Vol. 47, No. 6, June 1999
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL pages 1049-1059
C H A N G E S OF N U C L E A R M E M B R A N E FLUIDITY D U R I N G RAT LIVER R E G E N E R A T I O N Maria-Letizia Tomassoni', Elisabetta Albi and Mariapia Viola Magafi Institute of General PathologF, School of Medicine, University t f Perugia, 06100 Peru#a, Italy "To whom correspondence should be addressed. Tel: +39-75-5724937; E-mail:
[email protected] Abbreviations: PL, phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine;PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; N-SMase,neutral-sphingomyelinase; PH, partial hepatectomy; DPH, diphenylhexatriene; TMA-DPH, trimethylammonium-diphenylhexatriene
SUMMARY 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. Key words: nuclear sphingomyelinase.
membrane
fluidity,
liver
regeneration,
phospholipids,
neutral-
INTRODUCTION The nuclear membrane is a double bilayer across from the nuclear pore complexes which permit the nucleocytoplasmic exchanges (for review see l, 2). The outer nuclear membrane is continuous with the endoplasmic reticulum (3) while the inner is closely linked at the lamina (3). Rat liver nuclear membrane composition has been shown to change with cell maturation (4, 5) and to affect the nuclear membrane fluidity (5). Although the presence of the phospholipids (PL) in the subnuclear compartments is widely known (6-8), the nuclear PL are mainly localised at the nuclear membrane level (9-1 t). The nuclear PL have a different composition and turnover from that of the chromatin PL (12, 13). During rat liver regeneration an increase of chromatin PL synthesis has been shown in relation to DNA synthesis (14), whereas rate of synthesis of nuclear membrane PL do not changes throughout the cell cycle (14). These results have lead to the hypothesis that the chromatin PL have a role during DNA synthesis but the role of nuclear membrane PL is yet unknown. There is 1049
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Vol. 47, No. 6, June 1999
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
some evidence that the nuclear membrane can play a role in DNA replication (15-19) b'.:*,other works support an opposite opinion (20-22). However, the composition and the properties of the nuclear membrane during the cell cycle remain such to be learned. The end of this work is, therefore, to study the relationship between the nuclear membrane fluidity and the cell cycle. Since the partial hepatectomy provides a model of relatively synchronous cell cycle progression in the living animal (23, 24), we evaluated the nuclear membrane fluidity during rat liver regeneration. The membrane fluidity has been assayed by use of two fluorescent probes: the trimethylammonium-diphenylhexatriene (TMA-DPH), a polar probe anchored in proximity to the bilayer surface (25) and the diphenylhexatriene (DPH), an apolar probe which enters phospholipid bilayers (26). Therefore, the two molecules probe the lipid environment near the surface bilayer and the hydrophobic core, respectively. At the same time, we also determined, during rat liver regeneration, the biochemical composition of nuclear membrane and the activity of neutral-sphingomyelinase (N-SMase, E,C. 3.1.4.12), an enzyme whom two forms in rat liver chromatin and nuclear membrane have been recently shown in our laboratory to have different optimum of pH and Km (27) and which catalyses the hydrolysis of sphingomyelin (SM) to phosphorylchotine and ceramide, a key molecule of signal transduction (28).
MATERIALS AND METHODS
Thirty-day-old Sprague-Dawley rats (100-150 gr. body wt., S. Morini, Italy) of either sex were used. They were kept at a natural light-dark cycle and had free access to pelleted food and water. The partial hepatectomy (PH), which involved removal of approximately 70 % of the liver was performed under light ether anaesthesia according to the method of Higgins & Anderson (23) between 9.00 and 10.00 a.m. At 12, 18, 24, 30 hrs following surgery the rats were killed by cervical dislocation and the livers were quickly removed. The liver from animals sacrificed to zero time was used as control. Nuclei and Nuclear Membranes Isolation The nuclei and the nuclear membranes were isolated according to the procedure of Widnell & Tara (29) and of Kay & Johnston (30), respectively. The glucose-6-phosphatase activity was assayed as purity marker in the isolated nuclear membranes of control rats (0 hrs) according to Agutter et al. (31). The enzyme activity was 3.87 :k 1.10 ~tmol Pi released/mg prot./30 min according to other AA. (31, 32) confirming, thus, a good nuclear membrane preparation. Lipid Extraction The lipids, extracted by 20 vol. chloroform/methanol, (2:1, vol./vol.), were washed with 0.2 vol. of 0.5% NaC1 (33). Phospholipid Chromatography The phospholipids were separated on thin layer silica gel in a bidimensional system as previously described (5). Finally, the lipids were detected with iodine vapour and scraped into test tubes for phosphorus assay (34). Biochemical Determinations Protein, DNA, RNA and phospholipids (PL) contents were determined according to Lowry et al. (35), Burton (36), Schneider (37) and Bartlett (34), respectively. 1050
Vol. 47, No. 6, June 1999
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Neutral-Sphingomyelinase Activity Assay The neutral-sphingomyelinase (N-SMase) assay was carried out according to a modification (27) of the method previously described by Okazaki et al. (38). In the reaction 1.6 nmol of radioactive SM (choline-methyl [14C], 54.5 Ci/mol, NEN, Boston, Massachusetts, USA) was diluted by adding 78.4 nmol cold SM to a final specific activity 1.08 Ci/mol. The reaction mixture contained 0.1 M Tris-HC1 pH 7.6, 6 mM MgC12, 0 . 1 % Triton X-100, 0.8 mM [14C]-SM and nuclear membrane suspension corresponding to 150 p.g of proteins to a final volume of 0.1 ml. The incubation was performed at 37~ for 10 min. The reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1), 0.2 ml of 0.5 % NaCI was added to the tubes and vortexed. The tubes were centrifuged at 2000 x g for 10 min and the upper phase was removed and diluted in counting vials with 3 ml At0mlight (NEN, Boston, Massachusetts, USA). Fluorescence Anisotropy Determination The fluidity of nuclear membranes was investigated by use of two fluorescent probes: the diphenylhexatriene (DPH) and the trimethylammonium-diphenylhexatriene (TMA-DPH). The probe solutions and the incorporation were carried out as previously described (5). The fluorescence anisotropy (r) was determined in a Shimadzu F-5000 spectrophotofluorimeter at different temperature checked with a thermistor thermometer. Excitation and emission were set at 365 um and 430 urn, respectively. Fluorescence anisotropy (r), inversely related to the membrane fluidity and to the temperature (39), was calculated according to Shinitzky & Barenholz (26): r = (I//- I1) / (I//+ 2 Ix), where Ix and I//are the intensities of the emitted light oriented perpendicular and parallel to the excitation beam respectively. Statistical analysis Data, expressed as means :t: S.D., were compared by Student's t test; p
