Flip-flop of doxorubicin across erythrocyte and lipid membranes

Doxorubicin, an anticancer drug, is extruded from multidrug resistant (MDR) cells and from the brain by P-glycoprotein located in the plasma membrane and the blood-brain barrier, respectively. MDR-type drugs are hydrophobic and, as such, enter cells by diffusion through the membrane without the requirement for a specific transporter. The apparent contradiction between the presumably free influx of MDR-type drugs into MDR cells and the efficient removal of the drugs by P-glycoprotein, an enzyme with a limited ATPase activity, prompted us to examine the mechanism of passive transport within the membrane. The kinetics of doxorubicin transport demonstrated the presence of two similar sized drug pools located in the two leaflets of the membrane. The transbilayer movement of doxorubicin occurred by a flip-flop mechanism of the drug between the two membrane leaflets. At 37 degrees, the flip-flop exhibited a half-life of 0.7 min, in both erythrocyte membranes and cholesterol-containing lipid membranes. The flip-flop was inhibited by cholesterol and accelerated by high temperatures and the fluidizer benzyl alcohol. The rate of doxorubicin flux across membranes is determined by both the massive binding to the membranes and the slow flip-flop across the membrane. The long residence-time of the drug in the inner leaflet of the plasma membrane allows P-glycoprotein a better opportunity to remove it from the cell.     

Hydrophobicity and sorption of chlorophenolates to lipid membranes

We have studied sorption of ionized species of chlorophenols and pentahalophenols to lipid membranes using egg-phosphatidylcholine (egg-PC) vesicles and measuring their zeta-potential as a function of aqueous concentration of the phenolates. The zeta-potential isotherms can be understood in terms of a sorption model that is a combination of the Gouy-Chapman model of the electrical double layer at the membrane-water interface and the Langmuir model for sorption. Two intrinsic sorption parameters were determined: the linear partition coefficient beta m, which relates the membrane surface density of the phenolates to their aqueous concentration and the area of the adsorption site, Ps. The linear partition coefficient is the measure of the affinity of phenolates to the lipid membrane. It depends strongly on the molecular structure: 2,6-dichlorophenolate beta m = (0.45 +/- 0.08) x 10(-7); m; 3,5-dichlorophenolate beta m = (0.22 +/- 0.02) x 10(-6) m; 2,4,6-trichlorophenolate beta m = (0.63 +/-0.06) x 10(-6) m; 2,4,5-trichlorophenolate beta m = (0.11 +/- 0.01) x 10(-5) m; 2,3,5,6-tetrachlorophenolate beta m = (0.56 +/- 0.07) x 10(-5) m; 2,3,4,5-tetrachlorophenolate beta m = (0.55 +/- 0.06) x 10(-5) m; pentachlorophenolate beta m = (0.34 +/- 0.05) x 10(-4) m; pentafluorophenolate beta m = (1.00 +/- 0.13) x 10(-7) m and pentabromophenolate beta m = (0.19 +/- 0.04) x 10(-3) m. Ps was found to be independent of phenolate structure, Ps = 3.3 +/- 0.1 nm2. The membrane affinity of chlorophenolates was compared with the octanol-water partition coefficients of un-ionized chlorophenols. It was shown that the free energy of transfer of chlorophenolates from water into the lipid membrane can be divided into non-electrostatic and electrostatic contributions. The no-nelectrostatic contribution corresponds to the hydrophobicity parameter alpha = 3.94 +/- 0.0.08 kcal per nm2 of molecular surface area. The electrostatic contribution contains a term inversely proportional to the molecular radius of the phenolate ion which has the physical meaning of the work of transfer of the phenolate ion from water into the membrane. The polarity of the sorption region of egg-PC membranes is given in terms of the dielectric constant and was estimated to be 12.4 (range 10.5-13.4).     

pICln predominantly localizes at luminal surface membranes of distal tubules and Henle''s ascending limbs

We produced a highly specific antibody to the C-terminal peptide sequence of pICln. It recognized pICln with a 38-kDa molecular mass on SDS-polyacrylamide gel electrophoresis, coinciding with that previously reported. During native polyacrylamide gel electrophoresis, three immunoreactive bands (38, 70, and 130 kDa) were detected. The isoelectric point of pICln was calculated to be 4.0. Subcellular localization study showed the presence of pICln in the soluble and microsomal fraction. pICln can be easily solubilized from the membrane fraction with Triton X-100. From immunohistochemical observations, we found pICln to be obviously located on the luminal surface membranes of the distal tubules and Henle's loop ascending limbs, and it can also be found inside proximal tubular cells. The present results suggested that pICln functions as a "cytosolic anchor = membrane insertion" model, and it plays important roles in the "urine dilution segment" cells of nephrons.     

