Addition Of Melittin Research Articles

Electrochemiluminescence Imaging of Liposome Permeabilization by an Antimicrobial Peptide: Melittin.

The permeabilization of liposomes by melittin, an antimicrobial peptide (AMP), has been studied by an electrochemiluminescence (ECL) imaging strategy. The methodology consisted first of encapsulating ECL reagents in sealed giant asymmetrical liposomes (100 μm in diameter) made of DOPG/DOPC phospholipids (i.e., 1,2-dioleoyl-sn-glycerol-3-phospho-(1'-rac-glycerol) sodium salt/1,2-dioleolyl-sn-glycero-3-phosphocholine). Then liposomes were placed on an indium tin oxide electrode coated with poly-l-lysine to avoid any membrane poration/permeabilization through polarization of the electrode surface. Finally, the addition of melittin (from 10 μM to 100 nM in concentration) enabled the permeabilization of the lipid membrane followed by the liposome content release and subsequent light generation through the ECL reagents oxidation processes. Interestingly, at a melittin concentration of 10 μM, two successive leakages occurring on the same liposome could be imaged. Combination of ECL and photoluminescence imaging allowed comprehensive monitoring of the permeabilization and content release of a single liposome. This ECL imaging approach opens interesting perspectives to characterize the instant release of vesicle content upon permeabilization by AMPs or other membrane-active species.

Effects of melittin on laying performance and intestinal barrier function of quails

To study the effects of melittin on egg-laying performance and intestinal barrier of quails, 240 quails (aged 70 d) were randomly divided into 4 groups with 6 replicates (10 quails per replicate). They were fed with basal diet (group B), basal diet+0.08 g/kg melittin (group BA1), basal diet+0.12 g/kg melittin (group BA2) and basal diet+0.16 g/kg melittin (group BA3). The experiment lasted for 21 days. The eggs were collected every day. At the end of the experiment, duodenal, jejunal, and ileal tissues were collected, and the cecal contents were sampled. Intestinal antioxidant index, barrier function, and intestinal flora were analyzed. The results showed that the addition of melittin significantly increased the laying rate and average egg weight. Addition of melittin significantly increased the antioxidant function, mechanical barrier, immune barrier, and the villus height to crypt depth ratio of small intestine. Addition of melittin had no significant effect on the α and β diversity of cecal flora, but significantly increased the abundance of Bacteroidales at family level and genus level. Bioinformatics analysis of cecal content showed significant increase in COG functional category of cytoskeleton, and significant decrease in RNA processing and modification in group BA2. KEGG functional analysis showed significant decrease in steroid biosynthesis, caffeine metabolism, and cytochrome P450 pathways in group BA2. In conclusion, addition of 0.12 g/kg melittin to feed improved the laying performance and the intestinal antioxidant capacity and barrier function of quails but had no significant effect on the composition and structure of cecal microbial community. This study provides experimental data and theoretical basis for the application of melittin as a new quail feed additive.

Development and characterization of the electrospun melittin-loaded chitosan nanofibers for treatment of acne vulgaris in animal model

Acne is an inflammatory disease of the sebaceous glands. Melittin (Mel) is one of the principal toxic components of bee venom that can cause antibacterial and anti-inflammatory effects. Chitosan is a biodegradable polysaccharide has anti-inflammatory, antimicrobial, and regenerative properties. In this study, chitosan (Ch)/Mel 0.001 and 0.003% nanofibers were fabricated for topical treatment of acne vulgaris. Physicochemical properties of Ch/Mel were evaluated using FTIR and FESEM. Furthermore, encapsulation efficiency, tensile strength, water absorption capacity, enzymatic activity, drug release, antimicrobial, and cell cytotoxicity were assayed. Animal models were exerted to study the impact of Ch/Mel nanofibers on the treatment of Propionibacterium acnes. The FESEM results showed that nanofibers were successfully prepared in nano-scale fiber diameter. The FTIR confirmed the presence of Mel in structures. The addition of melittin to the polymer solution reduced electrical conductivity, induced viscosity and tensile strength of the fibers, and decreased the percentage of the swelling. The Mel loading into Ch/Mel 0.003% was reported at 86.74±1%. This Mel releasing process (89.65%) of Ch/Mel 0.003% was slowly completed during 72 h. The Mel retained its hemolytic activity after being loaded into the polymer structure. The drug toxicity study displayed the lack of any significant toxicity from Ch/Mel nanofibers on normal Human Dermal Fibroblasts (HDF). The Ch/Mel 0.003% achieved the highest growth inhibition in P. acnes in vitro and animal studies, this group showed the greatest reduction in inflammation and redness. The Ch/Mel 0.003% structure is proposed as a suitable topical drug delivery system for treating acne vulgaris.

