valinomycin and Hemolysis

The effect of postirradiation conditions on the haemolysis of y-irradiated (2.1 kGy) human erythrocytes was studied. Haemolysis was inhibited by incubation in mannitol and sucrose instead of saline, by hypertonicity of the medium and by calcium chelators. Dithiothreitol, butylated hydroxytoluene, deferoxamine, DIDS (in inhibitor of anion exchange) and furosemide (an inhibitor of K/Cl and K/Na/Cl cotransport) did not slow down the haemolysis. Apparently, the radiation-induced haemolysis is due to the formation of membrane pores leaky for electrolytes. From the inhibition of haemolysis by mannitol, apparent pore radius was estimated to be about 0.7 nm. The pores appear to be transient, the average pore number per cell being much less than unity.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Chelating Agents; Dithiothreitol; Dose-Response Relationship, Radiation; Erythrocytes; Furosemide; Glucose; Hemolysis; Humans; Osmolar Concentration; Spermine; Valinomycin

1. The rate of dehydration of K+ permeabilized red cells is influenced by their Cl- permeability (PCl). In instances of pathological K+ permeabilization, cell-to-cell differences in PCl may determine which red cells dehydrate most. The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample. 2. Previously available methods measure only the mean PCl of red cell populations. We describe a 'profile migration' method in which dilute red cell suspensions in low-K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate-limiting factor for cell dehydration. As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times. Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities. 3. Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform. Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5%. 4. Results obtained with this technique were analysed using the Lew-Bookchin red cell model. The calculated PCl was within the normal range described in earlier studies.

Topics: Chloride Channels; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Ionophores; Osmotic Fragility; Potassium; Valinomycin

The selectivity of the transmembrane permeability induced by polyene antibiotics was studied in human erythrocytes and related to the hemolytic potency of the drugs. The selectivity induced was differently, dependent on the antibiotic structure in aromatic (vacidin A, gedamycin) and nonaromatic heptaenes (amphotericin B, candidin). Aromatic heptaenes were more effective than nonaromatic in inducing permeability to K+. For both groups of antibiotics, permeability to K+ was not affected by substitution at the carboxyl group but important differences in the induction of permeability to H+, OH- and Cl- were found. The strongly hemolytic aromatic heptaenes vacidin A and gedamycin exhibited much higher protonophoric activity than the nonaromatic ones: amphotericin B, and candidin. The protonophoric properties of aromatic heptaenes were related to the presence of a free carboxyl group in the antibiotic molecule. Indeed the esterification or amidation of the carboxyl group of vacidin A or gedamycin eliminated the ability of the antibiotic to increase H+ conductance and consequently diminished their hemolytic activity to an important extent. Both groups of antibiotics differed also in the efficiency of anion permeability induction. Only unsubstituted aromatic heptaenes, at high concentration, induced Cl-/OH- exchange and conductive flux of Cl- in a concentration-dependent manner. Substitution at the carboxyl group of vacidin A or gedamycin eliminated this property. Amphotericin B as well as its carboxyl-substituted derivatives formed a pathway characterized by low K+ over Cl- selectivity, whatever the concentration. The hemolytic activity, related to K+ permeability increased by heptaenes was dependent on simultaneous increase of the permeability to anions, and net KCl influx. Carboxyl-substituted derivatives of aromatic heptaenes presenting a remarkably high selectivity for K+, had consequently a very poor hemolytic activity.

Topics: Amphotericin B; Anions; Anti-Bacterial Agents; Candicidin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Chlorides; Erythrocyte Membrane; Hemolysis; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Permeability; Polyenes; Potassium; Protons; Structure-Activity Relationship; Valinomycin

One of the most conserved sequences in various delta-endotoxins is the 30 amino acid long block I. Block I of cryIIIA delta-endotoxin contains a 23 amino acid amphiphilic alpha-helix termed alpha 5. The potential involvement of this alpha 5 helix in the toxic mechanism of delta-endotoxin was examined. For this purpose, a peptide corresponding to the alpha 5 segment and its proline incorporated analogue (P-alpha 5) were synthesized and characterized. The alpha-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for alpha 5 and P-alpha 5, respectively. To monitor the interaction of alpha 5 peptides with phospholipid membranes, they were selectively labeled at their N-terminal amino acids with the fluorescent probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or carboxyfluorescein. Fluorometric studies allowed the calculation of membrane surface partition constants, which were about 10(4) M-1 for both alpha 5 and P-alpha 5, and revealed that their N-terminals are located within the lipid bilayers. The shape of the binding isotherms indicated that alpha 5 aggregated in both zwitterionic and acidic vesicles. Functional characterization of the alpha 5 peptides was determined by assessing their ability to dissipate a diffusion potential from sonicated small unilamellar vesicles (SUV) composed of zwitterionic or acidic phospholipids and to lyse human erythrocytes. alpha 5 was much more active than P-alpha 5 in both assays. Moreover, membrane-bound alpha 5 was more protected from enzymatic proteolysis than P-alpha 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Bacillus thuringiensis; Bacillus thuringiensis Toxins; Bacterial Proteins; Bacterial Toxins; Cell Membrane Permeability; Circular Dichroism; Diffusion; Endotoxins; Erythrocyte Membrane; Hemolysin Proteins; Hemolysis; Humans; In Vitro Techniques; Molecular Sequence Data; Protein Structure, Secondary; Solubility; Structure-Activity Relationship; Valinomycin

