Reconstruction of erythrocyte shape during modified morphological response.

Changes in erythrocyte shape during morphological response modified by benzalkonium chloride (BzA) were studied in sucrose solutions. Fixation of the cells with glutaraldehyde- and formaldehyde-containing fixatives at some time points is usually inadequate to maintain the current cell shape. Considering the reconstruction of erythrocyte shape, which takes into account the mode of fixative action, we showed that the echinocyte-forming activity of BzA depends on the concentration of this surfactant. It can induce a direct spherostomatocyte-spheroechinocyte transition without altering the near-spherical shape of the cells. On the other hand, the reverse spheroechinocyte-spherostomatocyte transition was always accompanied by some flattening of the cells, although in some instances discoidal shape was not achieved. The data point to asymmetric shape transitions of erythrocytes in sucrose solution, which contradicts the continuum and bilayer-couple models of shape regulation. It seems that the nonuniform structure of native erythrocyte membrane plays a more important role in morphological transitions of these cells than suggested earlier.

[Phospholipids composition of the myocardium in patients with ischemic heart disease and its connection to arrhythmias].

The purpose of this study was to establish phospholipid composition of the myocardium in patients with ischemic heart disease, and to estimate possible correlation of biochemical parameters with myocardium extrasystolic activity. The patients (n = 28) including 15 patients with ischemic heart disease and 13 patients with secondary atrium septum defect (control group) were studied. During surgical intervention the right atrium myocardium bioptates were taken. Phospholipid metabolism was studied in the myocardium samples. At the eve of surgical intervention a holter monitoring was performed. Deep changes in the myocardium lipid metabolism were found, including accumulation of free and estherified cholesterol, lysophospholipids, and sphingomyeline. An increase of free cholesterol content was accompanied by accumulation of sphingomyeline. This can be an evidence of changes in the constitution of lipid rafts. Extrasystoles, particularly ventricular ones, in patients with ischemic heart disease might depend on accumulation of lysophospholipids as they took place simultaneously with it.

[Effect of modification of been venom components on its hemolytic activity and ability to decrease the volume of the human erythrocyte]. / Vliianie modifikatsii komponentov pchelinogo iada na ego gemoliticheskuiu aktivnost' i sposobnost' umen'shat' ob''em éritrotsitov cheloveka.

The crude bee venom was modified by different chemical substances. The inverse correlation was established between increase of the hemolytic activity and decrease of the contractive one under modification of the bee venom by NaOH and KOH. The negligible decrease of the contractive activity occurs in the acid conditions (pH 1,0), while hemolytic one is stationary. Dithiothreitol, mercaptoethanol, urea and high temperature (60-90 degrees C) did not affect activity of the bee venom.

Determination of time-dependent shape changes in red blood cells.

A kinetic method for detecting changes in red blood cell shape with time resolution on the order of seconds is described. The definition of shape index (SI) and the experimental conditions under which the measurements should be conducted are given, and the relationship between SI and morphological index, which is often used to quantitatively assess red blood cell morphology, is established. Further, we show that the method can be used to assess shape changes under a variety of experimental conditions known to cause major perturbations in the morphology of red blood cells (such as addition of lysolecithin or chlorpromazine) and to detect changes in shape during chemical fixation for observation with microscopy. We report here that the ability of glutaraldehyde to fix red blood cells without additional alteration in shape strongly depends on the morphology that is being fixed and the initial state of the cells.

Peculiarities of the osmotic response in dehydrated erythrocytes.

Human erythrocytes were dehydrated in concentrated sucrose solutions and rehydrated in isotonic NaCl solution. The osmotic responses of erythrocytes dehydrated in media with osmolarities over 1 M corresponded to the ideal osmotic behaviour. This was also true of the ghosts formed during rehydration of dehydrated erythrocytes irrespective of the initial osmolarity. Fresh erythrocytes as well as erythrocytes dehydrated in sucrose media with osmolarity of 0.5-0.6 M displayed nonideal responses (volume changes were lower than predicted). In contrast to the cells dehydrated at 0 degree C, the peculiarities of the osmotic response of the cells dehydrated within the osmolarity range of 0.7-0.8 M at 37 degrees C can be detected spectrophotometrically. The data obtained suggest that the nonideal osmotic response of human erythrocytes is probably due to a specific state of hemoglobin.

