Yukio Niimura

Adaptive changes in sulfoglycolipids of kidney cell lines by culture in anisosmotic media

(1) The effects of osmolarity environments on renal glycolipid composition were examined using established renal cell lines. The profile of glycosphingolipids of Madin-Darby canine kidney cells (MDCK) in culture with anisosmotic media showed that a hyposomotic medium reduced the concentration of GalCer I3-sulfate and LacCer II3-sulfate. (2) The concentrations of sulfoglycolipids were increased by maintaining the culture in a hyperosmotic media prepared by the addition of various sodium salts to the control isosmotic medium, while the contents of most of the neutral glycolipids were reduced. The hyperosomotic medium supplemented with nonelectrolytes, mannitol, sucrose or urea, also increased the concentration of sulfoglycolipids. (3) Both sulfoglycolipids were increased linearly with gradual increases of sodium chloride in the medium. Hyperosmolarity produced by the addition of a nonelectrolyte, mannitol, also increased the levels of sulfoglycolipids. In both series of media, the most prominent accumulation was observed in LacCer II3-sulfate. (4) The incorporation of radioactive sulfate into sulfoglycolipids was elevated in cells adapted to high NaCl or mannitol. The increase of the label was observed not only in MDCK but also in three other established cell lines of renal tubular origin, JTC-12, LLC-PK1 and MDBK. (5) It was established, using the culture system of homogeneous cell lines, that the mechanism of increasing the amount of sulfoglycolipids is independent of the integral regulatory mechanism of animals and resides in the renal epithelial cell itself. These results suggest that by culture in hyperosmotic media, the elevated level of intracellular cations stimulated the activity of GalCer and LacCer sulfotransferase, inducing the increased expression of sulfoglycolipids.

Accumulation of sulfoglycolipids in hyperosmosis-resistant clones derived from the renal epithelial cell line MDCK (Madin-Darby canine kidney cell)

1. Two clones (osmR-A and osmR-B) resistant to hyperosmotic media of 700 and 800 mosmol/l, respectively, were selected from Madin-Darby canine kidney (MDCK) cells. 2. When cultured in isosmotic medium (300 mosmol/l), the concentration of galactosyl sulfatide and lactosyl sulfatide in these hyperosmosis-resistant clones was 3.4-5.9 times higher than in the wild-type MDCK. The rate of incorporation of [35S]sulfate into sulfolipids of osmR-A and osmR-B was 1.9-6.7 times higher than MDCK. 3. The stimulation of incorporation into sulfolipids by hyperosmotic culture was completely inhibited by cycloheximide. The pulse-chase studies indicated decreased turnover rate of sulfolipids in osmR-A.

Modification of sphingoglycolipids and sulfolipids in kidney cell lines under heat stress: Activation of monohexosylceramide synthesis as a ceramide scavenger

Heat stress on Madin-Darby canine kidney cells increased ceramide content to 187% at 40 degrees C for 24 h, and the de novo synthesis from serine increased to 146%. Glucosylceramide (GlcCer) and galactosylceramide (GalCer) synthesis from ceramide, the first glycosylation step of sphingolipid metabolism in kidney cells, increased to 290% (GalCer) and 143% (GlcCer) after metabolic labeling with (14)C-glucose at 42 degrees C for 20 h. The more complex glycolipid lactosylceramide also increased to 151%, whereas sulfatide and ganglioside GM3 decreased to 21% and 43%, respectively. Sulfatide (SM4s) showed optimal sulfation at 37 degrees C, whereas cholesterol sulfate was optimally sulfated at 40 degrees C. The gene expression of ceramide glucosyltransferase (GluT), ceramide galactosyltransferase, and GalCer sulfotransferase (GST) after 24 h culture at 42 degrees C significantly increased to 714%, 221%, and 174%, respectively. Another kidney cell line, COS7, showed less activation of these transferases by heat stress. Although GST gene expression was higher under heat stress, SM4s synthesis decreased, which may have been due to increased GST sensitivity to a temperature higher than 37 degrees C. When we introduced the HSP70 gene into the expression vector and transfected the plasmid (pCDM-dHSP70) into kidney cells, GlcCer synthesis increased significantly. From these results, we speculated that HSP70 may play a role in GluT gene expression to increase GlcCer and decrease intracellular ceramide level.

