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.     

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.     

Effect of lead on electrophoretic mobility of rat erythrocytes

Lead often affects the erythrocyte membrane. The relationship between the changes in erythrocyte membrane and the anemia caused by lead is still unclear. Initially, the effect of lead injected intraperitoneally on the electrophoretic mobility of rat erythrocytes was investigated in order to study the relationship between them. As indices of lead exposure, hemoglobin (Hb) levels, hematocrits (Ht), δ-aminolevulinic acid dehydratase (ALA-D) activities and blood lead (blood Pb) levels in the injected rats were also examined. Exposure to lead significantly decreased the mobility of rat erythrocytes. The changes in mobility seemed to be less sensitive than those in ALA-D activity, however, the decreases in mobility were simultaneous with or prior to those in Hb level and Ht. The decreases in mobility were evident to some extent below a blood Pb level of 100 μg/100 ml and generally present at a level of 100 μg/100 ml and over. In the rats exposed to lead a significant negative correlation was found between the mobilities and the logarithms of blood Pb level.     

Interaction of ticlopidine with the erythrocyte membrane

The membrane effects of ticlopidine on the erythrocyte membrane were explored by the spin label method at the proteic and phospholipidic levels. This spectroscopic study was completed by polyacrylamide gel electrophoresis of proteins, measurement of the protection against haemolysis and observation of the erythrocyte shape changes induced by the drug. Two types of effects have been observed. At concentrations higher than 5 × 10^?4 M, ticlopidine is a weak denaturating agent of the membrane proteins. At concentrations of pharmacological interest, the main effect of the drug is a protection against hypotonic haemolysis, and an increase in the fluidity of the membrane phospholipidic core. This last result could explain in part the interesting pharmacological effect of ticlopidine on various circulatory troubles.     

Mechanism of action of disodium cromoglycate--mast cell calcium ion influx after a histamine-releasing stimulus.

Rat peritoneal mast cells release histamine and accumulate ⁴⁵Ca in a dose-dependent manner when concentrations of compound 48/80 ranging from 0·1 to 1·0 μg/ml are incubated with suspensions of the cells for 5 min at 37°. Influx of ⁴⁵Ca stimulated by compound 48/80 can be inhibited to varying degrees by prior addition of disodium cromoglycate. Inhibition was dependent on the concentration of both disodium cromoglycate and compound 48/80. The electrokinetic properties of intact rat mast cells are described; disodium cromoglycate caused a plasma membrane alteration possibly related to Ca²⁺ influx. Cromoglycate increased mast cell electrophoretic mobility but decreased the electrophoretic mobility of rat erythrocytes. The net electrophoretic mobility was a function of terminal sialic acid residues, ionic strenght, and pH. Binding of disodium cromoglycate to Ca²⁺ could not be demonstrated by a variety of sensitive physical techniques. The data support the theory that secretion of mast cell histamine is coupled to Ca²⁺ influx. It is suggested that disodium cromoglycate prevents mast cell histamine release by a plasma membrane alteration which prevents an increase in membrane permeability to Ca²⁺ stimulated by compound 48/80.     

Morphological and biochemical perturbations in rat erythrocytes following in vitro exposure to Fenvalerate and its metabolite.

Erythrocytes are a convenient model to understand the membrane oxidative damage induced by various xenobiotic-prooxidants. In this investigation, we have examined the potency of Fenvalerate (FEN) and its metabolite, p-chlorophenyl isovaleric acid (p-CPIA) to induce oxidative stress response in rat erythrocytes in vitro in terms of lipid peroxidation and effects on selected antioxidant enzymes. Susceptibility of erythrocytes to FEN exposure was further investigated in terms of morphological alterations by scanning electron microscopy and protein damage by gel electrophoresis of erythrocyte ghosts. Following in vitro exposure, FEN caused a significant induction of oxidative damage in erythrocytes at concentrations beyond 0.1 mM as evidenced by increased thiobarbituric acid reactive substances (TBARS) levels. The response was both concentration and time dependent. At higher concentrations, significant decreases in the activities of vital antioxidant enzymes viz., catalase, superoxide dismutase, glutathione transferase and glutathione reductase were also discernible clearly suggesting the potency of both, parent compound and its metabolite to induce oxidative stress in erythrocytes. Scanning electron micrographs of erythrocytes following FEN exposure at higher concentrations revealed various degrees of distortion in shape and ruptured membranes. Furthermore, gel electrophoresis studies revealed consistent and significant aggregation of only band 3 protein in erythrocyte membranes exposed to either FEN or p-CPIA at higher concentrations. These in vitro findings show that FEN and its metabolite have the propensity to cause significant oxidative damage in rat erythrocytes, which is associated with marked damage to membrane proteins. These data suggest that both structural and functional perturbations may ensue in erythrocytes following exposure to FEN at higher concentrations under in vivo situations.     

