Uptake of cadmium by isolated human red blood cells

Uptake of Cd by human RBC in vitro was studied. The uptake was found to be biphasic with a rapid initial phase followed by a slower second phase which was still increasing at the time of the last experiment (60 min). Both the phases were found to be independent of metabolically derived energy and unaffected by zinc in the incubation medium.     

Ethynylestradiol alters lipid composition and phosphatidylethanolamine metabolism in red blood cells

Treatment of female rats with ethinylestradiol at a dose of 60 micrograms/rat, daily for 21 days, produced marked changes in red blood cell lipids. Cholesterol was decreased by 22% and total phospholipids were increased by 13%, resulting in a 31% decrease in the cholesterol to phospholipid ratio. The mass distribution of phosphatidylcholine and phosphatidylethanolamine relative to total phospholipids was unchanged. Whereas control red cells incorporated preferentially fatty acids in phosphatidylcholine, ethinylestradiol stimulated their incorporation specifically in phosphatidylethanolamine, where increases occurred with palmitic acid (+75%), oleic acid (+68%) and arachidonic acid (+31%). Incorporation in phosphatidylcholine was unaffected with any of the 3 fatty acids. The stimulation of fatty acid incorporation in phosphatidylethanolamine is likely to reflect an estrogen-dependent increase in turnover rate of fatty acids in this phospholipid. Such alterations in lipid composition and fatty acid incorporation in red cell phospholipids may have significant effects on membrane function.     

Autoxidation as a cause of altered lipid distribution in extracts from human red cells

A characteristic alteration in the distribution of human red cell phospholipids represents an artifact due to autoxidation of the lipid extract. This alteration is manifested on silicic acid chromatography by a decrease mainly in the phosphatidyl ethanolamine and phosphatidyl serine fractions (probably because of their abundance of highly unsaturated fatty acids) and an increase in the phospholipid recovered with the more polar fractions, sphingomyelin and lysolecithin. No evidence was found for "lysocephalin" formation or plasmalogen breakdown in dry lipid extracts after autoxidation by exposure to air at room temperature for 24-35 hr. On thin-layer chromatography, however, the ninhydrin-positive streaking in the autoxidized samples may be erroneously attributed to the presence of "lyso" derivatives. When the alterations in lipid distribution described above are found, the possibility of this artifact should be considered.     

Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.     

Evaluation of methyl mercury chelating agents using red blood cells and isolated hepatocytes

The relative efficacy of thiol-containing mercurial scavengers was assayed by using cellular suspensions of erythrocytes or isolated hepatocytes. The blood cells incubated in a buffer (pH 7.4) containing 1 mM glucose (10% hematocrit) were exposed to 5 μM methyl mercuric chloride. In the absence of extracellular thiols the red blood cells took up more than 90% of methyl mercury from the surrounding medium during 5–10 min. This uptake was almost completely inhibited by dimercaptosuccinic acid (DMSA) (1 mM) and the same chelant could rapidly remove 80% of the mercury from ‘pre-loaded’ erythrocytes. Hepatocytes prepared according to the method of Seglen [11] in a suspension of 10⁶ cells/ml in a buffer containing 5 mM glucose and 5 mg/ml of bovine serum albumin were also exposed to methyl mercuric chloride (4 μM). Almost 50% of the mercurial was taken up by the cells slowly during the incubation period of 240 min. DMSA (1 mM) almost completely blocked the methyl mercury binding by the hepatocytes. 2-Mercaptopropionylglycin (Thiola) or mercaptosuccinic acid (MSA) was almost as effective mercurial scavengers as DMSA in hepatocytes and in red blood cells. Diethyldithiocarbamate (DDC) and dimercaptopropanol (BAL) were considerably less effective than DMSA to inhibit the mercurial binding to hepatocytes. Experiments in vivo have shown that DMSA is a better mercurial chelator than Thiola or MSA, whereas DDC and BAL may both be considered to be inapplicable in methyl mercury poisonings. Our cellular assay provides preliminary information of the efficiency of chelating thiols and may serve as a useful first approximation when planning further experiments.     

