Symmetrical distribution and rapid transbilayer movement of cholesterol in Mycoplasma gallisepticum membranes

The exchange of cholesterol between [¹⁴C]cholesterol-labeled Mycoplasma gallisepticum cells and an excess of sonicated egg phosphatidylcholine/cholesterol vesicles (molar ratio of 0.9) was measured. More than 90% of the radioactive cholesterol underwent transfer from intact cells to the vesicles. The kinetics of the transfer was biphasic. About 50% of the radioactive cholesterol was exchanged with a half-time of about 4 h. The residual was exchanged at a slower rate with a half-time of about 9 h at 37°C. Bovine serum albumin had a pronounced effect in enhancing both the fast and slow rates of cholesterol exchange, but did not affect the pool sizes significantly. The half-time for equilibration of the two pools in the presence of 2% albumin, calculated using a reversible two-pool method of analysis, was 6.2 h. The effect of albumin was also obtained with isolated membrane preparations and with cells treated with growth inhibitors, suggesting that this effect is independent of albumin preservation of cell viability. The rate enhancement of albumin was concentration dependent with maximal effects observed with 2%, where the rates of exchange of both the rapidly and slowly exchanging pools were twice as fast. The mechanism by which albumin may affect the exchange rates is discussed.     

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Kinetics of transfer of alpha-tocopherol between model and native plasma lipoproteins

The rate of spontaneous transfer of alpha-tocopherol, cholesterol and beta-carotene between model and native lipoproteins was measured to determine the mechanism and kinetics of equilibration of these lipids in plasma. Cholesterol and alpha-tocopherol transfer from apolipoprotein A-I/1-palmityl-2- oleoylphosphatidylcholine ( POPC ) recombinants to bovine brain ganglioside/ POPC single bilage vesicles with half-times of approximately 20 min and 70 min, respectively. Under identical conditions, there is no significant transfer of beta-carotene even after an 18-h incubation period. alpha-Tocopherol transfers from apolipoprotein A-II/dimyristoylphosphatidylcholine recombinants with a half-time of 40 min and an activation energy of 17.2 kcal/mol. Incubation of high-density lipoproteins containing alpha-[3H]tocopherol with low-density lipoproteins or very-low-density lipoproteins results in the equilibration of the labelled lipid between the lipoprotein classes in 1 h. A comparison of the rates of transfer indicates that alpha-tocopherol equilibrates 2-3-times more slowly than cholesterol but on a time scale much shorter than the lifetime of lipoproteins in the circulation. Thus, the distribution of alpha-tocopherol is not kinetically controlled but determined thermodynamically by the partitioning between the total amount of lipid in each compartment. The spontaneous transfer of beta-carotene is too slow for this equilibration to occur.     

Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells

Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.     

Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts

The kinetics of low density lipoprotein (LDL) cholesterol transport to the plasma membrane of Chinese hamster ovary (CHO) cells was studied. LDL was reconstituted with [3H]cholesteryl linoleate and added to CHO cells in a pulse-chase experiment. The internalization and lysosomal cleavage of reconstituted LDL (rLDL) [3H]cholesteryl linoleate to free [3H]cholesterol occurred with a half-time of 37 min after a 30-min lag. The rate of transport of released [3H]cholesterol to the plasma membrane was measured by brief (20-30 sec) cholesterol oxidase treatment of intact, adherent cells: the half-time of transport was 42 min. The similarity in the rate of free cholesterol release from rLDL and transport of this cholesterol to the plasma membrane suggests very rapid transport of rLDL cholesterol from the lysosome to the plasma membrane. Cells were shown to be intact throughout the cholesterol oxidase treatment by the absence of cell-derived lactate dehydrogenase (LDH) activity or K+ in the assay buffer.     

Transmembrane migration (''flip-flop'') of cholesterol in erythrocyte membranes.

After exchange with [14C]cholesterol-labelled plasma lipoproteins for 0.5-4h, erythrocytes were extracted with bile-salt solutions. The extracted cholesterol (mainly from the outside of the erythrocyte membrane) had the same specific radioactivity as the residual sterol. Thus cholesterol equilibrates rapidly (half-time less than 1 h) between the two sides of the membrane.     

Alterations in biosynthesis and homeostatis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [¹⁴C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [¹⁴C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.     

Alterations in biosynthesis and homeostasis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.     

