[COMPARATIVE STUDY OF MECHANICAL STRESS EFFECT ON HUMAN AND ANIMAL ERYTHROCYTES].

Sensitivity of human and animal (bovine, rat, rabbit, equine) erythrocytes to the effect of mechanical stress has been studied. Mechanical stress effect was demonstrated to result in a time-dependent (5-60 min) release of potassium cations out of mammalian erythrocytes and a partial hemolytic cell damage. Herewith the release levels of potassium ions and hemolysis did not coincide for erythrocytes of all the mammals except rabbit ones. The most sensitive to mechanical stress (60 min) by the parameters of hemolytic damage and potassium ion release were rat (32%) and bovine (66%) erythrocytes respectively, the lowest sensitive by both parameters were rabbit ones (about 20%). Implemented correlation analysis has demonstrated a statistically significant negative relation between the values of mechanical hemolysis of mammalian erythrocytes and surface-volumetric ratio of cells (rs = -0.900, P = 0.037). A feasible relationship between the content of phosphatidylethanolamine in mammalian erythrocyte membranes and the level of potassium cation loss under mechanical stress effect is under discussion.

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.

[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.