The effect of undecaprenol on bilayer lipid membranes

The influence of undecaprenol on phosphatidylcholine macrovesicular bilayer lipid membranes has been studied by electrophysiological techniques. The current-voltage characteristics, ionic transference numbers, the membrane conductance-temperature relationships and the membrane breakdown voltage were measured. The permeability coefficients for Na+ and Cl- ions, the activation energy of ion migration across the membrane, the membrane hydrophobic thickness and the membrane Young's modulus were determined. Undecaprenol increases membrane conductance, membrane capacitance, membrane ionic permeability and membrane elastic deformability, decreases the activation energy, membrane hydrophobic thickness and membrane electromechanical stability, and does not change membrane selectivity. The formation by undecaprenyl molecules of fluid microdomains modulating membrane hydrophobic thickness is postulated. The data suggest that the behaviour of undecaprenol in membranes is regulated by transmembrane electrical potential.     

Theoretical analysis of protein organization in lipid membranes

The fundamental physical principles of the lateral organization of trans-membrane proteins and peptides as well as peripheral membrane proteins and enzymes are considered from the point of view of the lipid-bilayer membrane, its structure, dynamics, and cooperative phenomena. Based on a variety of theoretical considerations and model calculations, the nature of lipid-protein interactions is considered both for a single protein and an assembly of proteins that can lead to aggregation and protein crystallization in the plane of the membrane. Phenomena discussed include lipid sorting and selectivity at protein surfaces, protein-lipid phase equilibria, lipid-mediated protein-protein interactions, wetting and capillary condensation as means of protein organization, mechanisms of two-dimensional protein crystallization, as well as non-equilibrium organization of active proteins in membranes. The theoretical findings are compared with a variety of experimental data.     

Theory of self-assembly of lipid bilayers and vesicles

A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.     

Energetics of permeation of thin lipid membranes by ions

The energy of an ion in a thin hydrocarbon membrane relative to its energy in a bulk aqueous phase is considered in terms of the electrostatic and surface components that may be expected to be involved. Except when diffusion activation energies are large compared to partition free energies, the latter will control permeation rate and the state of an ion having the lowest partition energy will be critical for its permeability. This minimum is found when an ion is surrounded with a thin layer of water. All ions of the same charge will tend to be at their lowest state in a sphere of water of the same size. It is concluded, therefore, that all ions of a given charge will have about the same permeability in lipid membranes.     

Optical and electrical studies on dansyllysine-valinomycin in thin lipid membranes

Dansyllysine-valinomycin, a fluorescent analogue of the ionophore valinomycin was synthesized and incorporated into black lipid membranes. Its concentration inside the membrane was measured fluorometrically and was also determined from electrical relaxation experiments, which were analyzed on the basis of a previously proposed carrier model. The results of both methods agreed within less than one order of magnitude. This appears satisfactory in view of the sources of error inherent in both procedures. A conductance increment per carrier molecule of about 3 - 10(-17) omega-1 was obtained for dansyllysine-valinomycin in diphytanoyllecithin membranes at 25 degrees C and 1 M RbCl in the aqueous phases. This is about 400 times smaller compared to unmodified valinomycin in monoolein membranes. The difference is mainly caused by the change in the membrane properties and to a smaller extent by the structural modification of the carrier.     

Assembly of antibodies in lipid membranes for biosensor development

An investigation of the incorporation of antibody in lipid films of a composition that has been used for biosensor preparation is reported. IgG that is incorporated into lipid monolayers prepared from 7:3 mixtures of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid is edge-active, and enters and penetrates the fluid region of the mixed-phase system when monolayers are held at low pressure (< 20 mN/m). It was found that there is an "exclusion pressure" observed in pressure-area (pi-A) curves that are collected for monolayers that contain antibody. This term refers to a specific threshold of lateral pressure (which is reached by monolayer compression) that can cause explusion of antibody from the interior of a membrane. Microscopic images of monolayers containing the fluorescent phospholipid nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE), or antibody labeled with tetramethylrhodamine isothiocyanate (TRITC), were used to determine the structure of membranes, and the location of effects on structure caused by IgG. Ellipsometric measurements of lipid monolayers that were cast onto silicon wafers by the Langmuir-Blodgett method were used to study the thickness of monolayers and to investigate the structural changes that occurred at the "exclusion pressure." Both the use of fluorescent antigen and ellipsometry indicated that antibody binding activity was present and was dependent on compression pressure. The effects of pH and ionic strength of subphase, antibody concentration, incubation time, and lateral pressure have been examined. The results may indicate the conditions that can be used to improve the incorporation of active IgG for preparation of biosensors that are based on lipid membranes.     

