Propranolol-induced alterations in rat erythrocyte membrane fluidity and apparent phase-transition temperatures. A depth-dependent process

Propranolol-induced alterations of membrane structure were studied in rat erythrocytes using electron spin resonance techniques. Propranolol produced a concentration-dependent change in membrane fluidity in hydrophobic membrane regions, while producing virtually no change in hydrophilic membrane regions. The changes were associated with depth-dependent alterations in "apparent" phase-transition profiles and transition temperatures. The effects of propranolol on these membrane characteristics were similar to those produced by cholesterol. Propranolol fluidized erythrocyte membranes in a depth-specific fashion, by virtue of its association with the rigid phospholipid acyl chains and cholesterol sterol rings in the hydrophilic regions of the membrane, which produced distant perturbations within the hydrophilic regions of the membrane.     

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus.

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.     

The effect of daunorubicin and glutaraldehyde treatment on the structure of erythrocyte membrane.

The effect of daunorubicin (DNR) and glutaraldehyde on erythrocyte membrane structure was examined by Electron Spin Resonance spectroscopy. Human erythrocytes were incubated with daunorubicin and then with glutaraldehyde to prevent drug efflux. We have demonstrated that DNR alone caused changes in membrane fluidity mainly in the hydrophobic regions of the lipid bilayer. When DNR-preincubated erythrocytes were treated with glutaraldehyde, the alterations in fluidity were observed in the polar regions as well as in the deeper regions of the cell membrane. The incorporation of drug and glutaraldehyde into human erythrocytes also caused conformational alterations in membrane cytoskeletal proteins and changes in the internal viscosity of the cells. The results suggest that glutaraldehyde in the drug-pretreated erythrocytes may lead to significant perturbations in the organization of the plasma membrane lipids and proteins.     

Microscopic studies of Candida albicans and Torulopsis glabrata after in vitro treatment with bifonazole. Freeze fracture electron microscopy

1-[(4-Biphenylyl)-phenylmethyl]-1H-imidazole (bifonazole, Bay h 4502, Mycospor) induced profound ultrastructural alterations in Candida albicans and Torulopsis glabrata as observed by freeze fracture electron microscopy. These alterations were characterized by (1) deformation and decrease in number of invaginations in the protoplasmic fracture face and corresponding ridges on the exoplasmic fracture face, and (2) separation of the plasma membrane from the cell wall, leaving a gap which frequently contained small vesicles. Moreover, parts of the inner half of the plasma membrane of C. albicans cells had been torn off and adhered to the exoplasmic fracture face. Cross-fractures of bifonazole-treated cells of C. albicans showed swollen mitochondria and deposition of lipid granules.     

Effect of chlorpromazine on proteins in human erythrocyte membranes as inferred from spin labeling and biochemical analyses

ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) show two types of label environment: a weakly immobilized component and a strongly immobilized component. Chlorpromazine (CPZ) markedly altered the spectra: at pH 8.0, 3 mM CPZ reduced the amplitude of the spectrum by 40%, and the weakly immobilized component was almost completely removed. In order to clarify the mechanisms of these spectral changes the protein release from erythrocyte membranes induced by CPZ has been followed. CPZ had a weak solubilizing effect on erythrocyte membranes: less than 1% of the membrane protein was released, mainly Band 6. By comparison with the protein release induced by low-salt treatment it was found that the "detergent-like" property of CPZ cannot explain the alterations in the ESR spectra. The nature of the spectral changes induced by CPZ was different from that of changes induced by lowering the pH to 4.5; correlated with other data this shows that changes in organization or conformation of membrane protein cannot explain the CPZ-induced alterations in the ESR spectra. These spectral changes appeared to be due to the reduction by CPZ of the nitroxide free radical. This was documented by the marked reduction of spin concentration of the labeled ghosts in the presence of CPZ resulting in a decrease in amplitude of the ESR spectrum of MSL-labeled erythrocyte ghosts induced by CPZ. The reduction by CPZ of the nitroxide free radical was compared with that induced by ascorbate. It was found that CPZ preferentially reduces the mobile component of the ESR spectrum of MSL-labeled ghosts. The action of CPZ in reducing free radicals may have consequences for patients receiving long-term treatment with phenothiazine derivatives.     

