⁺-K⁺-ATPase in a membrane fraction prepared from rat hearts. The relative order of potency of the two drugs in this respect does not reflect their relative potency as general cardiac depressants. It is concluded that Na⁺-K⁺-ATPase is not a primary target for the action of promazine and thioridazine in the rat heart. It is suggested that inhibition of Na⁺-K⁺-ATPase by these agents is secondary to more general alterations of the physical properties of the cell membrane.     

The study of the quercetin action on human erythrocyte membranes

Quercetin is a naturally occurring flavonoid that exerts multiple pharmacological effects. In our previous study, we showed that quercetin greatly affects the lipid membrane. In this report, a study of quercetin on human erythrocyte membrane has been performed to determine the influence of this flavonoid on the fluidity and the conformational changes of membrane proteins. An additional aim of the study was to find how quercetin presence affects the resistance of membrane to haemolytic agents. The results showed that incorporation of quercetin into the erythrocyte membranes caused the changes of the partition coefficient of the Tempo spin label between the water and polar head group phases. In the studies, the W/S ratio has been used as a monitor of changes in protein conformation and in the environment within the membrane. It was observed that quercetin caused an increase in protein-protein interactions in human erythrocyte membranes. Haemolytic action of quercetin in the dark was also investigated. This compound showed protective effect against hypotonic haemolysis. However, in the heat-induced haemolysis quercetin caused acceleration of haemolysis. Dark reaction of erythrocyte with quercetin resulted in a shrinkage of the cells and alteration of their shapes. From the results we have concluded that modification of erythrocyte membrane by quercetin proceeds via reaction with membrane lipids and proteins.     

The influence of the antiviral drugs amantadine and rimantadine on erythrocyte and platelet membranes and its comparison with that of tetracaine

The influence of the antivirus drugs amantadine and rimantadine and of the anionic analogue 1-adamantane-carboxylic acid on a range of properties of human erythrocyte membrane and of thrombocytes has been compared with the effect of the local anaesthetic tetracaine. At low antiviral drug concentrations the abilities of the drugs to induce erythrocyte shape change and suppress osmotic haemolysis were quantitatively proportional to their clinical potency (rimantadine more effective than amantadine at the same concentration). Rimantadine was also more effective than amantadine in suppressing influenza virus-erythrocyte fusion and viral induced haemolysis. The antiviral drug effects were qualitatively similar to those induced by tetracaine. At the quantitative level, tetracaine was more efficient than the antiviral drugs in inhibiting osmotic haemolysis, virus membrane fusion and platelet aggregation. In the absence of any specificity of the antiviral drug effects we argue for a lysosomotropic mode of drug action, i.e. that the drugs modify virus-membrane interactions by changing the endosomal or lysosomal pH.     

Protective Effects of Dilazep and its Derivative K-7259 on the Haemolysis Induced by Amphiphiles in Rat Erythrocytes

The effects of dilazep and K-7259, a dilazep derivative, on the haemolysis (as evidenced by release of haemoglobin) induced by palmitoyl-l -carnitine (PAL-CAR) or palmitoyl 1-α-lysophosphatidylcholine (PAL-LPC) have been determined in rat erythrocytes. At concentrations above the critical micelle concentration both PAL-CAR and PAL-LPC induced haemolysis; the concentrations of PAL-CAR and PAL-LPC producing 50% haemolysis were approximately 13 and 14 μm , respectively. The 50% haemolysis induced by PAL-CAR or PAL-LPC was attenuated by dilazep (1, 10 or 100 μm ) but not at the highest concentration used (1 mm ). K-7259 attenuated the 50% haemolysis induced by PAL-CAR or PAL-LPC at concentrations ranging from 1 μm to 1 mm . Similarly, dilazep (1 to 100 μm ) and K-7259 (1 μm to 1 mm ) significantly or insignificantly attenuated the 25% and 75% haemolysis induced by PAL-CAR or PAL-LPC. Neither dilazep nor K-7259 affected micelle formation by PAL-CAR or PAL-LPC, nor, at concentrations of 1 and 10 μm , did they attenuate the haemolysis induced by osmotic imbalance (hypotonic haemolysis). These results suggest that both dilazep and K-7259 protect the erythrocyte membrane from the damage induced by PAL-CAR or PAL-LPC. The protective effects of dilazep and K-7259 are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.     

