Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

Amphotericin B-induced apoptosis and cytotoxicity is prevented by the Na+, K+, 2Cl(-)-cotransport blocker bumetanide

Amphotericin B is the most commonly used antifungal drug although it exhibits poor effectiveness and considerable toxicity during treatment. It acts as a ionophore inducing cellular potassium efflux. The efflux of potassium, which is necessary for cell shrinkage during apoptosis, is counteracted by increased inward pumping of potassium ions. Modulation of potassium pump activity could therefore interact with programmed cell death depending on the nature of the disruption of cellular potassium homeostasis and subsequently affect the cytotoxicity of various drugs. We explored the role of apoptosis in amphotericin B-induced cytotoxicity in a mesothelioma cell line (P31) and investigated the role of K+ influx inhibitors of Na+, K+, ATPase and Na+, K+, 2Cl(-)-cotransport in these processes. Clone formation was used to determine the cytotoxicity of amphotericin B, ouabain (Na+, K+, ATPase blocker), and bumetanide (Na+, K+, 2Cl(-)-cotransport blocker), alone or in combination. Apoptosis was estimated by quantifying free nucleosomes. Amphotericin B (3.2 micromol/L, 3 mg/L) per se reduced the percentage of surviving clones to 64% and increased the number of nucleosomes by 31% compared to untreated control. When ouabain (100 micromol/L) was added to amphotericin B a less than additive effect on clone formation was seen but no reduction of nucleosomes was noted. Bumetanide (100 micromol/L) per se was not cytotoxic but increased cellular nucleosome expression. Bumetanide eradicated amphotericin B-induced reduction of formed clones and generated nucleosomes. In conclusion, the induction of apoptosis seems to be of significant importance in amphotericin B-induced cytotoxicity. Amphotericin B-induced cytotoxicity and apoptosis was eradicated by the Na+, K+, 2Cl(-)-cotransport inhibitor bumetanide. The changes of cellular K+ fluxes induced by bumetanide combined with amphotericin B needs further elucidation. Bumetanide could possibly be used in antifungal therapy to increase amphotericin B effectiveness doses without increasing its adverse effects.     

No evidence of oxidant events in amphotericin B cytotoxicity versus L. infantum promastigotes.

Amphotericin B is used for the treatment of systemic mycoses and visceral leishmaniasis. The objective of our study was to evaluate the impact of catalase, ascorbic acid and ketoconazole on the amphotericin B toxicity towards Leishmania promastigotes membrane by two flow cytometric tests, the membrane potential assay using a cationic dye, [DiOC5(3)], and the membrane permeability test using propidium iodide. The collapse of membrane potential appeared at amphotericin B concentrations weaker than those assessed by the membrane permeability test. The binding of amphotericin B to membrane sterol was not modified by catalase or ascorbic acid whereas amphotericin B-induced growth inhibition could be modulated by these products. The permeabilizing effect of amphotericin B on parasite membrane was strongly reduced in the presence of ketoconazole. These results confirmed the pore hypothesis of amphotericin B action and suggested that flow cytometric methods constituted a valuable alternative to conventional methods for assessing the effect of drugs on cellular membrane and evaluating parasite susceptibility to polyene antibiotics.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Inhibition by Cu2+ of amphotericin B induced lysis of erythrocytes

Amphotericin B-induced lysis of erythrocytes is diminished in the presence of Cu2+, but the prelytic amphotericin B-induced K+ leakage is unaffected. These results and the weak binding of Cu2+ to amphotericin B, demonstrated by circular dichroism and EPR studies, are consistent with the view that Cu2+ protects erythrocytes by increasing their resistance to lysis.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合治疗隐球菌性脑膜炎的临床回顾性研究

目的 观察两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合治疗非AIDS、非器官移植隐球菌性脑膜炎患者的疗效和安全性,探讨非AIDS、非器官移植隐球菌性脑膜炎患者诱导治疗的新方案.方法 采用回顾性病例对照研究,18例非AIDS、非器官移植隐球菌性脑膜炎患者诱导期分别接受两性霉素B脂质体、氟胞嘧啶和伊曲康唑联合(研究组n=7)与两性霉素B、氟胞嘧啶和氟康唑联合治疗(对照组n=11).比较两组治疗方案在临床症状、体征变化、病死率、病原学转阴率、脑脊液改变等方面的差异及血常规、肝肾功能损害等不良反应发生情况.结果 治疗前研究组和对照组脑脊液新型隐球菌培养阳性分别为5例和8例(P =0.676),治疗1周后分别为5例和8例(P =0.676),治疗2周后分别为2例和4例(P =0.572),治疗3周后分别为0例和1例(P=0.611),治疗4周后,无死亡病例,两组脑脊液新型隐球菌培养均为阴性,脑脊液生化两组之间差异无统计学意义,对照组较研究组丙氨酸氨基转移酶(156.82±41.30 vs 97.00±22.02,P=0.003)、尿素氮(8.45±3.18 vs 5.54±1.28,P=0.020)升高更明显.结论 两性霉素B脂质体、氟胞嘧啶及伊曲康唑联合治疗非AIDS、非器官移植隐球菌性脑膜炎患者疗效确切,安全性较好,可以作为诱导期治疗的选择.     

