ࡱ> Y[X7 A:bjbjUU 1Z7|7|\'lnnnf f f 8 , ,x(@@@CEEEEEE$. N!in"i}@@E2}}} 8@n@C}C}}Vn@ kVTf <H0x8"M08"} Markoti et al. Exposure to hypebaric pressure alters ganglioside expression in rat liver following partial hepatectomy Markoti et al. Ganglioside expression after partial hepatectomy Anita Markoti1, Vedrana ikea uli1, Tina Ti inovi Kurir2, Saaa Radi3, Tatijana Zemunik4, Marko Mesari5 1Department of Biochemistry, Split University Medical School, `oltanska 2, 21000 Split, Croatia; 2Department of Pathophisiology, Clinical Hospital Split, Spin ieva 1, 21000 Split, Croatia; 3Undersea and Hyperbaric Medicine, Naval Medicine Institute of the Croatian Navy, `oltanska 1, 21000 Split, Croatia, 4Department of Biology, Split University Medical School, `oltanska 2, 21000 Split, Croatia; 5Department of Chemistry and Biochemistry, Zagreb University Medical School, `alata 3, 10000 Zagreb, Croatia *Corresponding author Telephone: 385.21.557938 Telefax: 385.21.557625 E-mail:  HYPERLINK "mailto:markotic@bsb.mefst.hr" markotic@bsb.mefst.hr Markoti et al. Hepatocytes, which are normally quiescent in the adult animal, are readily induced to proliferate by partial hepatectomy (PH). All of extracellular signals that mediate regenerative process act initially at the plasma membrane, where binding of receptors to endocrine and paracrine agents and to elements of the extracellular matrix, transport of nutritiens, and interactions with neighboring cells occur (1). Glycosphingolipids (GSLs) are ubiquitous, highly conserved membrane components that take place in the cell surface recognition and modulation of function of a variety of membrane associated proteins (2). They are assembled as "rafts" or "glycosynapses" in the outer leaflet of the plasma membrane. Since liver regeneration appears to involve a large number of hormone-receptor systems and a number of cellular changes (i.e., oncogene expression, TGF-( expression), modulation of the ganglioside content of hepatocyte plasma membrane could provide a means for upregulating cellular responsiveness to a variety of factors. Remnant liver after PH demands an increased amount of oxygen for mitochondrial oxidative phosphorilation to restore hepatic energy (3). Therefore we assayed liver ganglioside expression after PH in the rats which were kept under different hyperbaric hausing conditions. Different immunostaining assays of high perfomance thin layer chromatograms (HPTLC), which had detected GM2, GM1b, GalNAc-GM1b, GM1a GD1a, GD1b, GT1b and neolacto-series gangliosides, indicated that ganglioside metabolism may play an important role in the process of liver regeneration. Male Wistar, 1 month aged rats were 15% partially hepatectomized. Rats which underwent partial hepatectomy were divided in three groups, each containing 10 animals. The preHB group was treated 54 h before partial hepatectomy with hyperbaric pressure (HB), three times during the experiment (45 min/day at 202650 pascals). Control group was Markoti et al. not treated before PH and recovered after partial hepatectomy at normal ambient conditions. Group postHB was treated after partial hepatectomy with HB, three times (45 min/day at 202650 pascals). The HB-exposure took place in a Comex hyperbaric chamber (Comex, Marseilles, France). All animals were sacrificed 54 h after operation with prolonged anesthesia (diethylether). Liver was dissected from 10 animals of each group, homogenized, and isolated according to standard procedures (4). Gangliosides were separated on silica gel 60 precoated high-performance thin-layer chromatography plates using chloroform/methanol/water (120/85/20, each by vol.) with 2 mM CaCl2. The immuno-overlay technique was carried as described previously (4). Neuraminidase treatment of neolacto-series gangliosides with (2-3)-substituted sialic acid was necessary prior to immunostaining with anti-nLcOse4Cer antibody, whereas (2-6)-sialylated neolacto-type gangliosides can be detected without enzyme treatment, because sialylation at the position 6 of the terminal galactose does not hinder recognition. Detection of GM1a-type gangliosides was performed with anti-GM1a antibody. To reveal the presence of GD1a, GD1b, and GT1b, the plates were incubated with 5 mU/ml V. cholerae neuraminidase (2 h, 37(C) prior to anti-GM1a immunostaining (4). Among all groups examined, weak band of GM2 ganglioside was visible only in group preHB (Figure 1, panel A, lane 3) and absent in three other groups. GalNAc-GM1b bands were present in hyperbaric treated rats with higher expression in the liver of preHB rats (Figure 1, panel B, lanes 3 and 4). Livers of preHB treated rats showed the greatest expression of GM1a, GD1a, GD1b and GT1b compared to other groups examined (Figure 1, panel C, lane 3). The most prominent difference in the expression of neolacto-series gangliosides (nLc) between the four groups of rats was abundant expression of nLc4 and nLc6 in preHB group Markoti et al. (Figure 1, panel D, lane 