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Topics: Amino Acid Sequence; Bacteria; Cell Membrane Permeability; Circular Dichroism; Computer Simulation; Diffusion; Escherichia coli; Hemolysis; Humans; Models, Molecular; Molecular Sequence Data; Molecular Weight; Peptides; Protein Structure, Secondary; Pseudomonas aeruginosa; Staphylococcus aureus; Structure-Activity Relationship; Valinomycin

After the addition of valinomycin into the incubation medium, the potassium content of rat erythrocytes rapidly decreases. The rate-limiting step of this reaction is a unidirectional efflux of anions through band 3 protein. The rate of this efflux in erythrocytes of spontaneously hypertensive rats (SHR) of the Wistar-Kyoto strain, is not altered. The loss of KCl by rat erythrocytes is accompanied by a decrease in intracellular water, cell shrinking and activation of Na+-H+i exchange. The rate of Na+-H+ exchange in the erythrocytes of SHR in the pre-hypertensive stage (4 weeks old) was decreased by 30%. There were no differences between 14-week-old and 28-week-old SHR and normotensive Wistar-Kyoto (WKY) rats. The half-maximal increase of the valinomycin-induced Na+-H+ exchange in erythrocytes of 14-week-old WKY and SHR was observed at KCl concentrations in the incubation medium of 25 and 40 mmol, respectively. The addition of activators of protein kinase A (dibutyryl-cAMP) or protein kinase C (beta-phorbol ester) resulted in an increase in the maximal rate of Na+-H+ exchange, and did not modify its dependence on K+o concentration. In all groups of SHR, the rate of valinomycin-induced H+ efflux from erythrocytes in the sodium-free medium was 1.5-2.5-fold higher than in age-matched WKY. Under these conditions (addition of valinomycin and inhibition of Na+-H+ exchange), haemoglobin release from erythrocytes of SHR, treated with hypotonic solution, was significantly decreased. We conclude that these differences are due to the alteration of the skeleton protein organization in the erythrocyte membranes of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biological Transport; Erythrocytes; Hemolysis; Hypertension; Hypotonic Solutions; Male; Potassium; Protein Kinases; Protons; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Sodium; Valinomycin

Use of liposomal drug delivery systems can enhance the therapeutic potential of membrane active anti-cancer and anti-infectious drugs. Thus, the therapeutic index of the important antifungal agent amphotericin B is markedly improved via incorporation of the drug into liposomes. The mechanistic basis of this effect seems to be an increase in the selectivity of the drug at the cellular level. Thus, free amphotericin B can readily partition into both fungal and mammalian membranes and can cause toxicity to both types of cells, giving rise to the notorious in vivo toxicity of this drug. By contrast, when amphotericin B is formulated in certain types of liposomes, the drug still readily partitions into fungal membranes but can no longer partition into animal cell membranes, thus markedly reducing its toxicity. Liposomes can also be used to reduce the toxicity of membrane-active antitumor drugs. Thus, the peptide ionophore valinomycin is far less toxic to animals when presented in liposomal form. Nonetheless, the drug retains useful antitumor activity in this form. The underlying basis of the enhanced therapeutic index of liposomal valinomycin is unknown at this time but is being explored. The development of membrane-active anti-tumor drugs, in conjunction with liposomal delivery systems, could be an important new approach in cancer chemotherapy. While no anticancer drug is likely to be free of toxic side effects, the toxicities engendered by membrane-active antitumor drugs are likely to affect a different spectrum of tissues and organs than those caused by "conventional" antitumor drugs. Thus membrane-active drugs could complement existing drugs and provide a valuable adjunct to therapy.

Topics: Amphotericin B; Animals; Candida; Cell Membrane; Chemistry, Pharmaceutical; Hemolysis; Infections; Kidney; Leukemia P388; Leukemia, Experimental; Liposomes; Microscopy, Electron, Scanning; Rubidium; Solubility; Valinomycin

The permeability characteristics of the erythrocyte membrane were critically evaluated in electrolyte and non-electrolyte (sucrose) media by ion-selective electrodes and radioactive polyol fluxes as well as by the novel technique of osmometry. K+ efflux demonstrated a linear osmotic susceptibility distinct from Na+ influx upon incubation in NaCl media of various tonicities. In non-electrolyte media, acidification of the medium, large fluxes of K+, sucrose and even haemoglobin (as manifest by hypertonic disruption) were consistent with enhanced porosity of the bilayer due to the field created by surface charge density leading to density fluctuations in the bilayer.