Localization of an immunodominant 64kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence

A 64 kDa lipoprotein (LP 64) haemagglutinin (pI 4.9-5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X-114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]-palmitate-labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post-infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one- or two-dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14C-labelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.     

Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.

Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.     

Transbilayer distribution of sterols in mycoplasma membranes: a review

The polyene antibiotic, filipin, binds to 3 beta-hydroxysterols. The initial rate of filipin-sterol association, monitored in a stopped-flow spectrophotometer, was first order in each reacting partner. The ratio of rate constants in intact mycoplasma cells relative to isolated, unsealed membranes provides an estimate of sterol distribution in the membrane bilayer. Cholesterol is distributed symmetrically in the bilayer of M. gallisepticum cells from the early exponential phase. However, in the M. capricolum membrane two-thirds of the unesterified cholesterol is localized in the outer leaflet; alkyl-sterols are distributed predominantly in the external monolayer. Cholesterol is translocated rapidly in the bilayer of M. capricolum cells. Exogenous phospholipids incorporated into the membrane had no effect on the cholesterol distribution in M. capricolum.     

Cellular cholesterol efflux. Role of cell membrane kinetic pools and interaction with apolipoproteins AI, AII, and Cs.

Efflux of [14C]cholesterol from various cells was monitored in the presence of discoidal complexes of egg phosphatidylcholine and purified apolipoproteins, containing either apoAI, AII, or Cs. Particles containing apoAI were more efficient acceptors than those containing apoAII or Cs when the donor cells were J774 macrophages. No differences were observed when the same acceptor preparations were exposed to Fu5AH rat hepatoma or rabbit aortic smooth muscle cells. The differential efficiency of apolipoproteins in stimulating cholesterol removal from J774 cells was maintained in a plasma membrane-enriched fraction isolated from the same cells. Nonlinear regression analysis of kinetic data obtained from J774 cells exposed to apoAI complexes indicated that cholesterol efflux was best fitted to a curve describing the release from two kinetic compartments. Approximately 10% of cholesterol was transferred from a rapidly exchangeable pool with a t1/2 ranging between 1.5 and 3 h, and the remaining fraction was released from a slower pool with a t1/2 of about 20 h. Modulation of cholesterol efflux from J774 cells by either varying the concentration or the apolipoprotein composition of the acceptors influenced the size of the pools and the t1/2 of the slow pool. Kinetics of cholesterol efflux from membranes isolated from J774 cells also best fit a two-compartment model and modification of the apolipoprotein composition of the acceptor induced a pattern of changes in pool size and half-time similar to that described for whole cells. In the three cell lines studied, we consistently resolved a slow pool with a half-time ranging between 15 and 20 h. In smooth muscle cells only the slow pool was evident, whereas in Fu5AH a very large fast pool was also resolved. In contrast to J774 cells, apolipoprotein composition of the acceptor did not influence the pools in these two cell lines. These results led us to propose a new model regarding the influence of multiple kinetic pools of cholesterol on the regulation of cholesterol desorption from the cell membrane.     

Metabolic heterogeneity of post-lipolysis rat mesenteric lymph small chylomicrons produced in vitro

The study was undertaken to investigate the metabolic rat of post-lipolysis mesenteric lymph small chylomicrons produced in vitro. Small chylomicrons doubly labeled with [3H]cholesterol (more than 70% in cholesteryl esters) and [14C]palmitate-labeled triglycerides were collected from rat mesenteric lymph during periods of fasting. Lipolysis was performed in vitro with lipoprotein lipase purified from bovine milk. More than 98% of the chylomicron-triglycerides could be hydrolyzed to fatty acids. Post-lipolysis chylomicrons were separated by zonal ultracentrifugation, characterized, and tested for biological behavior in intact rats. Following lipolysis the lipoproteins lost nearly all their triglycerides, apoA-I, and apoC, and were relatively enriched with cholesteryl esters, unesterified cholesterol, phospholipids, and apoB. Three preparations were tested for biological behavior: pooled (total) post-lipolysis chylomicrons (diameter approximately 250 A); particles at the ascending part of the zonal effluent (diameter approximately 300 A), and at the descending part (diameter approximately 200 A). After intravenous injection to intact rats, [3H]cholesteryl ester decay was very rapid with pooled lipoproteins and the 300-A preparation (t1/2 = 5-10 min). The 200-A preparation in contrast stayed in circulation much longer (t1/2 = 60-90 min). The study thus demonstrated metabolic heterogeneity of post-lipolysis small chylomicrons and indicated that some may form an LDL-like subpopulation with a plasma lifetime slower than "remnants" but faster than LDL.     