Erythrocyte membrane properties of the chronic alcoholic rat

There is growing evidence for essential or genetic hypertension to be associated with certain membrane abnormalities. We have published previous results on biochemical studies performed on erythrocyte membranes of the Okamoto-Aoki spontaneously hypertensive rat (SHR) and its normotensive control the WKY, reporting evidence of structural and functional alterations in the membranes. These changes could lead to increased calcium permeability and possibly compensatory increase in calcium pump activity that we observed concurrently. Chronic ethanol consumption resulted in mild hypertension in the rats used in the present study. The elevation in blood pressure is not associated with gross membrane changes in the erythrocyte. We noticed, however, that there is a slight elevation in the high affinity Ca2+/Mg2+-ATPase activities together with a trend towards higher osmotic fragility in the red cells of the ethanol-treated rats when compared with controls. These changes could be the result of concurrent reduction in plasma and membrane cholesterol contents also observed in the ethanol-treated animals.     

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.     

An ultrastructural evaluation of acute 1-nitronaphthalene induced hepatic and pulmonary toxicity in the rat

1-Nitronaphthalene is a mutagenic particulate of diesel exhaust which causes acute liver and lung toxicity in rodents. The studies presented here describe morphological changes in the lung and liver at several time intervals following a single injection of 1-nitronaphthalene (100 mg/kg i.p.) in male Sprague-Dawley rats using transmission and scanning electron microscopy. Although both the lungs and liver are injured by 1-nitronaphthalene, the lungs appear to be the primary target organ. Within 4 h of treatment, all 1-nitronaphthalene treated animals exhibited respiratory distress characterized by labored breathing, severe gasping and chromodacryorrhea. The primary ultrastructural alteration were hydropic changes in the non-ciliated bronchiolar (Clara) cells of the distal-most bronchioles of the lung. These were apparent as early as 1 h after 1-nitronaphthalene injection, while adjacent ciliated cells showed no alterations. Over a 24 h period, the bronchioles showed progressive ultrastructural changes leading to necrosis and exfoliation of both ciliated and Clara cells. Interstitial pneumonitis and edema were observed in all animals treated with 1-nitronaphthalene, and was usually associated with bronchioles containing necrotic epithelium. In the liver, ultrastructural changes were observed in the centrilobular hepatocytes at 8 h and consisted of cytomegaly, loss of continuous inner membrane and reduced matrix density of the mitochondria. At 48 h, cellular damage to centrilobular hepatocytes was severe and nearly all mitochondria were damaged. Elevated levels of alanine aminotransferase, aspartate aminotransferase and bilirubin were detected in the serum of animals treated with 1-nitronaphthalene at 8–48 h. In conclusion, 1-nitronaphthalene is a pulmonary toxicant with a unique progression of injury, which primarily damages Clara cells followed by ciliated cells. This disparity is likely due to a difference in the bioactivation of 1-nitronaphthalene. Furthermore, this systemic toxicant also has injurious effects on the centrilobular region of the liver which precedes lung injury.     

Perturbation of the fluidity of the erythrocyte membrane with ionophoric antibiotics and lipophilic anaesthetics.

The fluidity of the rat erythrocyte membrane was evaluated by measurement of excimer fluorescence of an intra-molecular forming fluorophore, 1,3-di(1-pyrenyl)propane. The polyene ionophoric antibiotics, amphotericin B and nystatin, were found to fluidize the erythrocyte membrane, as assessed by the increase in the excimer/monomer fluorescence intensity ratio, by 42 and 13%, respectively, compared with control samples. In contrast, of the peptide ionophoric antibiotics, valinomycin demonstrated about twice the effect which gramicidin A had on depressing the fluidity of the erythrocyte membrane. On the other hand, the general lipophilic anaesthetics, propanidid and althesin, led to an increase, by 70 and 32%, respectively, while the local anaesthetic, procaine, led to a decrease by 20%, in the fluidity of the erythrocyte membrane. These results were explained in the light of the partition coefficients determined for these drugs in decane and native membranes, their affinities for specific membrane components and the changes which they induce in the permeability properties of erythrocyte and other biological membranes.     