Some similarities between processes at biological membranes and lipid bilayers

Several processes at biological membranes can be simulated by experiments with artificial lipid bilayer membranes. Three selected examples are discussed: The uncoupler induced proton permeability of lipid bilayers, the initiation of action potential like voltage responses in lipid membranes, and the reconstitution of active cation pumps across planar lipid bilayers or lipid vesicles.     

Role of lipids in the permeabilization of membranes by class L amphipathic helical peptides

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.     

Infrared spectroscopy of supported lipid monolayer, bilayer, and multibilayer membranes

A study was designed to determine the effects of triptorelin (Decapeptyl C.R.), a GnRH-agonist in a depot formulation, on androst-16-en-3-one (androstenone), LH, oestradiol and testosterone and on carcass composition and muscle structure in boars. Treatment with this highly potent GnRH analog ensured peptide release for 4 weeks when given to 18 animals at 135 days of age (group 1). Control animals (n = 20, group 2) received a saline injection. Blood samples (n = 10) were collected by venipuncture from each boar from 7 days before hormone treatment to 28 days after treatment. During the treatment period, three samples of back fat were taken by biopsy to determine the levels of androstenone and skatole. Testis size was determined at slaughter at 165 days of age. The treatment of boars with triptorelin increased concentrations of LH, oestradiol-17 beta, and testosterone for one day, followed by a decrease to base-line concentrations. The result of sensorial testing for boar taint detection at slaughter showed a slight reduction in this trait (0.76 vs. 0.99). The concentration of androstenone in the body fat of all treated animals was lower than the generally accepted limit of 0.5 microgram/g for the absence of boar taint, compared with the concentrations from 20 control animals among which 13 showed levels higher than this limit. The hormonal treatment significantly reduced the weight and size of the tests. There was no effect on skatole levels in backfat (0.141 vs. 0.150 microgram/g). The GnRH-agonist did not influence the carcass composition of treated boars. The muscle meat percentage was not significantly altered in the experimental group (51.39 vs. 50.50%). Overall, these results indicate that the use of this form of GnRH-agonist in a depot formulation offers a new possibility for reducing concentrations of androstenone which is mainly responsible for undesirable boar taint.     

The effect of anaesthetics on the dynamic heterogeneity of lipid membranes

A randomized multicenter study was performed in order to investigate the acceptance of a low-dose OC (30 micrograms of ethinyloestradiol and 150 micrograms of desogestrel), using a 9 weeks on and 1 week off schedule (prolonged regimen, n = 198), compared to a traditional 3 weeks on, 1 week off schedule (standard regimen, n = 96). Haemoglobin and blood pressure remained the same in both groups during the study. No significant differences were found in body weight changes between the two groups. There was significantly more breakthrough bleeding and spotting in the group with prolonged regimen than in the group with standard regimen, but both breakthrough bleeding and spotting decreased during the trial. Irregular bleeding was significantly less in women who were already using OC, compared to "new starters." No serious side effects occurred. Significantly more women stopped the trial because of bleeding problems in the group with prolonged regimen, while there were significantly more women who stopped the trial because of headache in the group with standard regimen. After completing 12 months, or after premature withdrawal from the study, each women completed a questionnaire. Sixty-three per cent of the women preferred the studied alternative and twenty-six per cent preferred the traditional OC.     

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.     

Temperature-jump experiments on thin lipid membranes in the presence of valinomycin

Temperature jump relaxation experiments on planar lipid membranes in the presence of valinomycin were performed using the absorption of a strong light flash as an energy source for the generation of the T-jump. The relaxation of the current carried by valinomycin/Rb+ complexes was measured. The results were interpreted on the basis of a transport model which was also analyzed by voltage jump relaxation experiments. The study shows that the application of the T-jump technique provides valuable information about transport kinetics as well as the dynamics of the membrane structure. At the given experimental conditions the relaxation of the current is believed to reflect a temperature-dependent transition of the membrane to a new conformational state of low order. The relaxation could be resolved with the present technique only at low temperatures and for membranes of high microviscosity.