The Comparative Effect of Chlorhexidine and Cetrimonium Bromide on Erythrocyte Membranes

Abstract The effect of chlorhexidine (Chx) and cetrimonium bromide (Ctab) on the haemolysis of erythrocytes has been studied. A concentration dependent release of haemoglobin was observed in isotonic media, with total haemolysis at 80 nmol Chx per 10⁷ cells and 20 nmol Ctab per 10⁷ cells. The rate of haemolysis induced by Chx shows a biphasic pattern in contrast to the uniphasic pattern of Ctab. In concentrations below 10 nmol per 10⁷ cells, Chx produces more haemolysis than Ctab whereas the opposite effect is observed at higher concentrations. Chx and Ctab stabilize the erythrocyte membrane against hypotonic shock. The concentrations of Chx and Ctab giving maximal stabilization are 0.25 nmol per 10⁷ cells and 2 nmol per 10⁷ cells respectively. The normal biconcave disc form of the erythrocytes is converted to cup forms and in-vaginated spheres by Chx and Ctab. The binding of Chx to erythrocytes in isotonic media increases linearly with the total concentration up to about 25 nmol Chx per 10⁷ cells where the curve has a point of inflection. With more than 25 nmol Chx per 10⁷ cells the amount of Chx bound again increases linearly up to 120 nmol per 10⁷ cells. The slope of the curve above the point of inflection is approximately 4 times that of the curve below this point. No level of saturation of the binding is observed at the concentrations of Chx used in this study. The mode of action of Chx on erythrocyte membranes is discussed in the light of the present results.     

The effect of propranolol on the osmotic fragility of red cells and liposomes and the influence of the drug on glycerol transport across the membrane of red cells

Propranolol has been found to have different effects on the stability of red-cell membranes and model phospholipid membranes (liposomes). Its effect on red-cell membranes is biphasic. At lower concentrations the drug protects the cells from hypotonic haemolysis, whereas at high concentrations it promotes haemolysis. In contrast to red cells, liposomses were destabilized by propranolol. The lytic effect on lipid membranes was observed within the same concentration range of the drug at which it exerts an antihaemolytic effect on red cells. Propranolol has been shown also to perturb glycerol transport in red cells. It stimulates passive diffusion of glycerol into bovine red cells and inhibits facilitated diffusion in human red cells. The experimental results suggest that although both membrane lipids and proteins are involved in drug-membrane interaction, the crucial role in propranolol-induced red-cell membrane stabilization is played by the protein component.     

Effect of Nitazoxanide on Erythrocytes

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis‐like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca²⁺‐activity ([Ca²⁺]_i). The Ca²⁺‐sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca²⁺]_i was estimated from Fluo3‐fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin‐V‐binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48‐hr exposure to nitazoxanide (1–50 μg/ml) did not significantly modify [Ca²⁺]_i but significantly increased ceramide formation, decreased forward scatter (≥10 μg/ml), increased the percentage of annexin‐V‐binding erythrocytes (≥10 μg/ml) and, at higher concentrations (≥20 μg/ml), stimulated haemolysis. The stimulation of annexin‐V‐binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.     

Stimulation of Suicidal Erythrocyte Death by Ribavirin

Ribavirin is widely used in the treatment for viral disease such as chronic viral hepatitis. Side effects limiting the use of the drug include haemolytic anaemia. If challenged by stimulators of haemolysis, erythrocytes may enter suicidal death or eryptosis, thus preventing the release of haemoglobin into circulating blood. Eryptosis is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i). This study explored whether ribavirin modifies [Ca²⁺]_i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, haemolysis from haemoglobin release and [Ca²⁺]_i from Fluo‐3 fluorescence. A 48‐hr exposure to ribavirin (≥8 μg/ml) was followed by a significant increase in [Ca²⁺]_i, a significant decrease in forward scatter and a significant increase in annexin V binding. The annexin V binding after ribavirin treatment was significantly blunted but not abolished in the nominal absence of extracellular Ca²⁺. In conclusion, ribaverin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca²⁺.     