Higher expression of renal sulfoglycolipids in marine mammals

Patterns and contents of major acidic glycosphingolipids in the kidney of three marine mammalian species, the Steller sea lion (Pinnipedia), the rough-toothed dolphin and the broad-beaked dolphin (Odontoceti), were examined, and compared with those of terrestrial mesic mammals. The profile of major acidic glycosphingolipids was not significantly different between the terrestrial and marine mammals: predominant gangliosides were GM3 and GD3, and major sulfoglycolipids were SM4s and SM3. On the other hand, the total concentration (nmol/g wet tissue) of sulfoglycolipids was considerably higher in the marine mammals (2.3–3.0 times) than that in the terrestrial mesic mammals with comparable body weights. In contrast, there was no significant difference in the level of renal glycolipids-bound sialic acid between the marine and the terrestrial mammals. These results suggest that higher expression of renal sulfoglycolipids in marine mammals may contribute to the maintenance of osmotic balance of their body fluid against sea water.

Medium osmolarity-dependent biosynthesis of renal cellular sulfoglycolipids is mediated by the MAPK signaling pathway

Verots S3 and Vero317 cells were shown by metabolic labeling with (35)S-sulfate to contain many more sulfoglycosphingolipids than original Vero cells derived from African green monkey kidney. The activity of galactosyl ceramide sulfotransferase (GST) was shown to be 89- and 92-fold higher in Vero317 cells and Verots S3 cells, respectively, than that of the parent cells, whereas the activity of the degradation enzyme, arylsulfatase A, was unchanged among all the three cell strains. GST gene transcript levels in Verots cells were 14.3-fold higher than those in Vero cells. The cell adhesiveness to the culture plate under hypertonic stress was strengthened significantly in both mutant strains. Among the major sulfoglycolipids of the Verots S3 cell line, assigned as SM4s, SM3, SM2a, and SB1a, the incorporation of (35)S-sulfate into SM3, SM2a and SB1a was upregulated with the increasing tonicity of the medium. Sulfoglycolipids in these renal cells seemed to contribute to the membrane barrier against hypertonic media as shown previously in another renal cell line, MDCK (Niimura and Nagai, 2008). Sulfoglycolipid synthesis was suppressed with the p38 (MAPK) inhibitor SB203580 and/or with the MEK-1/2 (MAPKK) inhibitor PD98059, and with the tyrosine kinase inhibitor genistein, which also reduced the sulfoglycolipid synthesis in a dose-dependent manner. Further the administration of the MAPK/MAPKK inhibitors to the culture medium reduced significantly the viability of Verots S3 cells under hypertonic stress. These findings suggest that sulfoglycolipid synthesis in those renal cells may be regulated to adapt to the renal osmotic circumstances by the mediums osmolarity via the MAPK signaling pathway.

Effect of nutritional substrate on sulfolipids metabolic turnover in isolated renal tubules from rat

Effects of a glycolytic (glucose) and a gluconeogenic renal nutritional substrate (glutamine) on metabolic turnover of sulfolipids, determined as [35S]sulfate incorporation, were compared in renal tubules prepared from well-fed rats. The results showed that the effects of glucose and glutamine, at nearly physiological serum concentration, are quite contrary to each other. Glucose increased the turnover rates of relatively long chain ganglio-series sulfoglycolipids (Gg3Cer II3-sulfate and Gg4Cer II3,IV3-bis-sulfate) (1.7 to 2.4-fold), but not of cholesterol 3-sulfate (0.9-fold). In contrast, glutamine accelerated the turnover rates of relatively short chain sulfoglycolipids (glucosyl sulfatide, galactosyl sulfatide and lactosyl sulfatide) (1.3 to 2.7-fold), as well as cholesterol 3-sulfate (2.4-fold). The possible mechanism which causes these marked differences is also discussed.