Bee venom-induced shrinkage of erythrocyte ghosts.

When hemolysis of erythrocyte ghosts is induced by bee venom, their volume becomes significantly less than the initial volume of the cells. Treating isolated ghost membranes with bee venom has the same effect. An analogous phenomenon is caused by a mixture of purified melittin and phospholipase A2, but not by the separate action of these substances. The volume changes develop non-monotonously in time. A prolonged phase of spontaneous volume restoration follows the initial phase of shrinkage. Inhibitors of melittin-induced hemolysis (Mg2+, chlorpromazine) retarded the development of both shrinkage and restoration phases, but did not influence the extent of the volume changes. High concentrations of Ca2+ (10 mM) acted in a similar manner, but increased the extent of shrinkage at low concentration (0.1 mM). The extent of shrinkage was increased several-fold in isotonic nonelectrolyte solutions. Analysis of the data shows that bee venom-induced reduction in ghost volume is not due to osmotic effects or permeabilization and/or fragmentation of the membrane, but rather is attributed to membrane contraction, which presumably relates to the membrane cytoskeleton. In this respect venom-induced shrinkage resembles that of contractile events in non-muscle cells.

[Modulation of melittin-induced hemolysis of erythrocytes]. / Moduliatsiia melittin-indutsirovannogo gemoliza éritrotsitov.

Three main groups of chemicals influence melittin-induced hemolysis including neutral compounds and inhibitors and activators of hemolysis. Inhibitors include divalent cations Zn2+ and Ca2+, albumin, DIDS, etc.; their potency significantly increases if they are present at early stages of peptide-membrane interaction. The rate of melittin-induced hemolysis depends on time of preincubation with the cells in physiologic saline but does not depend on the presence of inhibitors or activators. Longer incubation increases the rate of hemolysis. These effects can be due to membrane inhibitory components with specific affinity to melittin which initially protect the membrane from its lytic effect; these components can dissociate from the cell surface after dilution and incubation in physiologic saline. According to the suggested model, characteristics of peptide-induced hemolysis of erythrocytes are determined by sequential stages of peptide-membrane interaction and depend on the formation of triple non-lytic complex comprising the membrane inhibitory component, the blocker, and the peptide; the complex inhibits destruction of the membrane.

Protection by chlorpromazine, albumin and bivalent cations against haemolysis induced by melittin, [Ala-14]melittin and whole bee venom.

The ability of the peptides melittin, [Ala-14]melittin (P14A) and whole bee venom to lyse red blood cells (RBC) and to cause shape transformation, binding, partitioning and changes in volume of the cells during haemolysis, as well as the action of the bivalent cations Zn2+ and Ca2+, chlorpromazine, albumin and plasma on the peptide-induced haemolysis of RBC in high ionic-strength solution, have been investigated. The protective effect of all inhibitors depends on whether they have been added to the media before or after the cells. When added before the cells they reduced significantly the rate of peptide-induced haemolysis and shape transformation. The effect was maximal when agents acted simultaneously after introduction of the cells into the media containing both inhibitors and peptides. Incubation of the cells in isotonic solution before the addition of peptides enhanced 2-3-fold the RBC susceptibility (i.e. rate of haemolysis) to lytic action of the same amount of peptides, and increased the order of the haemolytic reaction, although the power law coefficient did not exceed a value of 2 for all peptides, suggesting that haemolysis is attributable to the monomeric or dimeric forms of the peptides. Partition coefficients were of the order of approximately 10(6) M-1, and P14A possessed a value 3-fold larger compared with melittin and bee venom, which correlated with its enhanced haemolytic activity. The protective action of inhibitors against peptide-induced haemolysis has been explained on the basis of their ability to compete with peptide binding at an early stage of peptide-membrane interaction, and not as a result of inhibition of a pre-existing peptide-induced pore. Whereas melittin increased the volume of RBC during haemolysis, P14A, melittin in the presence of phospholipase A2 or bee venom, reduced the volume in a concentration-dependent manner. The present data reveal the significant role of the initial stage of peptide-membrane interaction and peptide structure in the mechanism of haemolysis. These data are not consistent with a lipid-based mechanism of peptide-induced haemolysis, indicating that the mode of peptide-protein interaction is an important and decisive step in the haemolytic mechanism. It should be noted that data (in the form of three additional Tables) on the ability of inhibitors to protect cells from haemolysis when inhibitor and peptide act simultaneously are available. They are reported in Supplementary Publication SUP 50178, which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9.