Effect of Dermaseptin on Erythrocytes

Dermaseptin, an antimicrobial peptide participating in the host defence against pathogens, interacts with the membrane of target cells, leading to membrane permeabilization and eventual cell lysis. Dermaseptin has previously been shown to trigger haemolysis. Prior to haemolysis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase in cytosolic Ca²⁺ activity [(Ca²⁺)]_i and formation of ceramide. This study explored whether dermaseptin modifies [Ca²⁺]_i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine exposure from annexin‐V binding, haemolysis from haemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca²⁺]_i from Fluo3‐fluorescence. A 48‐hr exposure to dermaseptin (50 μM) was followed by a significant increase in [Ca²⁺]_i, a significant increase ceramide abundance, a significant decrease in forward scatter and a significant increase in annexin‐V binding. The annexin‐V binding after dermaseptin treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. Dermaseptin triggers eryptosis, an effect at least partially due to entry of extracellular Ca²⁺.     

Cytotoxicity of myeloperoxidase-activated catechols: oxidative injury to the red blood cell.

The effects of two catechols (1,2-benzenediol and nordihydroguaiaretic acid) on the myeloperoxidase-Cl(-)-H2O2 antimicrobial/cytotoxic system of the human neutrophil were investigated. To determine the cytotoxicity of myeloperoxidase-generated oxygen metabolites (mainly chlorinated oxidants such as hypochlorite) and catechol oxidation products, the well characterized erythrocyte was used as a target. At relatively low concentrations (less than 10 microM), the catechols acted as redox catalysts by stimulating the generation of chlorinated oxidants. This is visualized as a promotion of haemolysis which reached a maximum and then decreased again with increasing concentrations of the catechol. In this respect, the dicatechol, nordihydroguaiaretic acid, was more potent. At higher concentrations, the catechols competed more effectively with Cl- as electron donors and the generation of chlorinated oxidants decreased with a consequent decrease in haemolysis. Above 200 microM nordihydroguaiaretic acid, complete haemolysis occurred which might be due to high membrane concentrations of the catechol due to its high lipid solubility. In contrast, high 1,2-benzenediol concentrations did not induce haemolysis. The catechols stimulated methaemoglobin formation in a concentration-dependent fashion with 1,2-benzenediol more potent than nordihydroguaiaretic acid. There was some correlation between membrane microviscosity and haemolysis which in turn did not correlate with haemoglobin oxidation. No direct correlation existed between intracellular methaemoglobin formation and the precipitation of haemoglobin oxidation products on the membrane. Disulphide crosslinks were not involved in the covalent polymerization of haemoglobin subunits.     

The Binding of CTAB,a Cationic Surfactant,to the Rat Erythrocyte Membrane

Abstract The adsorption of CTAB (cetyltrimethylammonium bromide) to the rat erythrocyte membrane was studied by determining the electrophoretic mobility of erythrocytes treated with CTAB and by SDS polyacrylamide electrophoresis of membrane proteins from erythrocytes treated with ¹⁴C-CTAB. At low concentrations of CTAB there was only a small reduction in the electrophoretic mobility of the erythrocytes. At lytic concentrations the electrophoretic mobility of the erythrocytes was markedly reduced. Alterations at the cell surface were found to be a more likely reason for the reduction in the electrophoretic mobility than interactions between the surfactant and charged groups at the cell surface. Very small amounts of radioactivity were found to be associated with the protein and sialoglycoprotein bands of the polyacrylamide gels. It is suggested that the adsorption of CTAB to the rat erythrocyte membrane does not involve electrostatic interactions between the surfactant and negatively charged groups of the sialoglycoproteins and that membrane proteins do not play a major role in the lytic events.     

