Changes of nuclear membrane fluidity during rat liver regeneration.

We have previously shown that the nuclear membrane fluidity is affected by lipid composition changes and that is very high, particularly in the hydrophobic core. The aim of this work is to study the modifications of nuclear membrane fluidity in relation to the cell cycle. Since compensatory hepatic growth is an informative and well characterised model for natural cell proliferation, the nuclear membrane fluidity, detected by two fluorescent probes, was studied at various regenerating times, ranging from 0 to 30 hours after partial hepatectomy. At 18 hours after partial hepatectomy the nuclear membrane fluidity increased and at 30 hours the higher values of hydrophobic core fluidity were observed. The behaviour of fluidity was related to the nuclear membrane neutral-sphingomyelinase activity and, then, to the content of sphingomyelin. Therefore, the significant changes of the nuclear membrane fluidity and of the neutral-sphingomyelinase activity found during rat liver regeneration suggested a their likely role in signal transduction pathways implying cell regeneration.

Changes of nuclear membrane lipid composition affect RNA nucleocytoplasmic transport.

We have previously shown that the nuclear membrane fluidity of rat liver, measured by fluorescence anisotropy of two probes, is higher in the hydrophobic core, with respect to the bilayer surface, in newborn rats compared to adult rats. The aim of the present research is to investigate whether the nuclear membrane fluidity influences RNA nucleocytoplasmic transport. To this end two experimental models were used: the fluidity of nuclear membrane isolated from adult rats was increased by a choline base exchange reaction, which is known to be accompanied by an increase of phosphatidylcholine unsaturated fatty acids, whereas that of nuclear membrane isolated from newborn rats was decreased by incubation with dimyristoylphosphatidylcholine-cholesterol liposomes. The RNA efflux, evaluated by using [3H]uridine, significantly increased in the adult nuclear membrane submitted to choline base exchange reaction, whereas a strong decrease in the newborn nuclear membrane enriched with cholesterol was found. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme which is correlated with mRNA transport, showed parallel variations. Therefore, for the first time, we have provided evidence that the nuclear membrane fluidity plays a regulatory role in RNA nucleocytoplasmic transport, although the mechanism by which this effect takes place remains to be clarified.

Effect of lipid composition on rat liver nuclear membrane fluidity.

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed.

Rat liver chromatin phospholipids.

To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08-0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.

Separation and purification of dolichol and dolichyl phosphate by anion-exchange paper chromatography: application to cultured cells.

Dolichyl phosphate, dolichol C80-105 (dolichol 17:dihydroheptadecaprenol-dolichol 21:dihydrohexeicosaprenol), and dolichol C55 (dolichol 11:dihydroundecaprenol) were separated by anion-exchange paper chromatography. Squalene, sterols, phospholipids, anionic glycolipids, and glycerol did not migrate as dolichyl phosphate, dolichol C80-105, and dolichol C55 under our elution conditions. However, since the Rf of triglycerides was similar to that of dolichol C80-105, saponification, prior to chromatography, removed traces of triglycerides. Silica gel thin-layer chromatography (TLC) allowed the separation of dolichol C80-105 from dolichol C55, whereas dolichyl phosphate was eluted with other lipids. Incubation of spontaneously transformed cells derived from rat astrocytes primary cultures with [2-14C]acetate, saponification of the extracted lipids, and anion-exchange paper chromatography revealed the presence of radioactive dolichyl phosphate and dolichol C80-105 (15 pmol/mg protein). Extraction of labeled dolichyl phosphate followed by acid phosphatase treatment and subsequent analysis on TLC confirmed the identity of dolichyl phosphate since all the radioactivity was associated with dolichol C55. Treatment of the transformed cells with 30 microM 7-ketocholesterol or 7 beta-hydroxycholesterol stimulated markedly (two- to threefold) the incorporation of [2-14C]-acetate in both dolichol C80-105 and dolichyl phosphate. These data demonstrate that anion-exchange paper chromatography is technically suitable for the separation and analysis of dolichol C55, dolichol C80-105, and dolichyl phosphate in cultured cells prelabeled with radioactive precursors.