Apparent activation volumes of hydrophobic ions and carriers in planar lipid bilayers

Summary The role of large amounts of membrane-bound water in regulating various functions of the membrane is not clear at present. We have investigated the effect of perturbing the interfacial water structure on the osmotic shrinkage properties, such as water permeability and extent of shrinkage of egg lecithin liposomes. Water structure was perturbed by a series of reagents which have been earlier reported to affect phase transition of dipalmitoyl phosphatidylcholine liposomes by perturbing interfacial water structure. Anomalous variations of osmotic shrinkage properties with concentration of structure maker and breaker reagents have been interpreted to arise from concentration-dependent structural transitions of the ordered water at the membrane-aqueous interface. Various modes of interaction of these reagents on interfacial structured water have been suggested. Influence of molecular size and functional groups on the molecule in actions of some structure makers and breakers were also observed.     

Amphotericin B-induced ion flux is markedly attenuated in phosphatidylglycerol membrane as evidenced by a newly devised fluorometric method

The effect of phospholipid head group on the membrane-permeabilizing activity of amphotericin B (AmB) was examined using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) liposomes. The activity of AmB was evaluated as K⁺ influx measured as pH change inside liposomes by fluorescent measurements of 2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein (BCECF). AmB showed prominent permeability in POPC liposomes, whereas hardly inducing ion flux in POPG membrane. POPC added to POPG liposomes as a minor constituent markedly enhanced membrane permeability, indicating the importance of a phosphonocholine group of PC for the drug’s activity.     

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.     

Cationic polypeptide-induced fusion of acidic liposomes

Fusion of acidic liposomes was induced by Mg²⁺, Ca²⁺, polylysine and polymyxin B. The extent of fusion and the concomitant change in liposome permeability induced by divalent cations depended on the concentration of liposomes in the suspension as well as on the cation concentration. In contradistinction, the extent of fusion and the change in permeability induced by the polypeptides depended only on the polycation concentration. The difference in the pattern of interaction, between the liposomes and the various cations, is a result of different binding affinities. The binding of the polypeptides to the liposomes, in contrast to divalent cations, is practically irreversible. The potential of polylysine to induce fusion of acidic phosphatidylethanolamine-devoid liposomes was used to demonstrate that in order to obtain fusion, both membranes involved must be susceptible, at least to a certain degree, to fusion by the proper inducer. When lysophosphatidylcholine substituted for phosphatidylcholine in phosphatidylethanolamine-rich acidic liposomes, extensive polylysine-induced fusion was obtained without concomitant spillage of the liposome contents.     

Fusion of planar bilayer phospholipid membranes with liposomes]

Lecithine-cholesterol liposomes containing amphotericin B ionoforic marker were used to study the interaction between liposomes and planar phospholipid membranes. The liposomes were shown to increase the permeability of the planar membrane, which may be explained in terms of membrane fusion. Bivalent cations (Mg2+ and particularly Ca2+), dicetylphosphate producing negatively charged groups on the membrane surface and the n-decane suspension in water promote the fusion, whereas the increase of the cholesterol content in the liposomes prevents it.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biologically active amphotericin B-calix[4]arene conjugates

In order to provide tools for investigations of amphotericin B ion channels, new conjugates bearing a calix[4]arene scaffold covalently linked to four amphotericin B molecules were synthesized. These macromolecules adopt a cone conformation that mimics the structure of a transmembrane pore. The antifungal activity of the conjugates 3 and 4 was superior or similar to that of native amphotericin B, with minimal inhibitory concentration values of 0.10 and 0.25 microM, respectively. Furthermore, the hemotoxicity of the new conjugates was considerably lower (at least 10 times) than the hemotoxicity of monomeric amphotericin B. Finally, the formation of ion channels in the lipid bilayer by the amphotericin B tetramer was monitored by measuring the K+ efflux from various liposomes.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Interaction of dopamine with artificial phospholipid membranes

The interaction of dopamine (DA) with phospholipid membranes has been investigated. The membrane current in planar bilipid membrane (BLM) modified by amphotericin B in voltage clamp conditions under alternating polarity was shown to symmetrically increase 1.2 times when DA was added outside the BLM. This implies a uniform change of charge on each membrane surface and hence the diffusion of DA within the BLM and its exposure on the internal side. The appearance of single threads and bundles of filaments within the internal liposomal cavities was observed in the ultrastructure of suspended thin-walled liposomes filled with globular actin after the introduction of DA into external solution. This reshaped liposomes into rod-like, spindle-shaped or angular structures. Actin serves as a marker for DA due to its property to polymerize itself under the influence of DA. Thus, the structural reorganization of liposomes manifests the presence of DA inside them and the induction of actin polymerization.     

A tormentor in the quest for plant p53-like proteins

Pretreatment with salicylic acid (SA), an inducer of plant disease resistance, enhanced the capacity of parsley cells for the induction of a rapid K⁺/pH response and the subsequent coumarin (phytoalexin) secretion. In SA-primed cells, a low elicitor dose induced these two responses to a similar extent as did a high elicitor dose in non-primed cells. These observations suggest that the SA-mediated augmentation of the early K⁺/pH response may contribute to the enhancement of subsequent coumarin secretion. As the amphotericin B-induced K⁺/pH response was not enhanced in SA-primed cells, it is concluded that signaling components that are improved by priming are located between elicitor signal perception and the plasma membrane transporters mediating the K⁺/pH response.