Melittin exerts opposing effects on short- and long-range dynamics in bicontinuous microemulsions

Bicontinuous microemulsions (BμEs) are a promising biomembrane mimetic system for investigating the behavior of antimicrobial peptides (AMPs) and their delivery to open wounds to combat antibiotic-resistant microorganisms. The properties of the BμE host are in turn affected by the guest AMP and can deviate from those of the unperturbed BμEs, especially at higher AMP concentrations. Here we report the effect of an archetypal AMP, melittin, over a wide range of concentrations, on the nanoscopic dynamics of BμEs formed by water/sodium dodecyl sulfate (SDS)/1-pentanol/dodecane, investigated using quasi-elastic neutron scattering (QENS). Two distinct motions are observed, namely, (i) the lateral motion of the surfactant on the surface of the oil channels and (ii) the internal motion of the surfactants. It is found that melittin restricts both the lateral and the internal motion, thereby acting as a stiffening agent. The lateral motion is more strongly affected, at low concentration of melittin. The lateral diffusion coefficient decreased sharply, approaching a constant value at higher melittin concentration. These results are in sharp contrast with the recent dynamic light scattering and neutron spin echo results which showed that at the length and time scales longer than those probed in the current work, melittin enhanced the long-range collective and local undulation motions of BμEs. Considered together, our results indicate that incorporation of melittin modulates the dynamics differently depending on the spatial and temporal regimes, in which the dynamics are being probed. The addition of melittin at low concentrations increased the magnitude of the zeta potential, but further increase of the melittin concentration decreased it. This suggests that addition of melittin at low concentrations led to increase in the surfactant concentration, but did not affect the negative charge per surfactant molecule, while further addition of melittin led to ion pairing of melittin with the oppositely charged surfactant. This study therefore demonstrates how the addition of melittin hinders the lateral motion of surfactants as a result of the strong association between melittin and SDS, suggesting that the release of AMPs from BμE-based delivery vehicles may be hindered.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells.

RNA interference (RNAi) offers the potential to selectively silence disease-related genes in defined cell subsets. Translation into the clinical routine is, however, still hampered by the lack of efficient carrier systems for therapeutic siRNA, endosomal entrapment presenting a major hurdle. A promising siRNA delivery system has previously been developed on the base of polyethylenimine (PEI) and the targeting ligand transferrin (Tf) to specifically reach activated T cells in the lung. In the present work, the focus is on optimizing Tf-PEI polyplexes for gene knockdown in primary activated T cells by improving their endosomal escape properties. Blending of the conjugate with membrane lytic melittin significantly enhanced endosomal release and thereby cytoplasmic delivery, while maintaining selective T cell targeting abilities and overall cell tolerability. The gathered data furthermore demonstrate that melittin addition also distinctly improves several other essential particle characteristics, such as siRNA encapsulation efficiency and stability in lung lining fluids. In conclusion, this results in a novel upgraded siRNA delivery system that is not only able to specifically deliver its payload to the desired target cells via receptor-mediated endocytosis, but also shows enhanced release from endosomal vesicles in order to initiate RNAi in the cytoplasm.

Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells.

Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant.

Potential of mean force for insertion of antimicrobial peptide melittin into a pore in mixed DOPC/DOPG lipid bilayer by molecular dynamics simulation.