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.     

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.     

Characterization of the initial response of engineered human skin to sulfur mustard.

We have used a new approach to identify early events in sulfur mustard-induced, cutaneous injury by exposing human, bioengineered tissues that mimic human skin to this agent to determine the morphologic, apoptotic, inflammatory, ultrastructural, and basement membrane alterations that lead to dermal-epidermal separation. We found distinct prevesication and post-vesication phases of tissue damage that were identified 6 and 24 h after sulfur mustard (SM) exposure, respectively. Prevesication (6 h) injury was restricted to small groups of basal keratinocytes that underwent apoptotic cell death independent of SM dose. Immunoreactivity for basement membrane proteins was preserved and basement membrane ultrastructure was intact 6 h after exposure. Dermal-epidermal separation was seen by the presence of microvesicles 24 h after SM exposure. This change was accompanied by the dose-dependent induction of apoptosis, focal loss of basement membrane immunoreactivity, increase in acute inflammatory cell infiltration, and ultrastructural evidence of altered basement membrane integrity. These studies provide important proof of concept that bioengineered, human skin demonstrates many alterations previously found in animal models of cutaneous SM injury. These findings further our understanding of mechanisms of SM-induced damage and can help development of new countermeasures designed to limit the morbidity and mortality caused by this chemical agent.     

Altered long chain fatty acids composition in Duchenne muscular dystrophy erythrocytes

BACKGROUND: Biochemical abnormalities, increased efflux of soluble enzymes and muscle proteins, and altered permeability of muscle membranes imply the presence of a disorganized erythrocyte membrane in Duchenne muscular dystrophy (DMD). The purpose of the present study was to investigate this hypothesis of a generalized membrane defect. MATERIALS AND METHODS: Twenty-five patients with the disease were analyzed for their erythrocyte lipid composition and for alterations in their fatty acid content compared to twenty-five healthy subjects. RESULTS: DMD patients showed a decreased concentration of total phospholipids compared to healthy volunteers, with striking fluctuations in concentrations of erythrocyte long chain fatty acids. Specifically, the unsaturated fatty acids such as oleic, linoleic and arachidonic acids were significantly decreased in the disease, whereas the saturated fatty acid, palmitic acid was increased in DMD patients compared to healthy controls. CONCLUSION: Our findings suggest an abnormal fatty acid composition and disorganization of erythrocyte membrane in patients with DMD associated with possible functional alterations.     

In vivo and in vitro influence of etretinate on erythrocyte membrane fluidity.

The molecular mechanisms underlying the action of synthetic retinoids have been studied intensively, but they are not fully understood yet. It is well known that retinoids exert their effects on gene expression via the retinoic acid receptor. Some observations suggest that the main aromatic retinoid etretinate (Tigason) exerts its therapeutic effect in psoriasis also through an action on the cell membrane. In this paper, we present the results of previously unreleased experiments (when Tigason was still in use) concerning the in vivo and in vitro influence of etretinate on erythrocyte membrane fluidity in psoriatic patients. Erythrocytes from healthy subjects and topically treated psoriatics were chosen as control groups. Membrane fluidity was measured by the electron paramagnetic resonance (EPR) spin-labelling technique. Erythrocytes from psoriatic patients had lower membrane fluidity, a lower antioxidant activity and a greater susceptibility to peroxidation than those from healthy subjects. After treatment with etretinate, a significant increase in erythrocyte membrane fluidity and in antioxidant activity as well as a decrease in lipid peroxidation were observed in erythrocytes from patients. Local therapy of psoriatic lesions had no influence on the improvement in membrane fluidity and antioxidant activity of erythrocytes. Incubation of erythrocytes from healthy controls and topically treated psoriatics with etretinate in vitro confirmed its fluidizing effect on erythrocyte membranes. These data may indicate that two mechanisms lead to an increase in erythrocyte membrane fluidity in psoriatic patients treated with Tigason: the first one, indirect, by improvement of the antioxidant defence system and cell protection against lipid peroxidation, and the second one, by a direct fluidizing effect of etretinate on the erythrocyte membrane.     