Nasal absorption in the rat: IV. Membrane activity of absorption enhancers

An erythrocyte model has been used to investigate the membrane activity of various agents which have been employed as enhancers of intranasal drug absorption. Enhancers were ranked according to haemolytic activity as follows: LPC-stearoyl > laureth-9 > STDHF > LPC-decanoyl > LPC-caproyl = DEAE-dextran = PBS (pH 7.0). The concentrations known to significantly enhance nasal absorption were generally, though not exclusively, in excess of that causing haemolysis, suggesting that membrane activity is involved in their mechanism of action, but that other mechanisms may also be important. In some cases, the effects on the erythrocyte membrane contrasted with those observed on the nasal epithelium in vivo, indicating the influence of the local environment of the nasal cavity on the activity of enhancing agents and the importance of histological studies in vivo.     

The influence of dimethylsulfoxide on the red cell membrane

Dimethylsulfoxide protects human red blood cells against haemolysis. This protection is caused by a drug-induced increase of the critical volume of the cells. Protection is abolished, when the cells are preincubated with dimethylsulfoxide in isotonic NaCl. Dimethylsulfoxide-induced K⁺ loss from red blood cells appeared to depend on the red cell volume. Presumably this phenomenon is related to volume dependent conformation changes at the cell surface, as described in recent literature. Further, dimethylsulfoxide causes a strong decrease of the red blood cell deformability, reflecting its profound influence on the physico-chemical properties of biomembranes. This effect also depends on the red cell volume. The results indicate that dimethylsulfoxide has similar effects on biomembranes as a wide variety of anesthetics. The effects of these anesthetics on biomembranes are usually attributed to a hydrophobic interaction with the membrane. It is shown, however, that a different interpretation of the results is also possible.     

Evolution of structural changes leading to haemolysis in human erythrocytes treated with SK&F 95018, an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties

SK&F 95018 is an antihypertensive compound with combined vasodilator and β-adrenoceptor antagonist properties, which, when given to dogs by intravenous infusion, rapidly produced symptoms of intravascular haemolysis. The haemolytic potency of SK&F 95018 was confirmed in vitro using human erythrocytes, was concentration dependent and was associated with dose-specific morphological changes as determined by scanning and transmission electron microscopy. Treatment of washed human erythrocytes with 0.5 mm-SK&F 95018 for up to 30 min resulted in gradual transformation from the biconcave discocyte to stomatocyte forms. Stomatocytes developed more rapidly on exposure to 2 mm-SK&F 95018, exhibited unilateral, multifocal invaginations by 2 min and evolved into spherocytic erythrocytes showing many membrane protuberances and invaginations. At the highest treatment level (10 mm) the crenated erythrocytes seen at time 0 transformed rapidly into spherocytes with many membrane-bound, surface projections that were retained in erythrocyte membrane ‘ghosts’. The membrane-active properties of SK&F 95018 were investigated in a phospholipid-membrane model (an aqueous dispersion of side-chain perdeuterated dipalmitoylphosphatidylcholine) by proton and deuterium nuclear magnetic resonance spectroscopy. The results suggest that SK&F 95018, with its molecular dual polarity, inserts into and effectively disrupts the intergrity of biological membranes by micellar reorganization of the bilayer plasmalemma. The slow change in shape from discocyte to stomatospherocyte at the lowest concentration (without the development of membrane-associated protuberances) suggests a disruptive effect on the erythrocyte osmotic balance by gradual cumulative drug insertion into the membrane. At higher concentrations this initial effect (leading to cell swelling) appears to proceed contemporaneously with micellar membrane reordering, producing membrane protuberances.     

Isaxonine base is a strong perturber of phospholipid bilayer order and fluidity--a differential scanning calorimetry and spin labeling study

The effects of the neurotropic drug isaxonine on fully hydrated dipalmitoyl-phosphatidyl-choline (DPPC) bilayers has been studied in the temperature range 0 degree-60 degrees, using differential scanning calorimetry and electron spin resonance spectroscopy, with two stearic acid spin labels. At low concentration (1% mol/mol), isaxonine is trapped in the polar interface and enhances the phospholipid multibilayers organization in the gel state. In contrast, at high concentration (30% mol/mol), the drug disorganizes the phospholipidic structures and may induce domain formation by phase separation. The strong interactions of isaxonine at the lipid-water interface change the ionization state of the stearic acid spin labels which become totally ionized. Then isaxonine acts as a modifier of the surface pH of the bilayer. The strong membrane effects of isaxonine may explain in part its pharmacological properties in vivo.     