[Anomalous transport of cations upon stimulation of a Ca-dependent K-channel in rehydrated erythrocytes]. / Anomal'nyi transport kationov pri stimuliatsii Ca-zavisimogo K-kanala v regidratirovannykh éritrotsitakh.

The Ca(2+)-dependent potassium transport in human erythrocytes dehydrated into hypertonic sucrose solutions and further rehydrated into isotonic media of NaCl and KCl, has been studied. Analysis of controversies between potassium release and volume changes showed that a new transport mechanism for monovalent cations might be activated in rehydrated cells which is silent in normal cells.

[The effect of Zn ions on erythrocyte hemolysis induced by melittin]. / Vliianie ionov Zn na gemoliz éritrotsitov, indutsirovannyi melittinom.

Divalent cations--Ca2+ and Zn(2+)--were shown to produce inhibition of melittin-induced hemolysis of erythrocytes in the hemolysis phase, the degree of blocking increasing with a rise in the cation concentration. Besides, Zn2+ ions altered, in a concentration-dependent manner, the initial stages of interaction between melittin and red cell membranes, resulting in a slowdown of the melittin insertion and hemolysis rate. A comparison of dependencies between the hemolysis rate and melittin and cells concentrations in the medium as well as the regularities of the blocking activity of cations under various conditions with the corresponding data concerning melittin-induced lysis of lipid vesicles indicates that melittin-induced hemolysis and lysis of lipid vesicles have little in common, thus being suggestive of an insignificant role of melittin-lipid interactions in the mechanism of cell hemolysis. The data obtained provide evidence that the erythrocyte membrane contains an intrinsic blocking mechanism whose efficacy can be synergistically enhanced by Zn2+ ions protecting the cells against the lytic effect of melittin.

[Change in erythrocyte volume and spectrum of membrane proteins induced by melittin, phospholipase A2 and bee venom]. / Izmenenie ob''ema éritrotsitov i spektra membrannykh belkov, indutsirovannoe melittinom, fosfolipazoi A2 i pchelinym iadom.

Human erythrocytes and erythrocyte ghost membranes were treated with melittin (M), phospholipase A2 (PLA2) and whole bee venom. Treatment by whole bee venom or M + PLA2 resulted in a significant decrease of the cell volume, cell spherulation and hemolysis. Used separately, M produced an increase in the cell volume during hemolysis, while treatment with PLA2 stimulated M-induced hemolysis without any effect on the cell shape or volume even at relatively high concentrations. Combined action of M and other agents revealed: stimulation of M-induced hemolysis by Triton X-100 used at concentrations by two orders of magnitude lower than lytic concentrations; saponin acted similarly but at concentrations closer to those known to lyse cells: chlorpromazine inhibited the hemolysis at sublytic concentrations. The effects of these agents on M-induced hemolysis was unaccompanied by a decrease in the cell volume. Erythrocytes or ghosts treated with M + PLA2 or bee venom showed altered SDS-electrophoretic protein patterns: bands 6 and 8 were markedly reduced, while new bands of relatively low molecular weights were identified. The absence of volume or protein pattern changes during detergent-stimulated M-induced hemolysis points to the specificity of M-membrane interactions in the presence of PLA2.

Cation-sensitive pore formation in rehydrated erythrocytes.

