Age-dependent changes of rat liver plasma membrane composition

The chemical composition of liver plasma membrane was studied in Wistar rats aged between 3 and 24 months. Results obtained indicate a significant age-dependent positive correlation of both the protein: phospholipid and cholesterol: phospholipid ratios, whereas the protein: cholesterol ratio seems to remain unaffected. Phospholipid analysis of liver plasma membrane reveals that only the phosphatidylcholine content has a significant negative correlation with age; all other phospholipid species remain basically unchanged.     

Abnormal lipid composition of microsomes from cirrhotic rat liver—does it contribute to decreased microsomal function?

We determined to what extent a change in the lipid composition of the smooth endoplasmic reticulum contributes to altered microsomal function in cirrhosis. Rats were rendered cirrhotic either by chronic exposure to phenobarbital/CCl₄ (MCIR) or by bile duct ligation (BCIR). Microsomal function was tested in vivo by the aminopyrine breath test (ABT), then microsomes were prepared and their phospholipid and cholesterol composition analysed. ABT was reduced by 35 and 41% in BCIR and MCIR, respectively. Cholesterol in microsomes was increased in both cirrhotic groups. (BCIR +154%, MCIR +75%) while total phospholipid content was not affected. As shown in other membrane systems, the phospholipid/cholesterol (PL/XOL) ratio showed an excellent inverse correlation with fluorescence anisotropy determined by diphenylhexatriene fluorescence polarization (r=–0.896). The PL/XOL ratio was significantly correlated with aminopyrine N-demethylation in vivo (r=0.649). Alterations in the composition of phospholipid groups (an increase in sphingomyelin in both cirrhotic groups, and a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine in BCIR) also contributed to increased membrane rigidity. We conclude that altered membrane fluidity contributes to diminished microsomal function but that other factors must also be involved since the PL/XOL ratio explained only 42% of the variance in aminopyrine N-demethylation.     

Pressure acclimation of the eel and liver membrane composition

Homeoviscous adaptation of membrane fluidity is known to exist in fish living at great depths. Is this adaptation also present in fish living near the surface but experimentally acclimated to high pressure? The composition of mitochondria-rich fractions extracted from the livers of eels acclimated for 15 days at 101 ATA was determined. The results show that pressure induced a significant increase (+100%) of total phospholipids (PL) and cholesterol without a change in their ratio. The increase of PL content was accompanied by a decrease in phosphatidylcholine in favour of phosphatidyl ethanolamine which, due to its preference for the H₁₁ form, is able to compensate for the loss in fluidity induced by pressure.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

In vitro modulation of rat adipocyte ghost membrane fluidity by cholesterol oxysterols

The effects of cholesterol and cholesterol-derived oxysterols (cholestanone, cholestenone, coprostanone and epicoprostanol) on adipocyte ghost membrane fluidity were studied using a fluorescence depolarization method. The fluorescence anisotropy of the treated membranes was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Cholestanone and cholesterol decreased membranes fluidity at both the concentrations tested (10 & 50 M) while the rest of the sterols did not exert any significant effect on membrane fluidity. In the presence of epinephrine, cholestanone partitioned more towards the lipid core but cholesterol partitioning was not affected. The fusion activation energies (E) obtained for membranes preincubated with cholestanone (8.6 kcal/mol) and cholesterol (8.2 kcal/mol) were not significantly different from that of untreated membranes (8.3 kcal/mol). Membranes preincubated with cholestanone and cholesterol did not exhibit any change in lipid phase throughout the temperature range (10–45°C) tested. The sterols were found to inhibit fisetin-induced phospholipid methylation in isolated rat adipocytes in the rank order of cholesterol > epicoprostanol > cholestanone=cholestenone=coprostanone, while basal methylations was unaffected. When adipocytes were preincubated with the sterols before the addition of fisetin, cholestanone and cholestenone showed 74% and 66% inhibition of maximal methylation respectively. These results indicated that cholesterol oxysterols interact differently with rat adipocyte membranes, with cholestanone interacting more with phospholipids located at the inner lipid bilayer (e.g. phosphatidylethanolamine) while cholesterol interacts more with phosphatidylcholine located at the outer lipid bilayer. This differential interaction may cause selective changes in membrane fluidity at different depths of the bilayer and thus may modulate the activities of membrane-bound proteins such as enzymes and receptors.     

Proteomic analysis of low-abundant integral plasma membrane proteins based on gels

To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na₂CO₃, chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fiftyseven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin. Received 15 March 2006; received after revision 17 May 2006; accepted 10 June 2006 L.-J. Zhang and X.-e Wang are contributed equally to this work.     

Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.     

