A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization.

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab')2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25-0.5 µL of intravenous Apilic antivenom/µg honeybee venom. In vivo injection of 0.1-1 µg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 µg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

ER stress and Rho kinase activation underlie the vasculopathy of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.

Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques.

Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response

Abstract Background Apis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases. Methods Africanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti- and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined. Results Promastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 μg.mL−1 and 1.4 μg.mL−1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL−1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-α, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-α, NO and H2O2. Conclusions The results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of ß-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Magnetic "fishing" assay to screen small-molecule mixtures for modulators of protein-protein interactions.

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 µM), with the latter two values being in close agreement with literature values.

Targeted ablation of prostate carcinoma cells through LH receptor using Hecate-CGbeta conjugate: functional characteristic and molecular mechanism of cell death pathway.

A Hecate-CGbeta conjugate (lytic peptide and beta-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.

Cholesterol inhibits the lytic activity of melittin in erythrocytes.

Although cell lysis by the hemolytic peptide, melittin, has been extensively studied, the role of specific lipids of the erythrocyte membrane on melittin-induced hemolysis remains unexplored. In this report, we have explored the modulatory role of cholesterol on the hemolytic activity of melittin by specifically depleting cholesterol from rat erythrocytes using methyl-beta-cyclodextrin (MbetaCD). Our results show that the hemolytic activity of melittin is increased by approximately 3-fold upon depletion of erythrocyte membrane cholesterol by approximately 55% without any appreciable loss of phospholipids. This result constitutes the first report demonstrating that the presence of cholesterol inhibits the lytic activity of melittin in its natural target membrane, i.e., the erythrocyte membrane. These results are relevant in understanding the role of cholesterol in the mechanism of action of melittin in the erythrocyte membrane.

Lipopolysaccharides in bacterial membranes act like cholesterol in eukaryotic plasma membranes in providing protection against melittin-induced bilayer lysis.

Melittin is a small, cationic peptide that, like many other antimicrobial peptides, lyses cell membranes by acting on their lipid bilayers. However, the sensitivity to antimicrobial peptides varies among cell types. We have performed direct binding and vesicle leakage experiments to determine the sensitivity to melittin of bilayers composed of various physiologically relevant lipids, in particular, key components of eukaryotic membranes (cholesterol) and bacterial outer membranes (lipopolysaccharide or LPS). Melittin binds to bilayers composed of both zwitterionic and negatively charged phospholipids, as well as to the highly charged LPS bilayers. The magnitude of the free energy of binding (deltaG degrees ) increases with increasing bilayer charge density; deltaG degrees = -7.6 kcal/mol for phosphatidylcholine (PC) bilayers and -8.9 to -11.0 kcal/mol for negatively charged bilayers containing phosphatidylserine (PS), phospholipids with covalently attached polyethylene glycol (PEG-lipids), or LPS. Comparisons of these data show that binding is not markedly affected by the steric barrier produced by the PEG in PEG-lipids or by the polysaccharide core of LPS. The addition of equimolar cholesterol to PC bilayers reduces the level of binding (deltaG degrees = -6.4 kcal/mol) and reduces the extent of melittin-induced leakage by 20-fold. LPS and 1:1 PC/cholesterol bilayers have similar high resistance to melittin-induced leakage, indicating that cholesterol in eukaryotic plasma membranes and LPS in Gram-negative bacteria provide strong protection against the lytic effects of melittin. We argue that this resistance is due at least in part to the similar tight packing of the lipid acyl chains in PC/cholesterol and LPS bilayers. The addition of bacterial phospholipids to LPS bilayers increases their sensitivity to melittin, helping to explain the higher sensitivity of deep rough bacteria compared to smooth phenotypes.

Solid-state NMR conformational studies of a melittin-inhibitor complex.

Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-IVIFDC-NH(2), with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. Solid-state NMR spectroscopy was used to study the interaction between melittin and the peptide inhibitor. Location of the binding site in the melittin-inhibitor complex was determined using lanthanide ions, which quench NMR resonances from molecular sites that are in close proximity to the unique ion binding site. Our results indicated that the inhibitor binding site in melittin is near Leu13, Leu16 and Ile17, but not near Leu6 or Val8. On the basis of these data we propose that the inhibitor binds to melittin in the vicinity of Ala15 to Trp19 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance, using rotational resonance NMR, between the [1-(13)C] of Leu13 in melittin and the [3-(13)C] of Phe4 in the inhibitor.

Conformational studies of a melittin-inhibitor complex.

The conformation of a melittin-inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in 1HT1 relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in alpha-helical structure for increasing ratios of inhibitor bound to melittin.

