Glycosphingolipid composition of a renal cell line (MDCK) and its ouabain-resistant mutant

Glycosphingolipids were isolated from a canine kidney cell line (MDCK) and its ouabain-resistant mutant (MDCK-OR) by solvent extraction, mild alkaline methanolysis, a DEAE-Sephadex column, and preparative TLC. The glycolipids were characterized by their mobilities on TLC, an analysis of carbohydrates as trimethylsilyl methyl glycosides and acetates of partially methylated alditols, as well as by treatment with specific glycosidases. In the neutral glycolipid fraction of both cell lines, galactosylceramide (GalCer), glucosylceramide (GlcCer), lactosylceramide (LacCer), digalactosylceramide (Ga2Cer), globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), and the Forssman antigen (IV3GalNAc alpha-Gb4Cer) were identified. The contents of Ga2Cer (4.4 nmol/mg protein), Gb3Cer (0.6), Gb4Cer (2.9), and IV3GalNac alpha-Gb4Cer (19.5) in MDCK-OR were 1.4- to 2.1-fold higher than those in MDCK, while the concentrations of GlcCer (5.3) and LacCer (1.4) in MDCK-OR were about half of those in MDCK. Among acidic glycolipids of MDCK-OR, galactosyl sulfatide (GalCer-I3-sulfate) and lactosyl sulfatide (LacCer-II3-sulfate) were increased to 1.9 (2.7-fold) and 0.2 nmol/mg protein (2.0-fold), respectively, as compared to MDCK. However, N-acetylneuraminosyllactosylceramide (GM3), the predominant ganglioside in both cell lines, was decreased to about one third of the level (1.5 nmol/mg protein) in the parent MDCK (4.7 nmol/mg protein). The fatty acid of the glycolipids in both cell lines consisted mainly of saturated acids of 16, 18, 22, and 24 carbons.     

Isolation and identification of nine sulfated glycosphingolipids containing two unique sulfated gangliosides from the African green monkey kidney cells, Verots S3, and their possible metabolic pathways

Verots S3 cells derived from the African green monkey kidney were revealed to contain nine types of sulfoglycolipids by incorporating [35S]sulfate. These sulfated glycolipids were separated by DEAE-Sephadex column chromatography and preparative thin-layer chromatography (TLC). The major sulfoglycolipids were characterized using TLC, gas-liquid chromatography (GLC), mass spectrometry, solvolysis, TLC immunostaining, and nuclear magnetic resonance spectra as follows: V1, SM4s (GalCer I3-sulfate); V2, SM3 (LacCer II3-sulfate); V3, SM2a (Gg3Cer II3-sulfate); V4, globopentaosyl ceramide sulfate (Gb5Cer V3-sulfate); V5, (Gg4Cer II3-sulfate, IV3-NeuAc); V6, SB1a (Gg4Cer II3, IV3-bis-sulfate); and V8, (Gg4Cer II3-NeuAc, IV3-sulfate). Both V5 and V8 were sulfated gangliosides comprising both N-acetyl neuraminic acid and sulfate, and this was the first report on V8. A minor component V7 was identified as SM1a (Gg4Cer II3-sulfate) based on its behavior in TLC, GLC, and liquid secondary ion mass spectroscopy. It was postulated that this substance was a precursor of V6 (SB1a) and V5 (Gg4Cer II3-sulfate, IV3-NeuAc), and to date, its presence has not been demonstrated in nature. Another minor component V9 was identified as glucosyl ceramide sulfate based on its migration in TLC and GLC. This renal cell line was shown to be an excellent model for studying the metabolism and function of sulfoglycolipids.     

Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses

Incorporation of (35)S-sulfate into the polar molecular species of sulfoglycolipids (SM4s) in Madin-Darby canine kidney cells increased in a hypertonic medium (500 mOsm/L) supplemented with sodium chloride. The unknown sulfoglycolipid (SX) was identified as GlcCer sulfate based on the results of TLC, GLC, and mass spectra. The synthesis of SX increased in the hypotonic medium unlike that of SM4s and SM3. TLC showed that hypertonic stress induced the accumulation of GalCer as a precursor of SM4s, whereas hypotonic stress increased GlcCer as a precursor of GlcCer sulfate. The level of ceramide as a precursor of both GalCer and GlcCer increased under hypertonic stress and decreased under hypotonic stress. Cerebroside sulfotransferase mRNA was shown to be elevated in the hyperosmotic condition but not in the hypotonic condition. The increase in SM4s under hypertonic stress was induced by the activation of both the ceramide galactosyltransferase and the cerebroside sulfotransferase genes, whereas the increase in GlcCer sulfate under hypotonic stress was caused by the accumulation of GlcCer as the result of activation of ceramide glucosyltransferase.     

Kidney lipids in galactosylceramide synthase-deficient mice. Absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I(3)-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt(-/-) and, as a control, Cgt(+/-) and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga(2)Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II(3)-sulfate (SM3) and gangliotetraosylceramide II(3),IV(3)-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt(-/-) mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice.     

Specificity of the glycolipid transfer protein from pig brain

Lipid specificity has been studied in the lipid transfer reaction facilitated by the glycolipid transfer protein from pig brain. The lipid transfer was measured by determining the transfer of a radioisotopically labeled lipid from donor liposomes to either acceptor liposomes or mitochondria. Whenever possible, the liposomes contained 1 mol % of the lipid whose transfer was under study. The transfer protein accelerates the transfer of glucosylceramide, galactosylceramide (GalCer), lactosylceramide (LacCer), galactosylceramide 3-sulfate, globotriaosylceramide, LacCer sulfate, sialosyl-LacCer, globotetraosylceramide, and globopentaosylceramide. An inverse relationship is found between the length of sugar chains in glycosphingolipids and the transfer rates. In addition to the glycosphingolipids, the transfer protein facilitates the transfer of galactosyldiacylglycerol, digalactosyldiacylglycerol, glucosyldiacylglycerol, and diglucosyldiacylglycerol. The protein does not facilitate the transfer of dimannosyldiacylglycerol. The transfer of periodate-oxidized and subsequently reduced derivatives of GalCer and LacCer is facilitated by the transfer protein. The derivatives of GalCer are transferred at lower rates than GalCer, whereas the derivatives of LacCer are transferred at higher rates than LacCer. The transfer protein does not facilitate the transfer of phosphatidylcholine, phosphatidylinositol, cholesterol, or cholesteryloleate. These results suggest that the glycolipid transfer protein from pig brain has specificity to hydroxyl groups present in the sugar residue directly linked to either ceramide or diacylglycerol. The presence of glucose or galactose linked to these hydrophobic moieties makes the glycolipid transferable by the protein.     

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.     

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.     

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.     

Enzymatic sulfation of galactosyl- and lactosylceramides in cell lines derived from renal tubules.

1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines.     

Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.     

Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Molecular Cloning and Characterization of Galactosylceramide Expression Factor-1 (GEF-1)

A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney.

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3':5'-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5-5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1-34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E1 (14 micronM) and isopropylnorepinephrine (10 micronM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H235SO4, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Protein patterns,osmolytes, and aldose reductase of L-929 cells exposed to hyperosmotic media

L-929 cells acclimated to media made hyperosmotic (600 mosmol/kgH₂O) by addition of NaCl, sorbitol, or mannitol show, on SDS-polyacrylamide gels, a markedly enhanced protein band at 40 kDa, most likely corresponding to the enzyme aldose reductase. The effect was not observed in cells acclimated to a medium rendered hyperosmotic by addition of proline. The major organic osmolyte accumulated is sorbitol in cells acclimated to high-sorbitol or high-NaCl medium, proline in cells acclimated to high-proline medium. Cells acclimated to any of these hyperosmotic media display unaltered Na⁺ levels and similarly increased K⁺ levels and decreased Cl⁻ levels. These results are interpreted in terms of the mechanisms involved in aldose reductase induction and in regulation of the enzyme activity in long-term acclimation to hyperosmotic media. © 1996 Wiley-Liss, Inc.     

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule.The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E₁ (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell.In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ was observed.The cell lines JTC-12, MDCK and MDBK, when incubated with H₂³⁵SO₄, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate.The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.