Antimicrobial peptides (AMPs) inactivate microorganisms by forming transmembrane pores in a cell membrane through adsorption and aggregation. Energetics of addition of an AMP to a transmembrane pore is important for evaluation of its formation and growth. Such information is essential for the characterization of pore forming ability of peptides in cell membranes. This study quantifies the potential of mean force through molecular dynamics (MD) simulation for the addition of melittin, a naturally occurring AMP, into a DOPC/DOPG mixed bilayer, a mimic of bacterial membrane, for different extents of insertion into either a bilayer or a pore consisting of three to six transmembrane peptides. The energy barrier for insertion of a melittin molecule into the bilayer was highest in the absence of transmembrane peptides and decreased for the number of transmembrane peptides from three to six, eventually approaching zero. The decrease in free energy for complete insertion of peptide was found to be higher for larger pore size. Water channel formation occurred only for insertion into pores consisting of three or more transmembrane peptides with the radius of water channel being larger for a larger number of transmembrane peptides. The structure of the pore was found to be paraboloid. The estimated free energy barrier for insertion of melittin into an ideal paraboloid pore accounting for different intermolecular interactions was consistent with MD simulation results. The results reported in this manuscript will be useful for the development of a model for nucleation of pores and a rational methodology for selection of synthetic antimicrobial peptides.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells.

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 μg/mL melittin + 2 μg/mL cisplatin) and A2780CR (combination treatment 2 μg/mL melittin + 10 μg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R2) and goodness of prediction (Q2), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

Antimicrobial Peptide Impacts the Lateral Diffusion and Bending Rigidity of Phospholipid Membrane

Antimicrobial peptides (AMPs) are the first line of defense system against foreign microorganisms. They are well known for their strong interaction with the cell membrane of pathogens. Quasielastic neutron scattering (QENS) and Neutron spin echo (NSE) measurements have been carried out on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane to understand the effect of melittin, an important AMP on the dynamical and mechanical behavior of membrane. Even at low concentration (0.2 mol%), the addition of melittin is found to influence the membrane dynamics significantly in a different way, depending on the phase of the lipid bilayers. In the gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase, it acts as a stiffening agent, hindering the lateral motion of the lipid molecules. Various interesting results on the impact of melittin on the phase behavior and lateral diffusion of the membrane will be discussed here. Their importance originate from the fact that many cellular functions, including cell signaling, membrane trafficking, and cell recognition are controlled to a great extent by the lateral diffusion of lipids in the membrane. NSE measurements indicate that incorporation of melittin modify the bending rigidity of the membrane, which is found to decrease with the concentration of melittin. Cholesterol is a vital component of erythrocyte membranes, which is a natural target for melittin. To understand the role of cholesterol, both QENS and NSE experiments have also been carried out on cholesterol-supplemented DMPC membrane in the absence and presence of melittin. Our measurements show that the destabilizing effect of melittin on the membranes can be mitigated by the presence of cholesterol. This study might provide new insights into the mechanism of action of antimicrobial peptides and their selective toxicity towards foreign microorganisms.

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from α-amylase of rice.

AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50 ) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 μM, respectively. AmyI-1-18 had little or no hemolytic activity even at 500 μM, and showed negligible cytotoxicity up to 1200 μM. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI-1-18 was added at a concentration equal to its 4×IC50 . Therefore, the antimicrobial activity of AmyI-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare.

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.

Mixtures of supported and hybrid lipid membranes on heterogeneously modified silica nanoparticles.

Simple supported lipid bilayers do not accurately reflect the complex heterogeneity of cellular membranes; however, surface modification makes it possible to tune membrane properties to better mimic biological systems. Here, 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (DETAS), a silica modifier, facilitated formation of supported lipid bilayers on silica nanoparticles. Evidence for a stable supported bilayer came from the successful entrapment of a soluble fluorophore within an interstitial water layer. A fluorescence-quenching assay that utilized a pore-forming peptide was used to demonstrate the existence of two separate lipid leaflets. In this assay, fluorescence was quenched by dithionite in roughly equal proportions prior to and after addition of melittin. When a hydrophobic modifier, octadecyltriethoxysilane, was codeposited on the nanoparticles with DETAS, there was a decrease in the amount of supported bilayer on the nanoparticles and an increase in the quantity of hybrid membrane. This allowed for a controlled mixture of two distinct types of membranes on a single substrate, one separated by a water cushion and the other anchored directly on the surface, thereby providing a new mimic of cellular membranes.

