Effect of temperature and pH on magnetic properties of human erythrocytes

Erythrocytes magnetic susceptibility depending on temperature and pH value of buffer were studied by analysing individual erythrocytes moving in a medium in the nonhomogeneous magnetic field near a transversely magnetized thin wire.     

Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.     

Effect of phosphatidylcholine chlorohydrins on human erythrocytes

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.     

Effect of chlorpromazine on the relation between temperature and the binding of ANS by human erythrocytes

It has been shown that under the effect of chlorpromazin (concentration to 2.10(-5) M) breaks observed on Arrhenius graphs of the rate constant of ANS binding with erythrocytes (k) at 28 and 36 degrees C are shifted to the region of low temperatures approximately to a similar interval (14-15 degrees C). The value k measured at 22 degrees C is not changed within the pH range of 5-7. It is concluded that breaks characterised the initial and final stages of temperature transition initiated in the zone of acid phospholipid of membranes.     

Effect of exogenous hydrogen peroxide on human erythrocytes

Circulating erythrocytes are drastically susceptible to peroxidative reactions. To examine the extent of the damage induced by exogenous H2O2 we limited the catalase activity in order to study the extent of lysis, the lipid peroxidation and namely the behaviour of membrane micro-viscosity. Our data showed that the erythrocytes can efficiently scavenge exogenous H2O2 without significant damage of the cells and/or their membranes. These findings could confirm the important role of the erythrocytes as extracellular-antioxidant defense.     

Effect of endo-beta-galactosidase on intact human erythrocytes.

Endo-beta-galactosidase, a glycosidase that hydrolyzes Gal beta 1-4 GlcNAc linkages in glycoconjugates, has been used to probe the plasma membrane of human erythrocytes. Coomassie blue staining of stroma components separated by sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that treatment of red cells with endo-beta-galactosidase converts Protein 3, the anion transporter of the erythrocyte, to a more compact staining band. No other components detected by Coomassie staining are affected. Following labeling of red cells with galactose oxidase + NaB3H4, 45 to 50% of the [3H]galactose residues can be released by endo-beta-galactosidase. In contrast, only 5% of the label incorporated by treatment with periodate + NaB3H4, can be removed. [3H]Galactose residues are released from three components: Protein 3, Band 4.5, and the megaloglycolipids. The susceptibility of these components to endo-beta-galactosidase, together with the high content of Gal and GlcNAc present in Protein 3 and the megaloglycolipids, suggests that the erythrocyte membrane contains several components with N-acetyllactosamine repeating units, a structure commonly found in connective tissue glycoconjugates.     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

Effect of temperature on kinetics and differential mobility of empty and loaded nucleoside transporter of human erythrocytes

The transmembrane equilibration of [3H]uridine was measured in human erythrocytes as a function of temperature using rapid kinetic techniques. Arrhenius plots of the maximum velocity of equilibrium exchange were continuous between 5 and 30 degrees C (Ea = 17-20 kcal/mol), but the increase in velocity with increase in temperature leveled off above 30 degrees C. This leveling off did not reflect heat inactivation of the carrier since transport activity was stable for 3 h at 37 degrees C. Transmembrane equilibration of uridine in equilibrium exchange and zero-trans modes at 5, 15, 25, and 35 degrees C conformed to appropriate integrated rate equations derived for the simple transporter. The nucleoside transporter exhibited directional symmetry, but the loaded carrier moved on the average 5 times more rapidly than the empty carrier at 15, 25, and 35 degrees C, but 25-40 times faster at 5 degrees C. This marked shift in differential mobility of loaded and empty carrier between 15 and 5 degrees C was entirely attributable to an impairment of mobility of empty carrier. The Michaelis-Menten constant for equilibrium exchange increased about 3-fold with increase in temperature between 5 and 35 degrees C. The van't Hoff plot of the values was approximately linear and yielded an estimate of the enthalpy of carrier:substrate dissociation of 7.8 kcal/mol.     

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC₅₀ values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.     

Effect of acidosis on bilirubin-induced toxicity to human erythrocytes

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0–8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0–7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.     

The influence of temperature and incubation time on deformability of human erythrocytes

Human erythrocytes have been heated and stressed in a novel and controlled manner using rectangular microcapillaries. Heated cells attached to the capillary wall were stressed by liquid flow. Under particular conditions of stress, temperature and incubation time the body of the cell could be pulled in the flow, retaining a connection with the glass by means of a narrow process or tether. The tethers appear as: regularly beaded, irregularly beaded or without beads depending upon the incubation conditions. We have outlined the incubation regimes necessary to achieve these different responses in the temperature range 48–55°C. The cells become less deformable as the incubation is continued beyond an optimum time. The behaviour of the tether is compared with that of a viscoelastic liquid. Circular dichroism studies of ghost membranes show that the denaturation of membrane proteins is partially reversible when incubation times are similar to those required to bring about a loss of deformability.