Propranolol-induced structural changes in human erythrocyte ghost membranes. A spin label study

The effect of propranolol on the structure of human erythrocyte membranes was studied using a spin labeling technique. Changes in electron spin resonance spectra of spin labeled membrane proteins were detected at concentration of the drug corresponding to its antihemolytic effect on intact erythrocytes. The character of spectral changes suggests that propranolol-induced alterations in organization of membrane proteins are connected mainly with perturbation of protein sites located on membrane surface. Propranolol also produces a decrease in order parameter of membrane lipids. The disordering effect is, however, small and detectable only at relatively high concentrations of the drug.     

Lipid emulsions as a novel system to reduce the hemolytic activity of lytic agents: mechanism of the protective effect.

The hemolytic activity of sodium oleate, a high lytic agent, was investigated in different surfactant solutions and lipid emulsion formulations. A new explanation of the protective function of these systems is proposed. It was found that the hemolytic activity of the lytic agent was greatly decreased in solutions and/or dispersions with the surfactant Cremophor EL, Solutol H16 and phospholipids, which can usually build a micellar or liposomal structure. In the case of F68, where the micelle formation is still controversial, the hemolytic activity of the lytic agent was practically not affected and complete hemolysis was observed. In contrast to this, all emulsion formulations, independent of the emulsifier type, showed a stable erythrocyte behavior. Additionally, in the case of lipid emulsions only, a larger amount of the lytic agent could be added without any remarkable increase in the hemolytic activity. As an explanation for these effects it is proposed that the lytic agent is either incorporated into the lipophilic core or intercalates between the emulsifier molecules at the interface. This decreases the direct contact of the lytic agent with the erythrocyte membrane. As a result, the erythrocytes will effectively be protected from hemolytic damage, which can otherwise be induced by such substances.     

Experimental study on prediction of drug toxicity. Drug-induced hemolysis and lysosomes lysis in vitro]

The effect of drugs on isolated rat liver lysosomes, erythrocytes and some erythrocyte enzymes was studied in vitro. Many tranquilizers, antihistaminics and antidepressants, which are known to have the greatest number of adverse reactions in clinical use, had a lytic effect on the particle membranes at concentrations above 2 X 10(-4) M. The present study revealed a good correlation between the p.o. LD50 (rat and mouse) and H30 (molar concentration of drug causing 30% hemolysis). It is suggested, therefore, that the labilizing effect of drugs on rat erythrocytes is useful for testing the lytic action of drugs in vivo.     

Temperature-dependent reaction of flufenamic acid with rat erythrocyte membrane

(1) Effect of flufenamic acid on hypotonie hemolysis of rat erythrocytes was investigated with special reference to temperature at which hemolysis took place. At low temperature such as 0 or 12°, flufenamic acid exhibited a dose-dependent inhibition of hemolysis at relatively low concentrations. This protective effect of flufenamic acid was, however, diminished by increase of temperature of hypotonie medium and finally it caused acceleration of hemolysis at 37°. Chlorpromazine was, in contrast with flufenamic acid, found to be protective at any temperature between 0 and 37°.(2) Flufenamic acid molecules were found to be adsorbed by rat erythrocyte either at 0 or 37°, but the manner of adsorption at 0° was quite different from adsorption at 37°. Kinetically, adsorption at 37° seems to be only passive, whereas some active bindingsites are assumed at the surface of erythrocyte membrane at 0°.(3) Effect on hypotonie hemolysis of a number of clinically useful drugs known as the membrane stabilizer were tested with respect to their temperature-dependency and they were found to be classified into two types; flufenamic acid type and chlorpromazine type. Drugs classified as the former were mostly acidic in the chemical nature and only anti-inflammatory drugs were found in this group. On the other hand, drugs of the latter were basic or non-ionic compounds and most of the tested tranquilizers, antihistamines, local anesthetics and non-ionic anti-inflammatory drugs belonged to this group.     

Effect of oxidative stress and erythropoietin on cytoskeletal protein and lipid organization in human erythrocytes.

Phenylhydrazine (PHX)-mediated damage in human red blood cells has been assessed by monitoring the release of tyrosine from cell proteins as well as using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PHX-treated red blood cells exhibited concentration- and time-dependent tyrosine release. ATP has no effect on the release of tyrosine. This observation is supported by SDS-PAGE pattern of RBC membrane proteins, which shows a correlation between tyrosine release and cytoskeletal protein degradation. PHX requires the presence of erythrocyte cytosolic fraction for the degradation, possibly due to the presence of a proteolytic enzyme in the cytosol. PHX treatment renders the membrane proteins susceptible to the proteolytic attack. Treatment of PHX-exposed erythrocyte with bee venom phospholipase A2 induces the translocation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer surface of the cell membrane. At the same time, phosphatidylcholine (PC) was translocated towards the inner surface, altering the membrane phospholipid asymmetry. Interestingly, increased tyrosine production followed by translocation of phospholipids across the red blood cell membrane by PHX treatment is completely inhibited by 0.2 units of erythropoietin (EP). Our findings suggest that exposure of red blood cells to an oxidant like PHX causes degradation of cytoskeletal protein by an ATP-independent proteolytic pathway and this in turn allows the transbilayer movement of phospholipids across the cell membrane. EP, by scavenging the hydroxyl radicals produced during interaction of PHX with red blood cells, protects the erythrocytes from oxidative attack.     