Rehydration of red blood cells (RBC) in isotonic media after dehydration in hypertonic electrolyte or nonelectrolyte saline leads to their posthypertonic hemolysis (PH). Ca2+ ions at a concentration of more than 5 mM stimulated hemolysis of RBC treated by hypertonic sucrose but not NaCl if rehydration was carried out in the presence of cations. Zn2+ produced a more complex response of stimulation followed by inhibition as a concentration is increased. Mg2+, Ca2+, Zn2+, EDTA and sucrose exhibited only inhibition when added to isotonic NaCl media immediately after onset of rehydration or later on. At low ionic strength inhibition produced by divalent cations was markedly reduced and sucrose was ineffective. An equimolar concentration of EDTA abolished the inhibition of PH by Zn2+ ions if they were introduced into the isotonic media after the cells, but activated hemolysis when rehydration was carried out in the presence of ions. The same divalent cations prevented shape transformation and hemolysis induced by melittin if they interacted with the plasma membrane prior to the addition of melittin. Subsequent chelation of cations by EDTA triggers the full sequence of events characteristic to the action of melittin alone and resulted in cell spherulation followed by hemolysis. Inhibition of melittin-induced hemolysis produced by all cations was reversible because EDTA abolished the action of divalent cations and even stimulated hemolysis in isotonic sucrose. Similarities in the mode of action of divalent cations and EDTA on posthypertonic hemolysis which is attributed to endogenous stimuli and melittin-induced hemolysis as far as the exogenous agent is concerned imply that in both cases common intrinsic mechanisms are involved in the process of cation-sensitive pore formation in erythrocyte membranes, while differences indicate that more complex pores are formed during posthypertonic injury.

[Characteristics of the generation of transmembrane current higher harmonics in lipid bilayers in glycerol and 1,2-propane diol solutions]. / Osobennosti generatsii vysshikh garmonik transmembrannogo toka v lipidnykh bisloiakh, nakhodiashchikhsia v rastvorakh glitserina i 1,2-propandiola.

It is shown that structural rearrangements of bilayer lipid membrane induced by glycerol and 1,2-propane diol result in noncompensated second harmonic of transmembrane current having quadratic dependence on input voltage and a weak dependence on frequency. The third harmonic has cubic dependence on input voltage and decreases with an increase in frequency. The mechanism is discussed according to which generation of non-compensated second harmonic is due to the formation of the intermediate lipid phase with viscoelastic properties differing from the initial bilayer in the process of structural rearrangements. The presence of this phase conditions the possibility of appearance of noncompensated second harmonic.

[Structural rearrangements induced by glycerol increase the permeability of bilayer lipid membranes for amphotericin]. / Pri strukturnykh perestroikakh, indutsirovannykh glitserinom, uvelichivaetsia pronitsaemost' bisloinykh lipidnykh membran dlia amfoteritsina.

It has been shown that structural rearrangements induced by glycerol in bilayer lipid membranes (BLM) containing cholesterol facilitate the transmembrane transport of amphotericin B molecules in the direction of glycerol gradient. The addition of amphotericin B to the same side with glycerol results in a change in bilayer selectivity from the cation to the anion one. Besides, the final conductivity is blocked by tetraethylammonium from the solution with no amphotericin B added. It testifies to the transport of amphotericin molecules to the opposite side of the membrane. The transport effect depends on the cholesterol content in bilayer, ionic strength of the medium and slightly depends on temperature. It is concluded that transport of amphotericin B in such conditions differs from the diffusive one and is due to the formation of intermediate lipid phases in the course of structural rearrangements of bilayers.

[Structural changes in the liposomes and the lamellar packing of liposomal lipids exposed to glycerin]. / Izmenenie struktury liposom i lamelliarnoi upakovki liposomal'nykh lipidov pri vozdeistvii glitserina.

Glycerol-induced structural changes in multilamellar liposomes (ML) were studied by light and thin-section electron microscopy. It is shown that high concentrations of glycerol induce non-bilayer rearrangements of liposomal lipids in ML. The data obtained are discussed regarding the possible mechanisms of cryoprotector effects on natural (biological) and artificial (liposomal) membranes.

[Effect of glycerol on the capacity and conductivity of lipid bilayer membranes]. / Vliianie glitserina na emkost' i provodimost' bisloinykh lipidnykh membran.

It is shown that glycerol addition to one side of BLM containing cholesterol leads to a significant decrease of its capacity, and the decrease rate is in indirect proportion to glycerol concentration. Washing out or addition of glycerol to another side results in a full or partial reconstitution of the capacity. The effect of glycerol on the BLM conductivity is manifested in its increase for membranes having specific conductivity above 2.10(-7) Om-1 X cm2. The peculiarity of glycerol is manifested in the fact that during definite period of time (20 +/- 30 min) the membrane is under "stress" condition with the characteristic current fluctuations, which may be compared with its medium meaning. Such condition is preserved up to the moment of BLM rupture. It is supposed that such effects are due to the big-scale reconstructions of membrane structure under the influence of glycerol.