3). In addition, very slow chromatographed fraction of nLc8 was present in preHB group while lacking in other groups (Figure 1, panel D, lane 3). Liver of preHBO group showed the highest expression of ganglioside GD1a, a marker of a peculiar transition phase of liver regeneration. Riboni et al. (5) found remarkably increase of total liver gangliosides till the 4th day after surgery, thereafter progressively decreasing to reach the values of controls at the 12th day. The qualitative pattern was characterised by the 95% relative increase of GD1a at the 4th day (5). Due to the highest expression of GD1a ganglioside in preHB group we could assume that pre HB treated rats have reached transition phase earlier then other groups examined. Same animals showed enhanced expression of the several gangliosides: GM2, GM1a, GD1a, GD1b, GT1b, GalNAcGM1b, nLc4, nLc6 and nLc8. Since galactosyl- or N-acetylgalactosaminyltransferase activities are responsible for the final step of synthesys of GM2, GM1a, GD1b, GalNAcGM1b, nLc4, nLc6 and nLc8 our results point at possible role of galactosyl- or N-acetylgalactosaminyltransferase in regulation of the liver growth after partial hepatectomy. The growth inhibitory activity of isolated plasma membranes and specific glycoproteins is lost when terminal galactosyl residues are removed by (-galactosidase digestion, suggesting that surface (1,4-galactosyltransferase and or its galactosylated substrates function during the control of cell growth (6). Transfected cells expressing mostly GM2 and GD1a as the result of stable transfection of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase showed high EGF receptor phosphorylation after stimulation with EGF (7) and the activity of EGF receptor could be inhibited by GalNAc-glycosylation (8). It is tempting to speculate that Gal and GalNAc Markoti et al. residues are built in gangliosides instead in important glycoproteins which could show supressing effect on liver growth in their Gal- and/or GalNAc-glycosylated forms. Proposed mechanism of liver growth regulation by switching from glycosylation of proteins to glycosylation of GSLs is only hypothetical and awaits further experimental proofs. Data shown resulted in the framework of the joined German-Croatian projects  Optimizing of factors which influence liver regeneration , no. TP-01/0216-04, A. Markoti, program TEST  Technological researching-developmental project; and  Expression of glycosphingolipids in regenerated rat liver , no. 0216013, A. Markoti, sponsored by the Croatian Ministry of Science and Technology. We express our warmest thanks to Prof. Dr. J. M(thing (Institute for Medical Physics and Biophysics, University of Mnster, Germany) for his kind gift of anti-glycosphigolipid antibodies used in this study. References 1. FISHMAN J B, CAHILL M, MORIN P, MCCRORY M, BUCHER N L, ULLMAN M D 1991 Specific gangliosides increase rapidly in rat liver following partial hepatectomy. Biochem Biophys Res Commun 174: 638-646 2. HAKOMORI S 2002 Inaugural Article: The glycosynapse. Proc Natl Acad Sci USA 99: 225-232 Markoti et al. 3. SHIMIZU Y, MIYAZAKI M, SHIMIZU H, ITO H, NAKAGAWA K, AMBIRU S, YOSHIDOME H, NAKAJIMA N 2000 Beneficial effects of arterialization of the portal vein on extended hepatectomy. J Surg 87: 784-789 4. MTHING J 2002 TLC and HPLC of Glycosphingolipids. In: El Rassi Z (ed) Carbohydrate Analysis by Modern Chromatography and Electrophoresis. Journal of Chromatography Library, vol 160, pp 423-482 5. RIBONI L, GHIDONI R, BENEVENTO A, TETTAMANTI G 1990 Content, pattern and metabolic processing of rat-liver gangliosides during liver regeneration. Eur J Biochem 194: 377-382 6. HINTON D A, EVANS S C, SHUR B D 1995 Altering the expression of cell surface beta 1,4-galactosyltransferase modulates cell growth. Exp Cell Res 219: 640-649 7. ZURITA A R, MACCIONI H J, DANIOTTI J L 2001 Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides. Biochem J 355: 465-472 8. WANG X Q, SUN P, O'GORMAN M, TAI T, PALLER A S 2001 Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3-mediated suppression of activation. Glycobiology 11: 515-522 Markoti et al. Legend to figure Figure 1. Immunostain with antibodies against GM2 (panel A), GalNAcGM1b (panel B), GM1 after V. cholerae neuraminidase treatment (panel C) and nLc after V. cholerae neuraminidase treatment. Ganglioside fractions from the liver of rats were applied in order: non-operated (line 1), operated and recovered under normal ambient conditions (line 2), treated with hyperbaric pressure before operation (line 3), and treated with hyperbaric pressure after operation (line 4). Ganglioside amounts corresponding to 30 mg liver wet weight were chromatographed together with 2 (g of GM2 ganglioside (lane S, panel A), 1 (g YAC-1 gangliosides (lane S, panel B), 2 (g of gangliosides from rat brain (lane S, panel C), and 5 (g of human granulocyte gangliosides (lane S, panel D). 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