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Vascular endothelial cells form the inner nonthrombogenic lining of the large blood vessel. Through back-exchange and fluorescence recovery after photobleaching experiments and using the two fluorescent lipids 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl]glycerophosphocholine and 1-acyl-2[6-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]hexanoyl]glycerophosphoethanolamine, we have recently shown that an energy-dependent and protein-dependent aminophospholipid translocase activity is present in the plasma membrane of cultured bovine aortic endothelial cells, which specifically transports phosphatidylethanolamine from the outer leaflet toward the inner leaflet of the membrane lipid bilayer. In the present study, using the same approach and 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl] glycerophosphoserine as the probe, it is shown that this conclusion is also valid for phosphatidylserine. Furthermore, evidence is presented indicating that this aminophospholipid translocase activity can be maintained, suppressed, and restored at will, depending on the conditions of cell incubation. Thus, the translocase activity is detected for cells maintained in their normal culture medium or in a serum-free incubation medium [Dulbecco's modified Eagle's medium (DMEM)] supplemented with the basic fibroblast growth factor, whereas inhibition is observed for cells exposed for at least 2 h to DMEM. The translocase activity is restored when these pretreated cells are further incubated at least for 1 h in the presence of serum or of basic fibroblast growth factor. In view of the importance of basic fibroblast growth factor as a mitogenic and differentiating agent for vascular endothelial cells, various growth factors were tested (acidic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, transforming growth factors alpha and beta, vascular endothelial growth factor, interferon gamma, tumor-necrosis factor, insulin, and interleukin 4). Only basic fibroblast growth factor was active in the maintenance and restoration of the translocase activity. With respect to the effects of serum, evidence is presented showing that high-density lipoproteins might play a role in the control of the translocase activity. However, the positive effects of basic fibroblast growth factor, serum and high-density lipoproteins on the translocase activity were suppressed when experiments were carried out in the presence of an anti-(basic fibroblast growth factor) IgG, thus indicating that in all cases, basic fibroblast growth factor was directly involved in the modulation of the aminophospholipid translocase activity present in the plasma membrane of bovine aortic endothelial cells.

Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacterium halobium, was used and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2GroPCho), dipalmitoyl- (Pam2GroPCho) and distearoyl- (Ste2-GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-1,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsin/phosphatidylcholine molar ratios ∼ 1:50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length dp∼ 3.0–3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau2GroPCho (ΔT∼ 40 K, mean hydrophobic thickness d∼ 2.4 nm), and to a lesser extent of Myr2GroPCho (ΔT∼ 23 K, d∼ 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam2GroPCho (ΔT∼ 0 K, d∼ 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (ΔT∼−13 K, d∼ 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsin/phosphatidylcholine molar ratios

The interaction with membranes of three anti-cancer drugs of the ellipticine family was studied by fluo- rescence quenching of membrane probes. The fluorescence of three probes, located at different levels in mem- branes, was quenched by addition of two types of ellipticine derivatives, one amphiphilic drug (9-methoxy- ellipticine) and two dipolar molecules (9-aminoellipticine and 9-hydroxyellipticine). By comparing the quenching curves obtained, the following can be proposed. a) 9-Methoxyellipticine can penetrate deeper in the lipid layers than 9-aminoellipticine and 9-hydroxy- ellipticine can. b) The three drugs are able to penetrate at least between the first methylene groups of the acyl chains of lipids in liposomes. c) In an isolated bacterial membrane, only 9-methoxyellipticine can be located in the region of the first methylene groups of lipids, the two dipolar drugs being adsorbed on the membrane surface. It was also shown that cholesterol hindered the penetration of 9-methoxyellipticine in the bilayer of liposomes.