Changes in the lipid composition of red blood cells in hyperglycemic rats

The effects of hyperglycemia (experimental diabetes) and insulin treatment were studied on cholesterol and total as well as individual components of phospholipids in red cell membrane from adult rats. While total phospholipid content did not change significantly, the individual components were selectively affected. Cholesterol content was reduced markedly during hyperglycemia. Insulin administration to hyperglycemic rats in general appeared to cause a reversal of the diabetic effects. A direct action of insulin on the red cell phospholipids and cholesterol metabolism was observed.     

Proteins involved in lipid translocation in eukaryotic cells

Since the first discovery of ATP-dependent translocation of lipids in the human erythrocyte membrane in 1984, there has been much evidence of the existence of various ATPases translocating lipids in eukaryotic cell membranes. They include P-type ATPases involved in inwards lipid transport from the exoplasmic leaflet to the cytosolic leaflet and ABC proteins involved in outwards transport. There are also ATP-independent proteins that catalyze the passage of lipids in both directions. Five P-type ATPase involved in lipid transport have been genetically characterized in yeast cells, suggesting a pool of several proteins with partially redundant activities responsible for the regulation of lipid asymmetry. However, expression and purification of individual yeast proteins is still insufficient to allow reconstitution experiments in liposomes. In this review, we want to give an overview over current investigation efforts about the identification and purification of proteins that may be involved in lipid translocation.     

Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells

The present study has examined the effect of elevated glucose levels on membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC). Defibrinated whole blood or RBC were incubated with varying concentrations of glucose at 37 degrees C for 24 h. RBC incubated with elevated levels of glucose showed a significantly increased membrane lipid peroxidation when compared with control RBC. A significant positive correlation was observed between the extent of glucose-induced membrane lipid peroxidation and the osmotic fragility of treated RBC. Glucose-induced membrane lipid peroxidation and osmotic fragility were blocked when RBC were pretreated with fluoride, an inhibitor of glucose metabolism; with vitamin E, an antioxidant; with para-chloromercurobenzoate and metyrapone, inhibitors of the cytochrome P-450 system; or with dimethylfurane, diphenylamine, and thiourea, scavengers of oxygen radicals. RBC treated with elevated glucose concentrations also showed an increase in NADPH levels. Exogenous addition of NADPH to normal RBC lysate induced membrane lipid peroxidation similar to that observed in the glucose-treated RBC. These data suggest that elevated glucose levels can cause the peroxidation of membrane lipids in human RBC.     

Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells

We have suggested that red blood cell proteolytic systems can degrade oxidatively damaged proteins, and that both damage and degradation are independent of lipid peroxidation (Davies, K. J. A., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 8220-8226. These ideas have now been tested in cell-free extracts of rabbit erythrocytes and reticulocytes. Exposure to oxygen radicals or H2O2 increases the degradation of endogenous proteins in cell-free extracts, as in intact cells. Various radical-generating systems (acetaldehyde or xanthine + xanthine oxidase, ascorbic acid + iron, H2O2 + iron) and H2O2 alone enhanced the rates of proteolysis severalfold. Since these extracts were free of membrane lipids, protein damage and degradation must be independent of lipid peroxidation. An antioxidant buffer consisting of HEPES, glycerol, and dithiothreitol inhibited the increased proteolysis by 60-100%. Mannitol caused a 50-80% reduction in proteolysis suggesting that the hydroxyl radical (.OH), or a species with similar reactivity, may be the initiator of protein damage. When casein or bovine serum albumin were exposed to .OH (generated by H2O2 + Fe2+, or COCo radiation) these proteins were degraded up to 50 times faster than untreated proteins during subsequent incubations with red cell extracts. Mannitol inhibited this increase in proteolysis only if present during .OH exposure; mannitol did not affect the degradative system. Although ATP increased the degradation of untreated proteins 4- to 6-fold in reticulocyte extracts, it had little or no effect on the degradation of proteins exposed to .OH. ATP also did not stimulate hydrolysis of .OH-treated proteins in erythrocyte extracts. Leupeptin did not affect the degradative processes in either extract; thus lysosomal or Ca2+-activated thiol proteases were not involved. We propose that red cells contain a soluble, ATP-independent proteolytic pathway which may protect against the accumulation of proteins damaged by .OH or other active oxygen species.     