Topics: Animals; Cell Membrane Permeability; Electrolytes; Erythrocyte Membrane; Gramicidin; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Models, Biological; Osmolar Concentration; Potassium; Rats; Sodium Chloride; Sucrose; Valinomycin

An acidic lipopeptide A21978C has previously been shown to have a powerful antibiotic activity against Gram-positive organisms. Due to its ability to increase the K+ permeability of bacterial cells and its specific calcium requirement, which is similar to a previously described ionophore CDA, its effect on planar bilayer membranes has been studied. Although it produces significant increases in the conductivity of lipid bilayers it is shown that this alone cannot account for its in vivo activity. Similarly, unlike the in vivo results, the Ca2+-induced increases in bilayer conductivity can be mimicked by Mg2+ and charged lipids. Results from a series of homologues differing in the length of the acyl moiety show a close similarity between bilayer conductance and LD50 trends from in vivo studies. A complex activity is proposed which depends upon incorporation in, rather than disruption of, the bilayer membrane.

Topics: Anti-Bacterial Agents; Calcium; Dose-Response Relationship, Drug; Hemolysis; Intercellular Signaling Peptides and Proteins; Lipid Bilayers; Peptides; Peptides, Cyclic; Permeability; Rubidium; Structure-Activity Relationship; Valinomycin

Spectrin in isolated erythrocyte membranes is known to undergo tetramer to dimer transformation upon hypotonic incubation at 37 degrees C. In the present study, we detect no such transformation in intact erythrocytes in which hypotonicity is achieved by valinomycin treatment followed by hypotonic swelling. The inhibition of spectrin tetramer to dimer transformation is attributable to intracellular hemoglobin, since the addition of hemoglobin to isolated membranes or spectrin extracts blocks a similar spectrin transformation. However, the inhibitory effect is not limited to hemoglobin; other proteins including heme-containing proteins and basic proteins such as cytochrome c, ribonuclease, and albumin are also effective. The magnitude of their effect is proportional to the increased pI value of these proteins. We conclude that the stabilizing effect of these proteins on spectrin tetramers under hypotonic conditions is partly due to their non-ideality, which excludes water from spectrin and thus increases the effective concentration of spectrin, and to their electrostatic interactions with spectrin. In addition, promotion of spectrin self-association by hemoglobin under hypotonic conditions increases the stability of membrane skeletons against mechanical shearing. More importantly, the hemoglobin effect on spectrin self-association is demonstrable at physiological hemoglobin concentration, pH, and osmolarity, suggesting that in intact red cells the spectrin dimer-dimer association, as well as the membrane skeletal structure, is strengthened by intracellular hemoglobin.

Topics: Chromatography, Gel; Cytochrome c Group; Erythrocytes; Hemoglobins; Hemolysis; Humans; Macromolecular Substances; Osmolar Concentration; Solutions; Spectrin; Valinomycin

Human erythrocytes suspended at 37 degrees C in hypertonic solution of either electrolytes or nonelectrolytes undergo hemolysis when the temperature is lowered toward 0 degrees C (Green, F.A., Jung, C.Y. 1977 J. Membrane Biol. 33:249). In the present studies this hypertonic cryohemolysis was profoundly affected by the pH of incubation, and was completely abolished at ph 5. In hypertonic NaCl, there was an apparent pH optimum at 6--6.5. In hypertonic sucrose, on the other hand, hemolysis increased progressively with increasing pH between 6 and 9. Amphotericin B inhibited hypertonic cryohemolysis in NaCl or KCl solution. No inhibiting effect of amphotericin B was observed when hypertonicity was due to sodium sulfate or sucrose. Valinomycin also inhibited hypertonic cryohemolysis in KCl, but did not affect the process in NaCl or sucrose solution. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) and phloretin interfered with this valinomycin effect, whereas phlorizin did not. These results indicate that dissipation of an osmotic gradient across membranes may be responsible for the inhibition of the hemolysis by these inophores. Iso-osmotic cell shrinkage induced by valinomycin in 150 mM NaCl solution did not result in cryohemolysis.

Topics: Amphotericin B; Cold Temperature; Cytochalasins; Glutaral; Hemolysis; Humans; Hydrogen-Ion Concentration; Hypertonic Solutions; Ionophores; Phloretin; Phlorhizin; Stilbenes; Valinomycin