A kinetics study of pig erythrocyte hemolysis induced by polyene antibiotics

The kinetics of the hemolysis induced by filipin is of the damage type, indicating the formation of large nonselective perforations of erythrocyte membranes. The process is relatively independent of the ionic composition of the incubation medium, and the differences between the hemolysis induced by filipin in pig and human erythrocytes are not significant. In a sucrose medium, filipin-induced hemolysis is inhibited in humans, whereas it is stimulated in pig erythrocytes. It is suggested that low ionic strength is the reason for the different modifications of complexation of filipin in pig and human erythrocyte membranes in a sucrose medium. The kinetics of the hemolysis induced in pig erythrocytes by amphotericin B and nystatin is of the permeability type, indicating the formation of selective channels in erythrocyte membranes and colloid osmotic hemolysis. The rate of the hemolysis, which is high in a KCl medium, is decreased in all the other media tested (CaCl2, MgCl2, potassium phosphate buffer, K2SO4, sucrose), although there are no changes in the kinetics of hemolysis. The results are interpreted as the formation of highly selective channels at a low concentration of the antibiotics. At increasing concentrations, channels of decreasing selectivity occur. The resistances of pig erythrocytes to amphotericin B and nystatin are lower than those of human erythrocytes.     

The mechanism of bile salt-induced hemolysis.

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

ATP utilizing reactions of human erythrocyte membranes and the influence of modulator proteins

The ATP production of human erythrocytes in the steady state (approximately 2 mmoles . 1 cells-1 . h-1, 37 degrees C, pHi 7.2) is maintained by glycolysis and the ATP consumption is essentially limited to the cell membrane. About 25% of the ATP consumption is used for ion transport ATPases. The bulk of the ATP consuming processes in intact erythrocytes remains poorly understood. "Isotonic" erythrocyte membranes prepared under approximate intracellular conditions after freeze-thaw hemolysis have high (Ca2+, Mg2+)-ATPase activities (80% of the total membrane ATPase activity). There is a great discrepancy between the high capacity of the (Ca2+, Mg2+)-ATPase in isotonic membranes and the actual activity in the intact cell. The (Ca2+, Mg2+)-ATPase of isotonic membranes has a "high" Ca2+-affinity (Ka less than 0.5 microM) and a "low" Mg-ATP affinity (Km approximately 760 microM). This state of (Ca2+, Mg2+)-ATPase is caused by the association of calmodulin and 30000 Dalton polypeptides (ATP affinity modulator protein). Hypotonic washings of isotonic membranes result in a loss of the 30 kD polypeptides. EGTA (0.5 mM) extracts derived from isotonic membranes contain the 30 kD modulator protein and restore the properties of the (Ca2+, Mg2+)-ATPase of hypotonic membrane preparations to the isotonic characteristics. The Mg-ATP affinity modulator protein is assumed to form a complex with calmodulin and (Ca2+, Mg2+)-ATPase.     

Effect of Cholesterol Content on Gramicidin S-Induced Hemolysis of Erythrocytes

Gramicidin S (GS) is a cyclo-decapeptide antibiotic with wide Gram+ and Gram− antimicrobial spectrum. However, its therapeutic application is very limited due to hemolytic activity of GS. The presence of cholesterol defines one of the most significant differences between eukaryotic plasma membranes and bacterial inner membranes. To find out the cholesterol effect on the GS hemolytic efficiency we compared GS-induced hemolysis of erythrocytes extracted from the blood of healthy donors against donors with atherosclerosis, “naturally” enriched with cholesterol. Our results show that increased cholesterol levels significantly attenuates yet does not abolishes the GS hemolytic activity. High levels of cholesterol content in erythrocyte membranes results in a decrease in the membrane fluidity and deformability leading to a decrease in the rate of GS interaction with membranes. The results obtained confirm that hydrophobic as well as electrostatic interactions must be involved in the binding of GS to cell membranes. Lipid peroxidation occurring within atherosclerotic erythrocytes leads to considerable decrease in the degree of GS-induced erythrocyte hemolysis in vitro. These results can be applied to the rational design of GS analogs with increased antibacterial efficiency but reduced hemolytic activity.     

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37 degrees C and 15 degrees C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K+ leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15 degrees C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37 degrees C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.     

Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.     