Ellipticine and some of its derivatives constitute an interesting class of substances displaying an antitumoral activity against both experimental and human tumors. We have recently shown that some of these molecules were able to interact rapidly and strongly with acidic phospholipids. In the present report, amphiphilic ellipticine and 9-methoxyellipticine were tested against synthetic dilauroyl phosphatidylglycerol and dipalmitoylphosphatidylglycerol as well as a sample of phosphatidyl-glycerol with methyl-branched fatty acids, to investigate the stoichiometry of these interactions and their consequences on the organization of the lipid phase. Through surface pressure and surface potential measurements, carried out at the air-water interface, it is shown that the two drugs strongly interact with phosphatidylglycerols, whatever the film surface pressure or the nature and physical state of the lipid acyl chains may be. Such interactions bring about large film expansions and a complete modification of the phase properties of the lipids. These interactions achieve, at saturation, drug/lipid associations near the 1/1 molar ratio, stabilized by both electrostatic and hydrophobic forces. The drug molecules can be viewed as inserted between the lipid molecules, the—NH+ group interacting with the phosphate function and the aromatic moiety being in contact with the lipid acyl chains. From compression isotherms of 1/1 drug/lipid mixtures, it can be inferred that the two drugs cause a large condensing effect on both the dilauroyl and dipalmitoyl derivatives of phosphatidyl-glycerol, as compared to the behaviour of the lipids alone when ionized. In the mixed films, both lipids would exist in a very similar and rather condensed state, intermediate between the liquid-expanded and gel states in which these two lipids can respectively exist on pure water. The possibility of membranes being one of the sites of action of ellipticines is discussed.

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12-(9-anthroyloxy)-stearic acid, a fluorescent compound which forms non-fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants KD and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 microM, saturating conditions), data indicate that the lipid remains in a liquid-expanded state. This is true even at a high surface pressure (pi approximately equal to 37 mN X m-1), above the apparent 'transition' which can be observed at 30-35 mN X m-1 on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion to the conclusion that this 'transition' would be a structural transition between two models of interaction: one, below the 'transition', where the polypeptide ring penetrates between the film-forming lipid molecules and another one, above the 'transition', were the antibiotic is adsorbed at the lipid-water interface with only its hydrocarbon chain penetrating the film.

C-6 esters of methyl alpha-D-glucoside and C-6, C-6' 'diesters of alpha, alpha'-D-trehalose with C18 and C32 threo and erythro mycolic acids (from chemical source) and of C80-erythro-mycolic acid (from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well. Throughout a monolayer study at the air-water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction. Compression isotherms of C32 esters suggested a transition between liquid-expanded and liquid-condensed states. Latent heats Qc and entropy changes delta S associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32-erythro-mycoloyl)-trehalose. On the other hand, short chain C18 esters form fluid phases (t greater than 10 degrees C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t = 20 degrees C). These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3-sn-phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes.

Fast lateral proton conduction was observed along the lipid/water interface using a fluorescence technique. This conduction can be detected for a large number of lipids, both phospholipids and glycolipids. The efficiency of the proton transfer is dependent on the molecular packing of the host lipid at a given surface pressure. The proton conduction which is present in the liquid expanded state is abolished by the transition to the liquid condensed state. The proton transfer is affected slightly by the ionic content of the aqueous subphase except in the case of calcium which can inhibit the conduction along phosphatidylglyceroethanolamine. We suggest that the transfer of the protons occurs along a bidimensional hydrogen-bond network formed from the polar head groups, their water molecules of hydration and the water molecules which are intercalated between the lipid molecules.

A fluorescent analogue of cord factor, a glycolipid toxin of mycobacteria, has been synthesized and its interactions with liposomes and isolated mitochondria have been studied. This compound, methyl alpha-D-6[12-(9-anthroyl)stearoyl]glucoside, is shown to be active against oxidative phosphorylation. When spread as a monolayer at the air-water interface, it forms a well organized phase and it strongly interacts with phosphatidylcholine. Addition of phosphatidylcholine liposomes or of isolated mitochondria to a water disperson of this fluorescent cord factor analogue results in a large increase of the fluorescence intensity. Moreover, the glycolipid probes for the temperature-dependent phase transition of the added suspensions. It is thus suggested that this cord factor analogue penetrates within mitochondrial membranes, a result which is discussed with respect to our previous conclusions concerning the way natural cord factors can interact with these organelles.

