Serum albumin enhances the uptake of [3H]cholesterol from phosphatidylcholine vesicles by cultured human fibroblasts.
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ABSTRACT:
SUBMITTER: Slotte JP
PROVIDER: S-EPMC1153667 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
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Project description:The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.
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Project description:Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.
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Project description:In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular ?-synuclein (?-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of ?-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of ?-syn can influence the distribution and secretion of ?-syn in human neuroblastoma cells. Different ?-syn variants, including ?-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted ?-syn, 0.1-2% was associated with vesicles. The major part of EV ?-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For ?-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT ?-syn. Moreover, such EV-associated ?-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T ?-syn displayed an increased association with EVs. Taken together, our data suggest that ?-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful ?-syn species and thereby contribute to the pathology in ?-synucleinopathies.
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Project description:The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [(3)H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC50s) consistent with those obtained with P. falciparum This assay provides a platform to use P. knowlesi in vitro for drug discovery.
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Project description:A one-step procedure has been developed for the preparation of [3H]bilirubin IX-alpha in good yield from unlabelled bilirubin. Irradiation of an aqueous solution of [3H]bilirubin IX-alpha in the presence of human serum albumin results in the covalent attachment of the bilirubin to the protein. Preliminary degradation studies have been carried out to locate the site of attachment of the bilirubin to the albumin.
| S-EPMC1161221 | biostudies-other
Project description:ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. AIMS:The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. METHODOLOGY:Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. RESULTS:Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-?-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. CONCLUSION:Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.
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Project description:An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low.
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Project description:Undesirable side effects remain a significant challenge in cancer chemotherapy. Here we report a strategy for cancer-selective chemotherapy by blocking acyl-CoA cholesterol acyltransferase-1 (ACAT-1)-mediated cholesterol esterification. To efficiently block cholesterol esterification in cancer in vivo, we developed a systemically injectable nanoformulation of avasimibe (a potent ACAT-1 inhibitor), called avasimin. In cell lines of human prostate, pancreatic, lung, and colon cancer, avasimin significantly reduced cholesteryl ester storage in lipid droplets and elevated intracellular free cholesterol levels, which led to apoptosis and suppression of proliferation. In xenograft models of prostate cancer and colon cancer, intravenous administration of avasimin caused the concentration of avasimibe in tumors to be 4-fold higher than the IC50 value. Systemic treatment of avasimin notably suppressed tumor growth in mice and extended the length of survival time. No adverse effects of avasimin to normal cells and organs were observed. Together, this study provides an effective approach for selective cancer chemotherapy by targeting altered cholesterol metabolism of cancer cells.
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Project description:Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation-Crosslinking-Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (-13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy.
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Project description:Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.
| S-EPMC1152915 | biostudies-other