Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells

Summary: Multidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-β-cyclodextrin (MβCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MβCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MβCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4° C) to higher density fractions in MβCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp functio

P-Glycoprotein in Proteoliposomes with Low Residual Detergent: The Effects of Cholesterol

Purpose: There is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents. Materials and methods: P-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis. Results: In the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol. Conclusions: Cholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activatio

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: Endothelial versus epithelial features

Summary: ECV 304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV 304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy, Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement, transmembrane electrical resistance, and activity of γ-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as T24 cells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV 304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endotheli

Comparing the Lipid Membrane Affinity and Permeation of Drug-like Acids: The Intriguing Effects of Cholesterol and Charged Lipids

Purpose: Lipid bilayers regulate the passage of solutes into and between cellular compartments. A general prerequisite for this passage is the partitioning of the solute into the bilayer. We investigated the relationship between bilayer partitioning and permeation of three drug-like acids in liposomal systems consisting of phosphatidylcholine alone or mixed with cholesterol or charged lipids. Materials and Methods: Bilayer partitioning was determined by equilibrium dialysis. Bilayer permeation was studied with a luminescence assay which is based on the energy transfer of the permeant to intraliposomal terbium(III). Results: The influence of the lipid composition on the pH-dependent membrane affinity was in accordance with the membrane rigidity and possible electrostatic interactions between the acids and the lipids. However, there was no direct relationship between membrane affinity and permeation. This seeming discrepancy was closer analyzed with numerical simulations of the permeation process based on the single rate constants for partitioning and translocation. The simulations were in line with our experimental findings. Conclusions: Depending on the single rate constants and on the geometry of the system, lipid bilayer permeation may positively, negatively or not correlate with the bilayer affinity of the permean

Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein-Related Activities

No Heading: Purpose.: P-glycoprotein (P-gp), a membrane ATPase expelling many structurally unrelated compounds out of cells, is one of the major contributors to multidrug resistance. It is enriched in cold TritonX-100 insoluble membrane domains (i.e., rafts). The purpose of this work was to characterize the ATPase activities of raft preparations from P388 cells overexpressing P-gp (P388/ADR) or devoid of P-gp (P388) and to establish a P-gp-enriched screening system for P-gp-interfering compounds. Methods.: Rafts were extracted with cold TritonX-100. The ATPase activity was characterized in 96-well plates using a fluorescence assay. Results.: The ATPase activity per mg protein was about five times higher in P388/ADR rafts than in crude membranes. The anti-P-gp antibody C219 inhibited 20% of the activity in P388/ADR rafts but only about 10% of the activity in P388/ADR crude membranes and had no effect on the activity of P388 rafts. The known P-gp-activating compounds verapamil, progesterone, and valinomycin revealed the typical bell-shaped activity/concentration profiles in P388/ADR rafts, indicative for activation at low compound concentrations and inhibition at concentrations >10 to 100 μM. The inhibitory effect was also observed in P388 rafts. Conclusions.: Extracted rafts are rich in functional ATPases. Rafts from P-gp-overexpressing cells display P-gp-typical ATPase activity and provide an easy, P-gp-enriched screening syste

Transfer of Lipophilic Markers from PLGA and Polystyrene Nanoparticles to Caco-2 Monolayers Mimics Particle Uptake

Purpose. The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Methods. Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy. Particle uptake was quantified either directly, by counting internalized nanoparticles after separation from the Caco-2 monolayers, or indirectly, by extraction of the lipophilic fluorescence marker. In vitro release studies of lipophilic markers from nanoparticles were performed in standard buffer systems and buffer systems supplemented with liposomes. Results. Instead of uptake of polystyrene and PLGA nanoparticles by Caco-2 monolayers an efficient transfer of lipophilic fluorescence markers from nanoparticles into Caco-2 cells with subsequent staining of intracellular lipophilic compartments was observed. Whereas in standard buffer no release of fluorescent marker from polystyrene and PLGA nanoparticles was observed, the release studies using liposome dispersions as receiver revealed an efficient transfer of fluorescent marker into the liposome dispersion. Conclusions. The results suggest that the deceptive particle uptake is caused by a collision-induced process facilitating the transfer of lipophilic fluorescent marker by formation of a complex between the nanoparticles and the biomembranes. Diffusion of the marker within this complex into lipophilic compartments of the cell strongly affects quantitative evaluation of particle uptak

Обеспечение пожаровзрывобезопасности и защита от чрезвычайных ситуаций особо опасных производств на территории Бурятии

Проведён аналитический обзор информации, знакомство с правовыми нормами и требованиями к пожарной безопасности на особо опасном объекте, велась разработка мероприятий по обеспечению пожарной безопасности на объекте, аналитический обзор современных методов пожаротушения на объекте.An analytical review of information, familiarity with the legal norms and requirements for fire safety at a particularly hazardous facility, the development of measures to ensure fire safety at the site, an analytical review of modern firefighting methods at the site

From Pharmacy to Pharmaceutical Sciences – The New Curriculum

Looking at the statistics of Pharmacy graduates, the picture has changed considerably over the last 25 years. In the 1980s at least 80% of the diploma students chose an occupation in a community pharmacy. Today graduates are employed in hospitals, industry, government, and in public health positions beside the traditional community pharmacy that still accounts for about 50%. This reflects the strategy of the Institute to develop 'Pharmacy' into 'Pharmaceutical Sciences', which has been pursued by the nomination of several professors in fields beyond classical pharmacy in the 1990s, while keeping the classical pharmacy chairs strong. This trend is ongoing with a recent nomination of a chair in Pharmacogenomics. As a consequence, a new concept for the training of pharmacists has been designed resulting in the Curriculum in Pharmaceutical Sciences that began in the year 2000

Immobilized Artificial Membrane (lAM)-HPLC for Partition Studies of Neutral and Ionized Acids and Bases in Comparison with the Liposomal Partition System

Purpose. To study the partitioning of model acids ((RS)-warfarin and salicylic acid), and bases (lidocaine, (RS)-propranolol and diazepam), with immobilized artificial membrane (lAM)-HPLC, as compared to partitioning in the standardized phosphatidylcholine liposome/buffer system. Methods. The pH-dependent apparent partition coefficients D were calculated from capacity factors (k′IAM) obtained by IAM-HPLC, using a 11-carboxylundecylphosphocholine column. For lipophilic compounds k′IAM, values were determined with organic modifiers and extrapolation to 100% water phase (k′IAMw) was optimized. Temperature dependence was explored (23 to 45° C), and Gibbs free energy (ΔG), partial molar enthalpy (ΔH) and change in entropy (ΔS) were calculated. Equilibrium dialysis was used for the partitioning studies with the liposome/buffer system. Results. For extrapolation of k′IAMw, linear plots were obtained both with the respective dielectric constants and the mole fractions of the organic modifier. All tested compounds showed a similar pH-D diagram in both systems; however, significant differences were reproducibly found in the pH range of 5 to 8. In all cases, ΔG and ΔH were negative, whereas ΔS values were negative for acids and positive for bases. Conclusions. In both partitioning systems, D values decreased significantly with the change from the neutral to the charged ionization state of the solute. The differences found under physiological conditions, i.e. around pH 7.4, were attributed to nonspecific interactions of the drug with the silica surface of the IAM colum

Immobilized Artificial Membrane (lAM)-HPLC for Partition Studies of Neutral and Ionized Acids and Bases in Comparison with the Liposomal Partition System

ISSN:0724-8741ISSN:1573-904