Ionization of the acidic phospholipid phosphatidylglycerol has been studied by measuring the surface potential of monomolecular films of the lipid as a function of the aqueous subphase pH and the concentration of monovalent cations (Li, Na, Cs). It is shown that the experimental data can be interpreted by means of the Gouy-Chapman theory in its simplest formulation, provided an adsorption of cations at the membrane surface is accounted for. This allows us to predict the ionization state of the lipid for given ionic conditions in the subphase. Above pH 4, for subphase ion concentration higher than 10 mM, or for ion concentrations above 0.1 mM at pH 5.6, phosphatidylglycerol is fully deprotonated. Within the limits of our theoretical approach, association constants of the cations to the lipid lie around 0.1-0.6 M-1.

It is shown that photo-induced cross-linking reaction between anthracene-labelled phospholipids can be used for studying, at a molecular level, their lateral distribution in bilayer structures. A simple and versatile method is proposed. It is based on the property of anthracene to form covalently bound dimers upon irradiation in the near ultraviolet (360 nm) and on the possibility of separating the lipid photo-dimers from the lipid monomers by thin-layer chromatography. Identification of the photo-dimers is easily achieved since, upon illumination at a shorter wavelength (250–280 nm), they partially dissociate to the native monomer molecules. The feasibility of the method was tested by checking the effects of cations (sodium, calcium) on the homogeneity of 1/1 mixtures of anthracene-phosphatidylcholine, i.e. 1-acyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholine (Anthr-PC) with anthracene-phosphatidic acid (Anthr-PA) and with anthracene-phosphatidylglycerol (Anthr-PG) in the form of liposomes. These lipids were anthracene-labelled by acylation of their glycerol backbone at the sn-2 position with the synthetic 9-(2-anthryl)-nonanoic acid. Data presented indicate a good miscibility of these lipids in the presence of sodium. For each lipid mixture, the lipid heterodimers were clearly identified and, quantitatively, they dominated the lipid homodimers, as expected for a regular distribution of the lipids in the 1/1 mixture. Addition of calcium ions to the lipid suspensions did not alter the miscibility properties of Anthr-PC and Anthr-PG. In contrast, calcium triggered a clear-cut phase separation in the Anthr-PC/Anthr-PA mixture as, in this case, only traces of the heterodimer form of the lipids remained observable on the chromatogram. The three anthracene-phospholipids, pure or mixed together, exhibit a clear-cut gel-to-liquid phase transition which was detectable by fluorescence intensity measurements. The analysis of, the corresponding phase-transition temperatures confirms, at a ‘macroscopic’ level, the effects of sodium and calcium on the mixing properties of the anthracene phospholipids which were revealed at a ‘microscopic’ level by the dimerization procedure.

In the present work, it is shown that the photobleaching technique as well as experimentation based on fluorescence recovery after bleaching can be extended to monolayers spread at the air-water interface. A mathematical model is derived which allows the determination of translational diffusion coefficients of species diffusing in such a system. Using 12-(9-anthroyl)stearic acid (anthroylstearate) as a fluorescent probe, dispersed either in dipalmitoylphosphatidylcholine or in dipalmitoylphosphatidylglycerol in various conditions of subphase ionic composition and surface pressure of the monolayer, including phase transition domains, we are led to the following conclusions: 1. Anthroylstearate molecules seem to aggregate in ‘microdomains’ where their fluorescence properties remain unchanged regardless of the compression states of the host monolayer. 2. In any case, a break in the diffusion constants appears on compressing films of both dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. In particular, this break coincides with the liquid expanded to gel phase transition of these lipids when it occurs. 3. Diffusion of anthroylstearate in dipalmitoylphosphatidylglycerol depends strongly on the subphase ionic strength and on the nature of cations: Na+, Mg2+, Ca2+.