Effect of Curcumin on Peptide-Membrane Interaction: In Kinetics and in Equilibrium

We performed a three-chamber aspirated GUV experiment to study the competitive binding between curcumin and melittin. GUVs were produced and aspirated in chamber 1. Chamber 2 contained 10 microM curcumin. Chamber 3 contained 10 microM curcumin and various concentrations of peptides: penetratin, melittin or magainin. Aspirated GUVs were first equilibrated in chamber 2 and then transferred to chamber 3, where we observed the response of the GUV to the addition of peptides. Without the peptides in chamber 3, the GUV should remain unchanged. The results can be described as the peptides replacing curcumin bound on the bilayer interface and shifting curcumin into the hydrocarbon region, except for magainin. The bound curcumin essentially blocked magainin from binding to the membranes. This can be explained as a kinetic effect because the effect size of a bound magainin is twice the effective size of a bound curcumin, whereas the effective sizes of penetratin and melittin are comparable to curcumin (this is an usual property of curcumin for its small molecular size). Thus for penetratin and melittin, the behavior of the peptides is the same with or without curcumin. On the other hand, the curcumin/peptide/lipid mixtures produce bilayers totally unlike either curcumin with lipid or peptide with lipid. Instead of steady membrane thinning by either melittin or curcumin alone, the addition of melittin to curcumin/DOPC actually thickening the bilayers. This is the first example of incompatible results from equilibrium experiment and from kinetic experiment. It implies that competitive binding of two different membrane-active molecules can be complicated, a caution for drawing conclusion about synergy effects.

Controlled Assembly of Vesicle Layers on Layer-by-layer Particles via DNA Hybridization

We report here on the formation of layers of large unilamellar vesicles (LUVs) superimposed on Layer-by-Layer- (LbL-) particles. DNA oligonucleotides were covalently attached to the outermost negatively charged polyelectrolyte layer of the particles and thus vesicles, with complementary lipophilic DNAs incorporated into the membranes, could be assembled in layers via sequence specific hybridization (see figure). Entrapment of calcein, NBD-rhodamine FRET fusion assay, FRAP, and cryo electron microscopy proved that LUVs attached to LbL-particles remained intact. The assembly was reversible, e.g. heating above the melting temperature of the DNA-hybrids led to the dissociation of the vesicle layer. Fusion of vesicles attached to the LbL-particles and leakage of the entrapped molecules was triggered on demand by addition of melittin. Using different DNA sequences, lipid anchors or compositions of the membrane can regulate the assembly of layers. The LUVs-LbL-particles have many advantages: a controlled and reversible assembly, small and defined size, easy manipulation, biocompatibility, and biodegradability of the particles, and the possibility of a triggered release of different reactants entrapped in different layers of vesicles. LUVs-LbL-particles can be potentially used in diagnostics or for the organization and regulation of reactions on nanoscale.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Melittin as a Permeability Enhancer II: In Vitro Investigations in Human Mucus Secreting Intestinal Monolayers and Rat Colonic Mucosae

Melittin has shown potential as a non-cytotoxic absorption enhancer in Caco-2 monolayers. Our objectives were to assess in vitro efficacy and cytotoxicity of melittin in two intestinal permeability models and investigate the potential mechanism by which melittin might enhance gastrointestinal absorption. The effects of melittin were examined in the mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12), using transepithelial electrical resistance (TER), transmission electron microscopy (TEM) and the MTT viability assay. The effects of melittin on TER, permeability and short circuit current (Isc) were also investigated in rat colon mucosae mounted in Ussing chambers. Ion transporting capacity of tissue was measured in response to secretagogues as surrogate markers of cytotoxicity. Melittin stability was examined by a means of a hemolytic assay. The mechanism by which melittin decreases TER across the rat mucosa was examined with a range of enzymatic inhibitors. Apical addition of melittin resulted in a reversible non-cytotoxic concentration-dependent decrease in TER across E12 monolayers, which was independent of the presence of mucus. Apical addition of melittin reduced TER and increased the permeability of [(14)C]-mannitol across rat colonic mucosae. The melittin-induced drop in TER in rat colon was significantly attenuated by W7 suggesting partial mediation by calmodulin. The rapid and reversible nature of melittin's permeation enhancing properties and its limited cytotoxicity in polarized intestinal epithelia, suggests a potential drug delivery role for the peptide in oral formulations of poorly absorbed drugs.

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2. The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors. Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4-70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling. Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.