Effect of methyl isocyanate (MIC) on rat erythrocytes

Changes in surface morphology of rat erythrocytes were studied by scanning electron microscopy after a single and repeated exposure of methyl isocyanate under static conditions. Marked morphological changes (echinocytes and spherocytes) were observed, followed by an increase in osmotic fragility of the erythrocyte membrane. The activity of the membrane bound enzyme adenosine triphosphatase (Na⁺, K⁺, Mg⁺⁺) was inhibited.     

Mapping of mammalian β-adrenoreceptors by use of macromolecular alprenolol derivatives: A comparison with amphibian erythrocyte receptors

The interaction of macromolecular alprenolol derivatives with β-adrenoreeeptors of rat heart, lung, and erythrocytes and frog erythrocytes has been studied. Macromolecular derivatives were prepared by covalently coupling alprenolol to dextrans containing a homologous series of spacer arms of various lengths. The affinity of these macromolecules for frog erythrocyte membranes increased with increasing length of spacer arm. In contrast, the affinity of these macromolecules for all mammalian membrane preparations was weak and insensitive to the length of the spacer arm. The inhibition of [³H]dihydroalprenolol binding to rat heart, lung, and erythrocyte membrane preparations by these macromolecular derivatives was more than 1000-fold less potent than inhibition by alprenolol. The results suggest different structural characteristics between mammalian and amphibian β-adrenoreceptors; however, apparently only small differences between mammalian receptors could be distinguished with these probes.     

The combined effect of IDA and glutaraldehyde on the erythrocyte membrane proteins

A number of investigators have been focusing their attention on the encapsulation of antineoplastic drugs within erythrocytes to diminish their side-effects. Glutaraldehyde is often used as crosslinking agent to link the drugs (including idarubicin, IDA) to the cells. The previous studies indicated that in glutaraldehyde-treated human erythrocytes the elevated level of drugs was observed but also the various changes in the organization of the red cells were noted. In this study, we continue our investigations on the interaction of IDA and glutaraldehyde on the erythrocytes and now we concentrate on the effect of these compounds with the erythrocyte membrane proteins. For this purpose, SDS-gel electrophoresis of the cell proteins was carried out. Additionally, analysis of the disturbances of erythrocytes shape and size, accompanied by the application of flow cytometry and microscopy examination, were undertaken. The fluorimetric method was used to estimate content of IDA in supernatants, after erythrocyte membranes incubation with different glutaraldehyde concentrations. It was observed that glutaraldehyde caused in gradually dependent manner an increase of percent of IDA linked to the cell membrane proteins. After this incorporation, perturbations in the content of the proteins in the cell membrane were observed. The protein aggregates and changes in the level of spectrin, band 3 protein and small mass proteins were noted. The use of flow cytometry and microscopy technique demonstrated also disturbances in the shape and size of erythrocytes. For all tested concentrations of glutaraldehyde, the changes were statistically significant.     

Fluoride-induced death of rat erythrocytes in vitro

Although fluoride (F) in low concentrations is essential for teeth and bone development, its excessive consumption causes numerous deleterious abnormalities in cellular metabolism and physiology often leading to cell death. The present study was performed to establish the toxic F effects inducing the death of rat erythrocytes in vitro. The cells were cultured in the presence of 0.5–16 mM NaF for 1, 5 and 24 h. The progression of erythrocyte death was monitored by cell viability (calcein assay), membrane integrity (hemolysis assay), alterations in the cell morphology (light microscopy) and size (flow cytometry forward scatter), plasma membrane scrambling (annexin V binding). To elucidate the molecular mechanisms underlying F-induced cell death, the cytosolic Ca²⁺ activity (Fluo-3 fluorescence) and ceramide formation (binding of FITC-labeled antibodies) were determined. Exposure of the rat erythrocytes to NaF considerably suppressed their viability and caused partial cell hemolysis within 24 h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1 h and swollen spherocytes within 24 h. The development of NaF-induced erythrocyte death was accompanied by progressive PS externalization at the outer cell membrane, ∼45% of the cells were annexin V-positive in response to 16 mM NaF within 24 h with a small cell population exhibiting necrotic features. The cell death was preceded by considerable accumulation of the free cytosolic Ca²⁺, with statistically significant increase in the number of Fluo-3-positive erythrocytes observed as early as during 1-h incubation with 0.5 mM NaF. NaF also induced moderate ceramide formation. Overall, exposure of the rat erythrocytes to NaF triggers rapid progression of their death in a dose- and time-dependent manner, with appearance of apoptotic cells after 1 and 5 h and transition to necrosis within 24 h. An increase in intracellular [Ca²⁺] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes.     