A unifying model of the cell proliferation emphasizing plasma membrane fluxes

Summary The regulation of cellular growth and proliferation is perhaps the most investigated and elusive problem in cell biology and seems to be possible to solve from almost any angle of study chosen. Among the non-systemic factors that have been discussed are genetic damage, genomic control, regulation by stimulatory and inhibitory peptide factors such as EGF, chalones, and fibronectin, protein kinase activition with tyrosine phosphorylation, adenylylcyclase and cAMP, cGMP, membrane perturbations and specifically in tumours the failure of the Pasteur effect in control of glycolysis, excessive membrane ATPase activity, and excessive hydrolytic and proteolytic activities at the cell surface. This article focusses on the central role of fluxes within the plasma membrane and re-examines the possibility that changes of flux of metabolites, ions, and reducing equivalents may be the common denominator regulating cellular proliferation.     

Age-related changes of the pattern of non-histone proteins in active and condensed fractions of mouse liver chromatin and hepatocarcinoma

Summary Electrophoretic analysis of histones and non-histone acid-soluble proteins in active (nuclease sensitive) and inactive chromatin from liver of young and old CBA mice and in age-related hepatocarcinomas showed a higher ratio of NHP: histones in active chromatin in old cells. Some liver- and hepatoma-specific fractions of non-histone proteins have been identified as chromatin matrix proteins.     

Effect of nerve stimulation on rat skeletal muscle. A study of plasma membrane

Summary The gastrocnemius muscle of the rat showed no morphological, histometric or plasma membrane changes, after sciatic nerve stimulation with a 5 mA current for 30 to 60 min, 10 mA for 30 min and 15 mA for 5 min. However, 10 mA for 60 and 200 min gave rise to mitochondrial and plasma membrane abnormalities. These changes were absent after a rest period. The results indicated the sciatic nerve stimulation at 10 mA for 60 and 200 min caused reversible changes in the rat skeletal muscle mitochondria and plasma membrane.     

Effect of insulin on lateral diffusion of pyrene in rat liver plasma membrane

Summary The yield of excimer formation by pyrene molecules inserted in rat liver plasma membranes is sensibly decreased in the presence of 1 nM insulin. This effect can be interpreted as indicating a decrease of the value of the translational diffusion coefficient of the dye within the membrane.This investigation was partially supported by Consiglio Nazionale delle Ricerche, Rome, Italy.     

The plasma membrane proton-translocating ATPase

Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.     

Effect of insulin on lateral diffusion of pyrene in rat liver plasma membrane.

The yield of excimer formation by pyrene molecules inserted in rat liver plasma membranes in sensibly decreased in the presence of 1 nM insulin. This effect can be interpreted as indicating a decrease of the value of the translational diffusion coefficient of the dye within the membrane.     

Effect of fasting on the lipid composition and enzyme activity of rat liver plasma membranes

After 24-h fasting, when the recovery of plasma membrane protein isolated from rat liver was unchanged, the enrichment in 5'-nucleotidase was decreased by 16%. Modifications of the lipid composition were also observed and resulted in a 27% decrease of the cholesterol/phospholipid molar ratio.     

Effect of fasting on the lipid composition and enzyme activity of rat liver plasma membranes

Summary After 24-h fasting, when the recovery of plasma membrane protein isolated from rat liver was unchanged, the enrichment in 5-nucleotidase was decreased by 16%. Modifications of the lipid composition were also observed and resulted in a 27% decrease of the cholesterol/phospholipid molar ratio.     

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T for a 3-h period at 37°C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.     

Differential changes in Cu,Zn and Mn superoxide dismutase activity in developing rat brain and liver

Summary The aim of our study was to assess the pattern of copper and zinc-containing superoxide dismutase (Cu, ZnSOD) and manganese-containing superoxide dismutase (MnSOD) activity from embryonic life to senescence in rat brain and liver. The two isoenzymes showed different profiles in the two organs examined. In particular, the cerebral MnSOD activity profile suggests a primary role during differentiation of this enzymatic form.     

Gamma-aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Summary Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for -aminobutyric acid (GABA), a high affinity system (Km=10.9 M) and a low affinity system (Km=1203 M). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, -alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMW uptake systems were clearly different from the GABA transport systems present in brain tissue.     

Carbon tetrachloride modulates the rat hepatic microsomal UDP-glucuronyl transferase activity and membrane fluidity

Summary Modulations in rat hepatic microsomal UDP-glucuronyl transferase activity have been observed during carbon tetrachloride (CCl₄) poisoning, with a large decrease in the enzyme cooperativity and increase in the membrane fluidity, occurring 30 min after administration. The results strengthen the possibility that an increase in microsomal membrane fluidity may be an early key event in liver injury induced by CCl₄.Acknowledgments. This work was supported by funds of the University of Athens.