PLA2 – a major regulator of volume‐sensitive taurine release in NIH3T3 fibroblasts

Release of the organic osmolyte taurine efflux from NIH3T3 cells is increased by osmotic cell swelling in hypotonic medium and decreased by osmotic cell shrinkage in hypertonic medium. Release of arachidonic acid is increased under hypotonic conditions if oxidation of the fatty acid via the 5-lipoxygenase (5-LO) system is prevented by the 5-LO inhibitor ETH 615-139 and is reduced under hypertonic conditions. Exposure to the amphiphilic bee venom peptide melittin, which has no effect on the kinetic properties of PLA2 but promotes substrate replenishment, induces release of arachidonic acid and taurine in NIH3T3 cells under isotonic conditions. The potentiating effect of melittin on arachidonic release and taurine efflux is substantially increased in osmotically swollen cells and almost abolished in osmotic shrunken cells. H2O2 potentiates the melittin-induced taurine efflux under isotonic conditions but has only a minor effect on the melittin-induced taurine efflux under hypertonic conditions. Bromoenol lactone and manoalide, known inhibitors of Ca2+-independent phospholipase A2 (iPLA2) and secretory phospholipase A2 (sPLA2), respectively, reduce arachidonic acid and taurine release from NIH3T3 cells under hypotonic conditions and following addition of melittin. It is suggested that iPLA2/sPLA2 activity is responsible for the volume-sensitivity of taurine release in NIH3T3 mouse fibroblasts.

Interaction of melittin with solid supported membranes

The interaction of the membrane active peptide melittin with solid supported lipid bilayers has been scrutinized by means of impedance spectroscopy and scanning force microscopy. Highly flexible lipid bilayers anchored ia a hydrophilic spacer terminated by a thiol group onto gold were used to investigate the influence of melittin on the electrical parameters of membranes by means of ac impedance analysis. A melittin induced increase in the capacitance and conductivity of the membrane was observed suggesting that melittin induces defects within these immobilized lipid bilayers. Experiments were performed in the absence and presence of EDTA to investigate the influence of remaining phospholipase A2 in the melittin sample as well as the sole impact of phospholipase A2 from Apis mellifera. It was shown that the most pronounced influence of melittin on the electrical parameters of bilayers was found when EDTA was absent, revealing a synergistic effect of phospholipase A2 and melittin. Morphological changes of solid supported membranes induced by the combined effect of melittin and phospholipase A2 were visualized by TappingMode scanning force microscopy in aqueous solution. Dissolution of the lipid layer and the formation of non-lamellar, discoid structures was observed within minutes after addition of melittin, supporting a “ carpet-like” mechanism of peptide action leaving mixed peptide lipid micelles behind. Scanning force microscopy allowed us to resolve the gradual action of melittin within one single scan.

Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells

Human monocytic leukemia cells (U937) were challenged with synthetic melittin, and arachidonic acid (AA)/acylated lipids from both cells (pellet) and media (supernatant) were analyzed by thin layer chromatography (TLC). From these data, melittin-mediated activation/inhibition of major phospholipases in U937 cells was related to pore formation, permeabilization and cytolysis as determined by light microscopy. Also, the effect of melittin on acylhydrolase activity in the cell-free sonicated lysates of U937 cells was examined. Here we report that synthetic melittin (1 μM) caused cytolysis of U937 cells within 10–15 min. Cellular hypertrophy (5 min) and aggregation (1 min) preceded cytolysis. TLC analysis of these lipids showed that total levels (cellular+medium) of diacylglycerol (DAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) decreased, while that of arachidonic acid (AA) increased continuously (5–30 min). However, levels of phosphatidylethanol (PEt) phosphatidic acid (PA) and phosphatidylserine (PS) were increased transiently at 5–10 min being maximal at 5 min. Taken together, the combined levels of PEt and PA (an end product of phopholipase D, PLD) were about 42-fold higher than the level of AA at 5–10 min. Enhancement of AA levels appeared to result from in vitro reactions of various acylhydrolases and their phospholipid substrates (free/membrane bound) liberated into the medium during pore formation/cell lysis. Incubation of sonicated cell lysates also enhanced release of AA, which decreased upon addition of melittin, indicating that melittin inhibited these acylhydrolases. A consistent decrease in the level of DAG showed that phospholipase C was unaffected. Hence, transient activation of PLD by melittin at the point of initiation of cytolysis, suggested a role for PLD in melittin-mediated membrane disruption/cytolysis by an uncharacterized signal transduction mechanism.