Cooperative binding of primycin and gramicidin on erythrocyte membranes. A cation transport study

In this paper the authors present a comparative study of the actions of the antibiotics primycin and gramicidin on the erythrocyte membrane permeability. It has been found that both antibiotics have a nonlinear effect on the membrane permeability. Above a threshold antibiotic concentration, which is characteristic of the type of the antibiotic, the cation permeability of the erythrocyte membranes increases sharply. In the range of nonlinearity the transport-kinetic curves level off before achieving the equilibrium radioactive ion distribution between the extra- and intracellular spaces. A stochastic model of the cooperative and aspecific incorporation of antibiotic molecules into the membrane explains the experimental findings. The authors conclude that membrane permeability increases at the places where two or more antibiotic molecules form aggregates in the membrane.     

The Interaction of Polyene Antibiotics with Thin Lipid Membranes

Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (R_m ≅ 10⁸ ohm-cm²) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (T_i) indicate that these membranes are cation-selective (T _Na ≅ 0.85; T _Cl ≅ 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing ≈36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced R_m to ≈10² ohm-cm²; concomitantly, T _Cl became ≅0.92. The slope of the line relating log R_m and log antibiotic concentration was ≅4.5. Neither nystatin (2 x 10^-5 M) nor amphotericin B (2 x 10^-7 M) had any effect on membrane stability. The antibiotics had no effect on R_m or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10^-5 M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect R_m or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl^-, acetate), and, to a lesser degree, cations (Na⁺, K⁺, Li⁺).     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40–50% of the existing cholesterol sites.As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9–10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

The interaction of short-chain aralkyl alcohols and amines with the erythrocyte membrane.

Erythrocytes in isotonic saline are hemolyzed by benzyl alcohol and by 2-phenylethanol, but not by the corresponding amines nor by the ring-or side-chain-hydroxylated analogs. All these compounds could however interact with the erythrocyte membrane since: a) they facilitated the hemolytic effect of benzyl alcohol and/or of phenylelytic effect of benzyl alcohol and/or of phenylethanol; b) they exerted a protective effect against controlled hypotonic hemolysis.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury

Exposure of human erythrocytes in a 50% hematocrit to 0.5-1 mM Hg2+ initiated immediate hemolysis which proceeded at a constant rate without any formation of lipid hydroperoxides. Treatment of 0.03% hematocrits with 0.4 ppm of Hg2+ or 40 ppm of methylmercury caused rapid hemolysis after a short lag period. The kinetics of the process were unaltered by saturation of the cell suspensions with oxygen, by its replacement with He or CO, or by variation in the level of vitamin E in the membranes. The results show that peroxidation of erythrocyte membrane lipids is not the cause of hemolysis induced by either Hg2+ or methylmercury.     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract of P. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37℃, and at pH 7.0; Hg^2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg^2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Interaction of polypeptide antibiotic gramicidin S with platelets

Gramicidin S (GS) is a cyclic decapeptide antibiotic active against both Gram‐positive and Gram‐negative bacteria as well as against several pathogenic fungi. However, clinical application of GS is limited because of GS hemolytic activity. The large number of GS analogues with potentially attenuated hemolytic activity has been developed over the last two decades. For all new GS derivatives, the antimicrobial test is accompanied with the hemolytic activity assay. At the same time, neither GS nor its analogues were tested against other blood cells. In the present work, the effects of GS on platelets and platelet aggregates have been studied. GS interaction with platelets is concentration dependent and leads either to platelet swelling or platelet shape change. Effect of GS on platelets is independent of platelet aggregation mechanism. GS induces disaggregation of platelet aggregates formed in the presence of aggregation agonists. The rate of the GS interaction with platelet membranes depends on membrane lipid mobility and significantly increases with temperature. The interaction of GS with the platelet membranes depends strongly on the state of the membrane lipids. Factors affecting the membrane lipids (temperature, lipid peroxidation and ionising irradiation) modify GS interaction with platelets. Our results show that GS is active not only against erythrocytes but also against other blood cells (platelets). The estimated numbers of GS molecules per 1 µm² of a blood cell required to induce erythrocyte hemolysis and disaggregation of platelet aggregates are comparable. This must be considered when developing new antimicrobial GS analogues with improved hemolytic properties. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.     

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.