Adriamycin Induces Protein Oxidation in Erythrocyte Membranes

Abstract: Adriamycin is an anthracycline antineoplastic agent whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. A current hypothesis for adriamycin cardiotoxicity involves free radical oxidative stress. To investigate this hypothesis in a model system, we applied the technique of immunochemical detection of protein carbonyls, known to be increased in oxidized proteins, to study the effect of adriamycin on rat erythrocyte membranes. Erythrocytes obtained from adriamycin-treated rats demonstrated an increase of carbonyl formation in their membrane proteins. Yet, in separate experiments when adriamycin was incubated with rat erythrocyte ghosts, there was no significant increase of membrane protein carbonyls detected. In contrast, isolated erythrocytes incubated with an adriamycin-Fe³⁺ complex exhibited a robust carbonyl incorporation into their membrane proteins in a time-dependent manner. The level of carbonyl formation was dependent upon the concentration of Fe³⁺ known to form the adriamycin-Fe³⁺ complex. When the time course between protein carbonyl formation and lipid peroxidation was compared, protein carbonyl detection occurred earlier than lipid peroxidation as assayed by thiobarbituric acid reactive substances formation. These results are consistent with the notion that oxidative modification of membrane proteins may contribute to the development of the acute adriamycin-mediated toxicity.     

Structure and Function of Snake Venom Cardiotoxins

Abstract

Cardiotoxins from elapid snake venoms are ‘three-finger’ shaped all β-sheet proteins with broad spectrum of biological properties. Members of this class possess strong membrane lytic property. Although, the amino acid sequences of cardiotoxins show more than 90% homology, the members of this class exhibit significant differences in erythrocyte lytic activity. Available evidence suggests that the ‘active site’ in cardiotoxins is constituted by non-polar and charged residues located in Loop 1 and Loop 2.     

Adsorption kinetics of plasma proteins on solid lipid nanoparticles for drug targeting

The interactions of intravenously injected carriers with plasma proteins are the determining factor for the in vivo fate of the particles. In this study the adsorption kinetics on solid lipid nanoparticles (SLN) were investigated and compared to the adsorption kinetics on previously analyzed polymeric model particles and O/W-emulsions. The adsorbed proteins were determined using two-dimensional polyacrylamide gel electrophoresis (2-DE). Employing diluted human plasma, a transient adsorption of fibrinogen was observed on the surface of SLN stabilized with the surfactant Tego Care 450, which in plasma of higher concentrations was displaced by apolipoproteins. This was in agreement with the "Vroman-effect" previously determined on solid surfaces. It says that in the early stages of adsorption, more plentiful proteins with low affinity are displaced by less plentiful with higher affinity to the surface. Over a period of time (0.5 min to 4 h) more interesting for the organ distribution of long circulating carriers, no relevant changes in the composition of the adsorption patterns of SLN, surface-modified with poloxamine 908 and poloxamer 407, respectively, were detected. This is in contrast to the chemically similar surface-modified polymeric particles but well in agreement with the surface-modified O/W-emulsions. As there is no competitive displacement of apolipoproteins on these modified SLN, the stable adsorption patterns may be better exploited for drug targeting than particles with an adsorption pattern being very dependent on contact time with plasma.     

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.     

Effect of calcium on the hemolytic activity of Stichodactyla helianthus toxin sticholysin II on human erythrocytes

Sticholysin II (St II) is a toxin from the sea anemona Stichodactyla helianthus that produces erythrocytes lysis at low concentration and its activity depends on the presence of calcium. Calcium may act modifying toxin interaction with erythrocyte membranes or activating cellular processes which may result in a modified St II lytic action. In this study we are reporting that, in the presence of external K⁺, extracellular calcium decreased St II activity on erythrocytes. On the other hand an increase of intracellular calcium promotes Sty II lytic activity. The effect of intracellular calcium was specifically studied in relation to membrane lipid translocation elicited by scramblases and how this action influence St II lytic activity on erythrocytes. We used 0.5 mmol/L calcium and 10 mmol/L A23187, as calcium ionophore, for scramblases activation and found increased St II activity associated to increase of intracellular calcium. N-ethyl maleimide (activator) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (inhibitor) were used as scramblases modulators in the assays which produced an increase and a decrease of the calcium effect, respectively. Results reported suggest an improved St II membrane pore-forming capacity promoted by intracellular calcium associated to membrane phospholipids translocation.