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.     

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.     

Disposition of intracellular cholesterol in human fibroblasts

We have examined the intracellular distribution of unesterified cholesterol in cultured human fibroblasts. Intact cells were treated with cholesterol oxidase to selectively transform cell surface cholesterol to cholestenone. Isopycnic centrifugation of homogenates showed that the cholestenone had a peak buoyant density of 1.13 g/cm3. The approximately 10% of total cholesterol which remained unoxidized was distributed in two peaks of roughly equal size: a sharp peak at approximately 1.09 g/cm3 and a broad peak centered at 1.18 g/cm3. When intact cells were incubated with exogenous [3H]cholesterol, the radiolabel entered the nonoxidizable pool in a temperature-dependent fashion with a half time of 3 h at 37 degrees C. This label initially was associated with the dense but not the buoyant peak of nonoxidized cholesterol. After 40 h, the buoyant peak also became labeled; both peaks then had a specific activity slightly less than the surface cholestenone. The buoyant density of the unoxidized cholesterol did not coincide with markers for the Golgi apparatus, endoplasmic reticulum, or lysosomes. However, two ingested markers of pinocytosis, calcein and horseradish peroxidase, comigrated with the dense peak of unoxidized cholesterol. That the size of the unoxidized cholesterol pool was greater in cells deprived of serum lipoproteins than in fed cells suggested that none of the intracellular cholesterol need be ascribed to ingested sterols. The mass of unoxidizable cholesterol was not diminished when cholesterol biosynthesis was inhibited by lovastatin in lipoprotein-deprived cells. Furthermore, the newly synthesized radiolabeled cholesterol resistant to cholesterol oxidase did not migrate with intracellular cholesterol mass on sucrose density gradients. The newly synthesized cholesterol amounted to about 10% of the total unoxidized sterol. These data indicate that most of the intracellular cholesterol was not newly synthesized. We conclude that a) approximately 90% of fibroblast cholesterol is associated with the cell surface; b) the bulk of intracellular cholesterol, approximately 10% of total, is derived from internalized (endocytic) plasma membrane; and c) the most recently synthesized cholesterol, approximately 1% of the total, is in a discrete organelle.     

Secretion of the newly synthesized cholesterol by rat hepatocytes in primary culture

We used monolayer cultured rat hepatocytes as an experimental model to study the secretion of the newly synthesized cholesterol by the liver. Cellular cholesterol was labeled by exposing cultured hepatocytes to [14C]acetate prior to the study of secretion. Secretion of the newly synthesized cholesterol was measured by extracting cholesterol in the culture medium and assaying for the radioactivity of [14C]cholesterol. We found that: (a) cultured hepatocytes could secrete newly synthesized cholesterol in serum-free medium; (b) secreted [14C]cholesterol was bound to macromolecule(s) and the secretion rate was not affected by cycloheximide for up to 5 h; (c) serum added to the culture medium greatly enhanced hepatic cholesterol secretion; (d) serum high-density lipoproteins were most effective, lipoprotein-deficient serum (d greater than 1.21) less effective in stimulating cholesterol secretion, whereas low-density and very-low-density lipoproteins had little effect; (e) when the serum-free culture medium was fractionated by ultracentrifugation, a major portion of the secreted [14C]cholesterol was found in the high-density lipoprotein fraction; (f) part of the medium [14C]cholesterol also turned up in the high-density lipoprotein fraction when lipoprotein-deficient serum was added as the acceptor; (g) secreted [14C]cholesterol was found only in free form, although some of the cellular [14C]cholesterol was found as esters.     

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg/ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t 1/2) for at least one-third of the cell cholesterol of 3.2 +/- 0.6 and 14.3 +/- 1.5 h, respectively. Plasma membrane vesicles (0.5-5.0 micron diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t 1/2 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 +/- 0.5 and 11.2 +/- 0.7 h, respectively. These t 1/2 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rates indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 +/- 0.1 and 2.9 +/- 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t 1/2 values for cholesterol efflux from these cell lines.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.