Redox dynamics of alpha-tocopherol in erythrocyte membranes of elderly patients with asymptomatic primary hyperlipidemia.

We investigated the redox dynamics of alpha-tocopherol in plasma and erythrocyte membranes in elderly patients with asymptomatic primary hyperlipidemia divided into three groups (hypercholesterolemia, hypertriglyceridemia and low high-density lipoprotein cholesterol levels) and in healthy elderly subjects to assess the antioxidative status of alpha-tocopherol. alpha-Tocopherol and alpha-tocopherolquinone were determined by high-performance liquid chromatography using a redox detection mode. In the erythrocyte membrane, there was no difference in the alpha-tocopherol concentration between hyperlipidemic and healthy subjects. The alpha-tocopherolquinone/alpha-tocopherol ratio in plasma and erythrocyte membrane, and the alpha-tocopherol in erythrocyte membrane/alpha-tocopherol in plasma ratio were significantly lower in elderly patients with hypercholesterolemia or hypertriglyceridemia. These findings suggest that the uptake ratio in erythrocyte membranes and the antioxidative activity of alpha-tocopherol in both plasma and erythrocyte membranes are decreased in elderly hyperlipidemic patients. These decreases may promote membrane lipid peroxidation or accelerate atherosclerosis.     

Possible artefacts in the in vitro determination of antimalarial activity of natural products that incorporate into lipid bilayer: apparent antiplasmodial activity of dehydroabietinol,a constituent of Hyptis suaveolens

Dehydroabietinol isolated from Hyptis suaveolens (L.) Poit. was found to inhibit growth of chloroquine-sensitive as well as chloroquine-resistant strains of Plasmodium falciparum cultivated in erythrocytes in vitro (IC 50 26-27 microM). However, erythrocytes exposed to dehydroabietinol were transformed in a dose-dependent manner towards spherostomatocytic forms with concomitant formation of endovesicles, as disclosed by transmission electron microscopy. The erythrocyte shape alterations caused by dehydroabietinol correlated well with its apparent IC 50 value. Thus, dehydroabietinol incorporates into the erythrocyte membrane, and since invasion and survival of Plasmodium parasites is known to depend on the function of the erythrocyte membrane, the observed antiplasmodial effect of dehydroabietinol is presumably an indirect effect on the host cell. Because of these findings, microscopic investigations should be generally used to support claims of antimalarial effects of apolar natural products.     

Effects of brazilin on lipid and phosphatidyl fatty acid composition of erythrocyte membrane in streptozotocin-induced diabetic rats

In diabetes, the abnormal increase of the membrane cholesterol/phospholipid ratio (C/PL) is considered to be the main reason for the decreased membrane fluidity, which then results in impaired erythrocyte deformability and subsequent microcirculatory disturbances. In this study, we examined the effects of brazilin on lipid and phosphatidyl fatty acid composition of erythrocyte membranes in streptozotocin induced diabetic rats. Treatment of brazilin (10 mg/kg or 100 mg/kg for 2 weeks, i.p) altered phospholipid and cholesterol contents in diabetic erythrocyte membranes. The C/PL ratio of brazilin treated groups decreased compared with that of diabetic control group while no change was observed in normal erythrocytes. In streptozotocin induced diabetic rats, alterations in phosphatidyl fatty acid composition of erythrocyte membranes were observed and brazilin could reverse these alterations. Arachidonic acid level retumed to a normal level while linoleic acid level remained unchanged by the treatment of brazilin. The results suggest that brazilin might increase erythrocyte membrane fluidity which plays a key role in regulating erythrocyte deformability, thereby it could exert positive effects on microcirculatory disturbances.     

