Michel Deleers

Neurotoxic cations induce membrane rigidification and membrane fusion at micromolar concentrations.

The effect of the neurotoxic cations aluminum, cadmium and manganese on membranes was examined in sonicated unilamellar vesicles containing phosphatidylserine and compared to the effect of Ca2+. Fusion of membranes was monitored by assessing the resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-y)phosphatidylethanolamine and N-(lissamine-rhodamine B-sulfonyl)phosphatidylethanolamine. Self-quenching of high concentrations of carboxyfluorescein in liposomes was used to demonstrate the release of molecules entrapped in liposomes to compare the kinetics of leakage and intermixing of lipid. Rigidification of membranes was evaluated by monitoring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene embedded in membranes containing phosphatidylserine and dipalmitoylphosphatidylcholine. Cation-induced lipid intermixing of vesicles membranes and release of dye from the vesicles occurred in the same concentration range. With aluminum, these effects were observed with concentrations less than 25 microM. Significant rigidification of vesicle membranes was apparent with less than 25 microM of Al3+. Similar effects could only be observed with concentrations of Cd2+ and Mn2+ at least one order of magnitude higher (200 and 400 microM, respectively).

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles.

The interaction of Al3+, Cd2+ and Mn2+ with phosphatidylserine-containing lipid vesicles was studied. Phase separation of vesicles was investigated by monitoring fluorescence quenching of the phospholipid analogue 1-palmitoyl-2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminocaproyl)phosphatidylcholine (C6-NBD-PC). Aggregation was determined by turbidimetry and leakage of vesicles content during fusion was monitored by the fluorescence of released 6-carboxyfluorescein. Al3+ demonstrated quenching at less than 30 mumol/l with a maximum effect at 100 mumol/l. Al3+-induced aggregation and dye release from the lipid vesicles were observed in the same concentration range. The effect of Cd2+ and Mn2+ on quenching was much less pronounced and could only be demonstrated in the 0.1-1 mmol/l range. Increasing amounts of phosphatidylcholine or phosphatidylethanolamine in the vesicles decreased both Al3+-induced quenching and aggregation, whereas cholesterol only slightly increased aggregation without affecting quenching.

Piracetam-induced changes to membrane physical properties: A combined approach by 31P nuclear magnetic resonance and conformational analysis

Piracetam, Nootropil (2-oxo-1-pyrrolidine acetamide), is a drug promoting erythrocyte deformability. To establish the mode of action of this compound, we have investigated its influence on the organization of model phospholipid membranes. 31P NMR data show that the drug induces a structural modification in liposomes made of phosphatidylcholine and phosphatidylethanolamine. Our conformational analysis results have allowed the interpretation of the effect of piracetam on these model membranes: the specific interaction between the drug molecules and the phosphate headgroups induces a new organization of the lipids favouring formation of mobile drug-phospholipid complexes that exhibit an isotropic-type signal in the 31P NMR spectra.

Ultrastructural, physico-chemical and conformational study of the interactions of gentamicin and bis(beta-diethylaminoethylether) hexestrol with negatively-charged phospholipid layers

Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

Like diphtheria toxin and the N‐terminal (M r 23 000) region of fragment B, CB1 (M r 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (M r 23 000 and 13 000) share a common segment (M r 6300) containing the predicted amphipathic, α‐helical, transverse lipid‐associating domain (M r 2750) of fragment B[J. Cell Biol. (1980) 87, 837–840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172–176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C‐terminal region of CB1 is proposed.

Specific interaction between concanavalin A and glycolipids incorporated into planar bilayer membranes.

Summary Changes in conductance of glycerol monooleate planar membranes containing glucosylceramide, lactosylceramide or human brain gangliosides were measured after the addition of Concanavalin A into the aqueous phase. A 380 and 31-fold increase of conductance values was observed respectively with glucosylceramide and lactosylceramide, whereas gangliosides had no significant effect. Modifications of membrane conductance were suppressed by the addition of methyl α-D glucopyranoside, a Concanavalin A inhibitor. It is concluded that the lectin molecule interacts with specific glycolipids incorporated into the membrane model and that this interaction may be related to the position of the glucose molecule in the carbohydrate moiety of the lipids.

Glucose induces membrane changes detected by fluorescence polarization in endocrine pancreatic cells

Abstract Pancreatic endocrine cells prepared from rat islets were labelled with 1,6-diphenyl-1,3,5-hexatriene and examined in a microviscosimeter. Glucose caused a dose-related decrease in fluorescence polarization. This decrease was observed within 1 min after increasing the concentration of glucose. These findings suggest that glucose affects membrane viscosity in pancreatic endocrine cells. At a glucose concentration of 16.7 mM, the estimated viscosity was 15 ± 3 per cent lower than basal value (2.01 ± 0.12 P).

Ionophore-mediated calcium exchange diffusion in liposomes

Abstract The exchange diffusion of 45 Ca in multilamellar liposomes containing the antibiotic ionophore A23187 is enhanced in a dose-related fashion at increasing concentrations of external Ca 2+ or at increasing A23187/lipid molar ratios. An increase in fluidity of the lipid bilayer augments the permeability to Ca by facilitating both the formation and mobility of the Ca-ionophore complexes.

Evidence for a specific interaction between GT1 ganglioside incorporated into bilayer membranes and thyrotropin

Recent studies suggest that a ganglioside or a ganglioside-like struc- ture may be an important component of the thyro- tropin (TSH) membrane receptor [ 11 ,121. The inter- action of TSH with the specific ganglioside would result in a conformational modification such that a subunit of the hormone molecule would penetrate the membrane and initiate the adenylate cyclase stimulation [11,12]. The present report demonstrates a specific inter- action between TSH and GTr ganglioside incorporated in a planar bilayer membrane. The membrane conduc- tance changes support the hypothesis that after specific binding to GTr ganglioside, the TSH molecule undergoes a conformational change which leads to a penetration of the hormone in the lipid layer. 2.

Increase in CO3H- influx and cellular pH in glucose-stimulated pancreatic islets

When rat islets are preincubated with fluorescein diacetate and examined in a spectrofluorometer, the intracellular pH rapidly increases by 0.21 pH units in response to a rise from 1.7 – 16.8 mM glucose. This coincides with a marked increase in 14Co3H— net uptake by the islets, suggesting that the glucose‐induced increase in H+ generation rate is compensated for by stimulation of CO3 H—/Cl− exchange.