1-Palmitoyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholine (Anthr-PC), a non-perturbing phospholipid probe [de Bony, J. and Tocane, J. F. (1983) Chem. Phys. Lipids 32, 105–121], has been designed in order to obtain insight into the membrane lipid organization at a ‘microscopic’ level, in terms of lateral distribution both in model and in natural membranes [de Bony, J. et al. (1984) Eur. J. Biochem. 143, 373–379; FEBS Lett. 174, 1–6]. In the present study, the molecular motions of this new fluorescent probe embedded in a lipid matrix have been investigated by fluorescence anisotropy techniques in steady-state and time-resolved modes. The results indicate that long axis rotation, monitored by the out-of-plane mode of rotation of the fluorophore, is fast even in the phospholipid gel state. It is moderately sensitive to the phase transition. The data suggest that this rotation is anisotropic. Cholesterol exhibits little effect on this rotation. The rotation of the long axis itself is sensitive to the transition. It is hindered as inferred from measurements at wavelength where both the in-plane and out-of-plane motions contribute to the depolarization of the emitted fluorescence light. Cholesterol restricts this motion. The behaviour of the free 9-(2-anthryl)-nonanoic acid is not significantly different from that of Anthr-PC. These results are discussed with respect to the influence of orientational constraints on the photodimerization process when this lipid probe is used to monitor phospholipid lateral distribution.

Methyl alpha-D-glucoside and alpha, alpha'-D-trehalose acylated on their primary hydroxyl group by alpha-alkyl-beta-hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was a strongly cooperative phenomenon. The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.

9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non-fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5-(N-hexadecanoyl)-aminofluorescein, 12-(9-anthroyloxy)-stearic acid, 9-(2-anthryl)-nonanoic acid and a synthetic anthracene-phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 - 2 x 10(-9) cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene-labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.

Fast lateral proton conduction along the lipid/water interface has recently been experimentally demonstrated in our laboratory [Teissie, J., Prats, M., Soucaille, P. & Tocanne, J.F. (1985) Proc. Natl Acad. Sci. USA, in the press]. The present study gives a more precise description of the way various physical parameters can affect this process. The dependence of the distance covered by the proton on time is demonstrated to be quadratic. Increasing the speed of stirring in the injection compartment or the amount of injected acid or the contact between the monolayer and the acidic subphase increased the efficiency of the proton transfer. Raising the strength of the buffer in the bulk phase inhibited proton conduction. Results from experiments where the transfer of protons from the bulk phase to the interface was modified, suggested the occurrence of an 'energy barrier' limiting the access of protons from the bulk phase to the lipid polar head region.

9-(2-Anthryl)-nonanoic acid is a new fluorescent and photoactivable probe, which has been designed for studying the lateral diffusion rate and the lateral distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. It is shown that this anthracene fatty acid is metabolically incorporated into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of the eukaryotic Chinese hamster ovary cells in culture. Under our culture conditions (Eagle's minimal essential medium plus delipidized fetal calf serum) this incorporation proceeded with a very good rate (up to 45 mol/100 mol, after two days culture) and could be easily modulated depending on the way the cells were fed with the anthracene fatty acid. It occurred to a similar extent at the sn-1 (55 ± 5%) or at the sn-2 (45 ± 5%) position on the phospholipid glycerol backbone, without any degradation or elongation. No double labelling at the sn-1 and sn-2 positions was detected. Although incorporation of the anthracene fatty acid affected the cell growth rate (generation time of 48 h compared to a generation time of 21 h for control cells) it did not bring about cell mortality. This incorporation took place not only into the phospholipids but also into the triglycerides with, as a consequence, the appearance of strongly fluorescent lipid vesicles inside the cells. It affected the whole cell fatty acid composition by slightly increasing the amount of palmitic acid and markedly decreasing the amount of stearic and oleic acids.

Ellipticine is the type of a group of substances used against human cancer. It was shown that ellipticine and 9-methoxyellipticine strongly interact with monolayers of negatively charged phospholipids. In the present paper it is shown by spectrophotometric methods that ellipticine interacts with suspensions of a model membrane with a 1/1 charge neutralization, and with a natural membrane, both negatively charged. At a physiological pH, ellipticine undergoes protonation in the presence of these membranes. Its apparent pK is increased by 1, and its spectral behaviour, when the pH is changed, indicates that the drug is in a less polar environment, suggesting that hydrophobic bonds link the drug and the lipids. The existence of such bonds is suggested by the fact that the drug is not significantly released from anionic membranes when the pH is increased to 10, at which value ellipticine is not ionized. The consequences of these ionic and hydrophobic interactions are briefly discussed.