Effect of heparin and hypobaric hypoxia on the blood electrolyte composition, ATPase activity and the membrane charge of erythrocytes

The effects of hypoxia (an "altitude" of 6000 m) and heparin on contents of ions of sodium, potassium, calcium, and magnesium in the plasma and erythrocytes, on ATPase activity and erythrocyte membrane charge in albino rats were studied. Heparin was shown to decrease levels of ions of sodium, potassium, in the plasma and erythrocytes, to increase the negative charge of erythrocytes and membrane permeability for potassium ions. Changes in concentrations of sodium, potassium, calcium and magnesium ions after keeping the rats in a barochamber are more marked on the 2nd day than on the 4th day. Na+, K+-ATPase activity of erythrocyte ghosts first decreased, but on the 4th day increased. Heparin action tended to compensate for changes in contents of the studied ions, ATPase activity and erythrocyte membrane changes as well as changes in gradients of potassium, calcium and magnesium ions concentrations in the erythrocyte-plasma system caused by hypobaric hypoxia.     

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

BACKGROUND AND PURPOSE

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.

EXPERIMENTAL APPROACH

Erythrocytes from healthy donors were subjected to incubation with dapsone and DDS-NHOH for varying times and the band 3 protein tyrosine-phosphorylation process, band 3 aggregation, membrane alteration and IgG binding were all examined and compared with erythrocytes from two patients receiving dapsone therapy.

KEY RESULTS

The hydroxylamine derivative, but not dapsone (the parent sulphone) altered membrane protein interactions, leading both to aggregation of band 3 protein and to circulating autologous antibody binding, shown in erythrocytes from patients receiving dapsone therapy. The band 3 tyrosine-phosphorylation process can be used as a diagnostic system to monitor membrane alterations both in vitro, assessing concentration and time-dependent effects of DDS-NHOH treatment, and in vivo, evaluating erythrocytes from dapsone-treated patients, in resting or oxidatively stimulated conditions.

CONCLUSIONS AND IMPLICATIONS

DDS-NHOH-induced alterations of human erythrocytes can be directly monitored in vitro by tyrosine-phosphorylation level and formation of band 3 protein aggregates. The latter, together with antibody-mediated labelling of erythrocytes, also observed after clinical use of dapsone, may lead to shortening of erythrocyte lifespan.     

Cellular immunotoxicity of amyl nitrite

Functional deficits in lymphocyte interaction following occasional or chronic exposure to inhaled nitrites may be a potential contributing but not the etiologic factor in the acquired immunodeficiency syndrome (AIDS). We evaluated the effect of amyl nitrite vapors on mononuclear cell function and demonstrated functional deficits and structural alterations in these cells. In this closed, in vitro system, exposure of cells to amyl nitrite for up to 30 minutes did not effect cell viability. The functional deficits demonstrated were: inhibition of lymphocyte erythrocyte (E) rosette formation, a suppression of lymphocyte mitogen (phytohemagglutinin) and antigen (cytomegalovirus) transformation, a block in the S, G2 and M phases of cell cycling and diminished cell cytotoxicity to CMV infected cells. These effects on cellular function were demonstrated following 5, 10, and 15 minutes of amyl nitrite vapor exposure; some effect on all cellular functions was demonstrated at 5 minutes. The structural alterations seen on scanning and transmission electron micrographs were: reduction of filapodia, smoothing of the cell profile, cytoplasmic protrusions with pseudopod-like extensions, an increase in rough endoplasmic reticulum with swollen cisternae, alterations in size and distribution of golgi components and exocytotic vesicles in the outer membrane of the nuclear envelope. These vesicles and increased membrane proliferation suggests an effect on the membrane synthesis mechanism in these cells. These effects may be a potential factor in the alterations of phenotypic markers on T lymphocyte populations, as well as, a potential contributing factor in the functional deficit of mononuclear cells in patients with AIDS.