Inhibition of membrane-active peptides by fatty acid-peptide hybrids.

Nine fatty acid-peptide hybrid molecules were constructed using the general formula CH3(CH2)nCO-Phe Asp Cys-amide and tested for their ability to inhibit cell lysis induced by the membrane-active peptide melittin. All of these molecules, where n = 4-14, inhibited the action of melittin to some extent, but the longer carbon chains were most effective. Several potential inhibitors were also constructed with conservative substitutions in the peptide portion of the molecule. All were effective to varying degrees. We concluded that in the hexapeptide inhibitor published by Blondelle et al. (1993), the role of the first three residues is only to provide hydrophobic interaction with the melittin and has no particular amino acid sequence specificity. Some of these inhibitors were found to inhibit the lytic activity of a melittin analogue which had only superficial sequence similarity to melittin and also a truncated form of melittin, indicating the generality of the action of the inhibitors.

Cyclosporin A, but not FK506, increases arachidonic acid release and inhibits proliferation of pituitary corticotrope tumor cells.

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

The lytic activity of the bee venom peptide melittin is strongly reduced by the presence of negatively charged phospholipids or chloroplast galactolipids in the membranes of phosphatidylcholine large unilamellar vesicles.

We have investigated the dependence of the lytic activity of the bee venom peptide melittin on the lipid composition of its target membrane. The lysis of large unilamellar liposomes, measured as loss of the fluorescent dye carboxyfluorescein, in the presence of melittin was strongly reduced when the negatively charged lipids phosphatidylglycerol (PG) or phosphatidylserine (PS), or the plant chloroplast lipids monogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG) were incorporated into egg phosphatidylcholine (EPC) membranes. This reduction was evident at concentrations below 10 wt% of the additional lipids. It was not due to reduced binding of melittin to the vesicles. It was also not related to a reduced insertion depth of the peptide into the bilayer, as shown by quenching of the intrinsic tryptophan fluorescence of the peptide by the aqueous quencher sodium nitrate. Fourier transform infrared spectroscopy (FTIR) revealed specific interactions of the peptide with the headgroups of the inhibitory lipids. The phosphate peak in PG was shifted by two wavenumbers after the addition of melittin. There was no shift in EPC or PS. Instead, in PS the COO- peak was strongly distorted in the presence of melittin. These data indicate ionic interactions between the basic peptide and the negative charges on the membrane surface. The galactolipids are uncharged. Here the evidence points to hydrogen bonding between melittin and OH-groups of the sugar headgroups. Liposomes containing DGDG were the only case where we found evidence for changes in fatty acyl chain motion due to the presence of melittin, from the CH2-scissoring peaks.

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.

Peptide inhibitors of melittin action.

The sequence of peptides necessary to inhibit melittin-induced lysis was studied using 13 peptide analogues of the inhibitor Ac-IVIFDC-NH2. Although this inhibitor is a disulfide-linked dimer, inhibition was equally effective if the thiol SH was blocked or replaced by methionine or lysine. The substitution of phenylalanine with other aromatic residues preserved activity, as did the replacement of aspartic acid by asparagine. The results suggest that the cytolytic activity of melittin can be inhibited by a short peptide of four hydrophobic residues followed by two other nonspecific residues. Fluorescence studies showed that the inhibitor caused a blue shift in the Trp emission spectrum. A spin label attached to the N-terminus of the inhibitor significantly quenched the fluorescence. These data confirmed the involvement of Trp 19 with the inhibitor, also predicted by molecular modeling of the probable binding site. Density gradient studies with large unilamellar vesicles indicated that the inhibitor prevented melittin from reacting with the lipid bilayer.

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries.

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D-amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.

Identification of inhibitors of melittin using nonsupport-bound combinatorial libraries.

A strategy has been developed for the identification of inhibitors of toxins or regulatory proteins. This approach is based on blocking the access of such proteins to their biological targets during their solution transport. This approach uses the strength of nonsupport-bound synthetic combinatorial libraries (SCLs) for the study of acceptor-ligand interactions. A non-receptor assisted toxin, melittin, was selected for the present study to illustrate this application of the SCL approach. Hexapeptide SCLs were assayed for their ability to inhibit the cytolytic activity of melittin toward bacterial and erythrocyte cells. Over 20 inhibitory hexapeptides were identified following the screening and deconvolution processes from millions of sequences. The identified inhibitory peptides appeared to interact directly with melittin. These interactions appear to decrease melittin's ability to undergo lipid- and/or polysaccharide-induced conformational changes, and are demonstrated by fluorescence and circular dichroism spectroscopy.

[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.

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.