Effect of cadmium ions on dioxygen affinity and polyphosphate activity of human red blood cells

The effect of cadmium ions on the dioxygen affinity, the time-dependent depletion of intracellular polyphosphates, and the elongation of human red blood cells (RBC's) was examined. The incubation of RBC's in the presence of 1 mM Cd2+ at 37 degrees C for more than one hour results in a decrease of the p50 value by 2.5-3.0 mmHg in comparison to controls. The p50 of stripped (phosphate-free) hemoglobin is not affected by the presence of 1 mM Cd2+ (p50 = 4.8 mmHg at pH 7.2 and 37 degrees C). Experiments with RBC cryolysates demonstrate an apparently competitive effect of 2.3-bisphosphoglycerate (DPG) with cadmium ions on the dioxygen affinity. From 31P NMR spectra, 31P T1 relaxation, and 31P T2 relaxation behavior a more direct evidence for DPG-Cd2+ complexation is obtained. 31P NMR spectra of RBC cryolysates also indicate DPG-Cd2+ complexation. The hydrolysis of free polyphosphates in RBC's incubated at 37 degrees C as monitored by 31P NMR spectra can be noticed after a three-hour lag phase (constant polyphosphate level). This lag phase is lengthened from three hours to four hours in the presence of Cd2+ ions. RBC elongation, as a measure of deformability, decreases slightly upon incubation with 1 mM Cd2+.     

Steroid-derived phospholipid scramblases induce exposure of phosphatidylserine on the surface of red blood cells

A series of methyl 7alpha,12alpha-bis(phenylurea) cholate derivatives with different cationic substituents at the 3alpha-position were prepared and evaluated for an ability to increase the level of endogenous phosphatidylserine (PS) on the surface of red blood cells (erythrocytes). Some of the compounds induced large fractions of erythrocytes to expose sufficient PS to become stained by the protein annexin V-FITC. In addition, the compounds were found to bind PS in homogeneous solution, and to promote the translocation of fluorescent NBD-labeled phospholipids across vesicle membranes, which supports the hypothesis that cholate-induced exposure of endogenous PS on the erythrocyte surface is due to the ability of the cationic cholates to promote anionic phospholipid flip-flop.     

Bioassay for thymic extracts: guinea pig spleen lymphocytes-rabbit red blood cells rosette method

An assay is described for assessing the potency of thymic extracts by the capacity of guinea pig spleen lymphocytes to form rosettes with rabbit red blood cells. Its sensitivity and reproducibility have been evaluated by statistical analysis of the data obtained testing different batches of the calf thymus extract TP-1.     

Carnitine and acetylcarnitine in red blood cells

Carnitine and acetylcarnitine were found to be present in human erythrocytes. Their presence was not as a factor of leucocyte contamination. Carnitine is present within the erythrocyte at a level comparable to that of the plasma, whilst acetylcarnitine is more concentrated within the cell. Red blood cell carnitine and acetylcarnitine do not freely exchange with plasma but intra-erythrocyte acetylcarnitine has a significant relationship to the plasma levels.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation

One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs.RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 °C or 37 °C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 °C was relatively innocuous; however, oxidative injury was evident at 37 °C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 °C, but seemed to have exacerbated the severity of cellular injury at 37 °C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation.The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 °C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

Energy of dissociation of lipid bilayer from the membrane skeleton of red blood cells

The association between the lipid bilayer and the membrane skeleton is important to cell function. In red blood cells, defects in this association can lead to various forms of hemolytic anemia. Although proteins involved in this association have been well characterized biochemically, the physical strength of this association is only beginning to be studied. Formation of a small cylindrical strand of membrane material (tether) from the membrane involves separation of the lipid bilayer from the membrane skeleton. By measuring the force required to form a tether, and knowing the contribution to the force due to the deformation of a lipid bilayer, it is possible to calculate the additional contribution to the work of tether formation due to the separation of membrane skeleton from the lipid bilayer. In the present study, we measured the tethering force during tether formation using a microcantilever (a thin, flexible glass fiber) as a force transducer. Numerical calculations of the red cell contour were performed to examine how the shape of the contour affects the calculation of tether radius, and subsequently separation work per unit area W(sk) and bending stiffness k(c). At high aspiration pressure and small external force, the red cell contour can be accurately modeled as a sphere, but at low aspiration pressure and large external force, the contour deviates from a sphere and may affect the calculation. Based on an energy balance and numerical calculations of the cell contour, values of the membrane bending stiffness k(c) = 2.0 x 10(-19) Nm and the separation work per unit area W(sk) = 0.06 mJ/m2 were obtained.