Lipid composition and molecular species of sialic acids of a mouse cell line (JLS-V9) and its ouabain-resistant mutant☆

Some properties of a mouse cell line (JLS-V9) and its ouabain-resistant mutant clone (JLS-V9OR) were compared. Specific activities of (Na+ + K+)-ATPase (EC 3.6.1.3) of the cell homogenate, particulate fraction and the plasma membrane fraction were 1.12, 1.72 and 8.75 mu mol/h per mg protein, respectively, in the parent cell and 0.97, 1.68 and 9.36 mu mol/h per mg protein in the mutant cell. The half-maximal concentration of ouabain for the inhibition of the (Na+ + K+)-ATPase was 3.4 . 10(-5) M in the parent cell and 4.0 . 10(-4) M in the resistant clone. The contents of phospholipid and cholesterol on a basis of protein in the mutant were 135 and 105% of those in the parent cell. The mutants monohexosylceramide (HexCer), lactosylceramide (LacCer), trihexosylceramide (GbOse3Cer) and sialyllactosylceramide (GM3) were 111, 145, 274 and 114% of those of the parent cell. Sialic acids of GM3, analyzed by GC-MS, were 98% N-acetylneuraminic and 2% N-glycolylneuraminic acids in the parent cell, whilst 69% N-acetylneuraminic and 31% N-glycolylneuraminic acids in the mutant clone. The in vivo syntheses of these glycolipids were confirmed by the incorporation of radioactive galactose. No significant difference in fatty acid composition was observed between the two cell types. Neutral glycolipids contained mainly 24:0, 24:1, 22:0 and 16:0, whilst in GM3 18:0 and 18:1 predominated. These results show that the lipid content per mg protein is elevated in the ouabain-resistant cell compared to the parent cell.

Novel biosynthesis of monogalactosyl-alkylacyl glycerolipid in Mop8 fibroblast cells transfected with a ceramide galactosyltransferase gene

Our recent study showed the activation of ceramide (Cer) glycosylation and Cer-related genes in the mouse fibroblast under heat stress. We investigated whether the Cer level was affected or not when the Cer galactosylation was introduced into a fibroblast cell line Mop8 by transfection of Cer galactosyltransferase (GalT, alternatively called UGT8) gene. When cells were labeled with 14C-galactose, the parental Mop8 cells showed only glucosylceramide (GlcCer) in the monohexosylceramide region on 2D-high-performance thin layer chromatography (HPTLC), whereas the transfectant cells (namely MopGT) expressed a spot corresponding to GalCer in addition to GlcCer. MopGT cells showed a significant decrease in intracellular Cer. These results suggest contribution of Cer glycosylation to the cellular regulation of Cer, which is known as a pro-apoptotic factor. Incidentally, another novel spot (which we referred to as GTX) moving more slowly than GalCer was observed in MopGT by the metabolic labeling. Comparison with the previous report that, in CHO cells transfected with the brain GalT gene, galactosyl-alkylacyl glycerol was stably expressed in addition to GalCer and was easily hydrolyzed with alkaline treatment to produce galactosyl-alkyl glycerol as the lyso form [van der Bijl P et al. (1996) Biochem J 317: 589-597] suggested that our GTX corresponded to galactosyl-alkyl glycerol. Indeed, when GTX prepared by the HPTLC was analyzed by mass spectrometry (MS), m/z 501, 527 and 529 were predominantly observed as [M+Na]+, which were consistent with monogalactosyl-monoalkyl glycerol containing an alkyl chain of 16:0, 18:1 and 18:0, respectively. Galactosyl-alkylacyl glycerol (namely, GTXorg) was also prepared by preparative TLC without alkaline treatment. MS spectrum of GTXorg showed molecular ions corresponding to 16:0 alkyl and 16:0 acyl chains, 18:1 alkyl and 16:0 acyl chains, and 18:0 alkyl and 16:0 acyl chains, respectively. These findings support the view that substrate recognition of the ceramide-binding site of GalT is not very specific, accepting glycerolipids as substrates. Correspondence to: Ken-ichi Nagai, Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University, 2-11-1 Kaga Itabashi-ku, Tokyo, Japan, Tel: 03-3964-1211, E-mail: knagai@med.teikyo-u.ac.jp