Toxic effects of the anticancer drug epirubicin in vitro assayed in human erythrocytes

Epirubicin is a cytotoxic drug used in the treatment of different types of cancer and increasing evidence suggests that its target is cell membranes. In order to gain insight on its toxic effects, intact red blood cells (RBC), human erythrocyte membranes and molecular models were used. The latter consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes found mainly in the outer and inner monolayers of the human erythrocyte membrane, respectively. The results obtained by X-ray diffraction displayed that epirubicin induced structural perturbations in multilayers of DMPC. Differential scanning calorimetry (DSC) showed that epirubicin disturbed the thermotropic behavior of both DMPC and DMPE vesicles, whereas fluorescence spectroscopy demonstrated alterations in the fluidity of DMPC vesicles and the erythrocyte membrane. Scanning electron microscopy (SEM) revealed that epirubicin changed the normal discoid form of RBC to echinocytes and stomatocytes. Electron paramagnetic resonance (EPR) disclosed that this drug induced conformational changes in the erythrocyte membrane proteins. These findings demonstrate that epirubicin interacts with lipids and proteins of the human erythrocyte membrane, effects that might compromise the integrity and function of cell membranes. This is the first time that its toxic effects on the human erythrocyte membrane have been described.     

Cell culture as a test system for toxicity

Appropriate cell culture systems provide a useful additional method of screening for toxicity, in spite of the obvious problems of relating in vitro effects of test compounds at the cellular level to their effects in the whole animal. It is shown that a wide range of chemically disimilar molecules have a reversible inhibitory activity on the growth of primary cultures of monkey kidney cells. The potency of these compounds correlates with their lipid solubility, suggesting that the cell membranes may be their main site of action. Support for this is obtained by the correlation of inhibitory activity and the ability of the same compounds to stabilize the erythrocyte membrane against hypotonic haemolysis which is known to be a direct effect of interaction with the erythrocyte membrane. It is suggested that the ability of the food additive butylated hydroxytoluene to act as a potent inhibitor of cellular growth may account for its reported ability to prolong the life span of mice.     

The binding of the radioprotective agent cysteamine with the phospholipidic membrane headgroup-interface region

The interaction of the aminothiol radioprotector cysteamine (beta-mercaptoethylamine) (CYST) with dipalmitoylphosphatidylcholine (DPPC) artificial membranes has been studied by differential scanning calorimetry (DSC), turbidimetry and spin labeling. This hydrophilic molecule displays a biphasic, concentration-dependent binding to the phospholipidic head groups at neutral pH. In the CYST/DPPC molar ratio 1:160-1:2 (mole/mole) an increasing ordering effect is observed. At high concentrations (over 3:1 ratio), this ordering effect decreases. With the symmetric disulfide dimer cystamine, the biphasic effect is not shown and the membrane rigidity decrease is obtained only at concentration ratio higher than 1:1. The charge repartition of the cysteamine molecule has been shown to be disymmetric, +0.52 e on the NH3 group and +0.19 e on the SH extremity, [38] whereas the cystamine molecule is electrostatically symmetrical. These properties could be related to their membrane effects. With cysteamine, at a low concentration, an electrostatic bridging between the negatively charged phosphate groups of the polar heads induces the increase in membrane stability: the molecules behave like a divalent cation. At high concentrations a displacement of the slightly charged SH extremity by the amine disrupts the bridges and induces the decrease in rigidity: the drug behaves like a monovalent cation. Due to its symmetric charge and its double length, such an effect is not observed with cystamine. This study could bring further information about the interactions between cysteamine and polyelectrolytic structures (ADN for example) and about the radioprotective properties of this drug.