Unique disialosyl gangliosides from salmon kidney : Characterization of V3αFuc, IV3βGalNac, II3(αNeuAc)2-Gg4Cer and its analogue with 4-O-acetyl-N-acetylneuraminic acid

Four unidentified acidic glycolipids (X3-X6) were isolated from the kidney of the Pacific salmon on an anion exchange column and by high performance liquid chromatography using a silica bead (Iatrobeads) column. Based on methylation analysis, chemical and enzymatic degradation, proton nuclear magnetic resonance spectroscopy and mass spectrometry, the glycon structure of X5 and X6 was identified as a unique disialosyl fucosyl-N-acetylgalactosaminyl ganglio-N-tetraose:Fucα3GalNAcβ3Galβ3GalNAcβ4[NeuAcα8NeuAcα3] Galβ4Glcβ1Cer. NMR showed that X3 and X4 were analogues of X5 and X6 and contained O-acetyl groups on C4 of the outer N-acetylneuraminic acid, first disialosyl gangliosides containing 4-O-acetyl-N-acetylneuraminic acid. The ceramides of X3 and X5 contained predominantly C24: 1, and X4 and X6 contained saturated fatty acids (C14: 0, C16: 0 and C18: 0), whereas the long chain base was exclusively sphingenine. The concentrations of X3 and X4 were 0.13 and 0.16 nmol/g of kidney respectively and those of X5 and X6, were 0.07 nmol/g each.

Effects of Ouabain and Interferon on the Production of Murine Leukemia and Vesicular Stomatitis Viruses in Ouabain-Sensitive and Resistant Cells

Treatment of cells with ouabain inhibits protein synthesis and cell growth as a result of specific inhibition of Nat.Kt-dependent adenosine triphosphatase (ATPase) (3,10, II). Ouabain also inhibits replication of some enveloped viruses such as Sendai virus (13) and Sindbis virus (12) as it depresses virus-specific RNA and protein synthesis. In a previous paper (16) we reported that treatment of mouse cells (K3b and JLS-V9) with ouabain reduced murine leukemia virus (MuLV) production more rapidly than the host cell-directed protein synthesis. In the present report we describe the isolation of an ouabain-resistant JLS-V9 cell line (JLS-V90R) and the effects of ouabain and interferon on MuLV production in these cells. JLS-V9 cells which are chronically infected with Rauscher MuLV, the conditions for culturing the cells and the estimation of MuLV production by reverse transcriptase determination were described previously (16). The multiplication of JLS-V9 cells was reversibly inhibited in a dose-dependent fashion by ouabain in concentrations ranging from 1 X 10-4 M to 1 X 10-3 M (16). To select an ouabainresistant line, JLS-V9 cells were incubated intermittently with 5 X 10-4 M ouabain for about one month. Then the ouabain dose was increased to 1 X 10-3 M. After 2 months a variant cell clone, JLS-V90R, which grew well in the presence of 1 X 10-3 M ouabain was isolated. As shown in Fig. 1(a), JLS-V90R cells showed almost the same growth rate as the parent JLS-V9 cells regardless of the presence of ouabain, while the growth ofJLS-V9 cells was significantly reduced in the presence of ouabain. Furthermore, the resistance to ouabain was not lost during cultivation for over 2 months in the absence of ouabain. To analyze the mechanism of ouabain resistance, we tested the sensitivity to ouabain of Nat.Kr-dependene ATPase in JLS-V9 and JLS-V90R cells. As shown in Fig. l(b), Nat.Kv-dependent ATPase from cell homogenates of JLSV90R cells showed a significantly decreased sensitivity to ouabain. The same results were obtained when partially purified membrane fractions were used instead of cell homogenates (data not shown). These results suggest that the resistance of cell multiplication to ouabain is mainly due to the decreased sensitivity