Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse.

Polycystic kidney disease is a disorder marked by aberrant renal tubular epithelial cell proliferation and transport abnormalities. Sphingolipids are ubiquitous membrane components implicated in several cellular functions including cell membrane sorting, signaling, growth, ion transport, and adhesion. To investigate a potential pathogenic role for sphingolipids in cystic kidney disease, we studied the sphingolipid content and associated enzymatic activities of the kidneys from cpk/cpk mice and their phenotypically normal litter mates. The neutral glycolipids, including glucosylceramide and lactosylceramide, displayed a striking increase in 3-week-old cpk/cpk mice as did the acidic lipid, ganglioside GM3. However, a correspondingly significant decrease in sulfoglycolipid and ceramide concentration was observed in the cpk/cpk kidneys. Glucosylceramide synthase activity was higher in the kidneys of the cpk/cpk mice than in those of the controls. Kinetic analysis of the glucosylceramide synthase revealed the presence of an endogenous activator in the cystic kidney. A marked decrease in sulfotransferase activity was observed in both whole kidney homogenates and in microsomal preparations that was consistent with the decrement in sulfolipid content. The increase in GM3, glucosyl- and lactosylceramide may therefore be the result of impaired sulfolipid synthesis at the 3-week time point. While sulfolipid and glucosylceramide concentrations are not different at 1 and 2 weeks of age, ceramide concentrations in cystic kidneys are significantly reduced compared to kidneys from phenotypically normal mice. These results suggest that sphingolipids may play a potential role in the proliferative and transport abnormalities associated with cystic renal disease and the development of azotemia.

A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin-induced diabetes mellitus.

Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.

Decreased formation of inositol trisphosphate in Madin-Darby canine kidney cells under conditions of beta-glucosidase inhibition.

Recent work has demonstrated the enhancement of hormone-stimulated inositol trisphosphate formation in renal epithelial cells under conditions of glucosylceramide depletion. The role of glucosylceramide metabolism was explored further by exposing Madin-Darby canine kidney (MDCK) cells to the beta-glucosidase inhibitor conduritol B epoxide, which produced time-dependent and concentration-dependent increases in glucosylceramide levels and decreased bradykinin-stimulated inositol trisphosphate formation from isolated MDCK cell membranes. These data provide further support for an association between glucosylceramide levels and hormone-stimulated inositol trisphosphate formation.

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.

Failure of L-carnitine to protect mice against hyperammonemia induced by ammonium acetate or urease injection.

Reports indicate that L-carnitine administration before 100% lethal dose of ammonium acetate suppresses the symptoms of ammonia toxicity and prevents death in mice. However, we have been unable to confirm this observation. The cause of discrepancy between our results and the results of others was investigated with two models of hyperammonemia in mice: 1) that induced by intraperitoneal injection of urease and 2) that induced by intraperitoneal injection of ammonium acetate. L-Carnitine administration failed to protect mice against ammonia toxicity induced by intraperitoneal injection of urease. Mortality in mice treated with L-carnitine 30 min before injection of ammonium acetate was similar to that of controls pretreated with saline. Ammonia and urea levels in plasma, liver, and brain were also similar in both groups. However, the values were significantly lower than those in mice denied either pretreatment before the ammonium acetate challenge. These results indicate that pretreatment acts to reduce blood and tissue ammonia simply by diminishing the rate of absorption of the challenge, owing to the dilution of ammonium acetate upon mixing with the contents of the peritoneal cavity. Thus, any protocol that does not compare results of a putative protective agent with those obtained with an equal volume of solvents or saline runs the risk of ascribing protective property to the agent when the protection may, in fact, have been afforded by the solvent.

Characterization of mucous cell synthetic functions in a new primary canine gastric mucous cell culture system.

Mucin is a critical component of the protective layer secreted by gastrointestinal mucous cells. A detailed understanding of the molecular processing of gastric mucin and the physiology of its secretion has been limited by the lack of an adequate model for their study. We have developed a primary culture system of canine gastric mucous cells that has permitted us to study their synthetic and secretory functions. It was found that [3H]glucosamine used for metabolic labeling studies was incorporated into both mucin and lipid components of gastric mucus. To measure mucin with this model, a new immunoassay was developed to quantitate canine gastric mucin. Mucin was purified from the canine stomach, a polyclonal antibody was generated, and an enzyme-linked immunosorbent assay for gastric mucin was established. Mast cells were frequent contaminants of the gastric mucous cell preparation, and two methods were developed to limit their contamination. A new culture system has been developed for the study of gastric mucous cells. These cells synthesize and secrete both mucin and phospholipids. This system will permit us to study the molecular processing of mucin and the physiology of its production and release.

The carbohydrate composition of mucin in colonic cancer.

Mucins synthesized in colonic cancer are known to be different from those in the normal colon; however, the biochemical differences between these mucins have not been defined. We have purified mucins from samples of nonneoplastic (normal) human colon and colon cancer and found that the carbohydrate content of the cancer-associated mucins is 48% of that in the normal colon, including significant reductions in galactose, N-acetylglucosamine, N-acetylgalactosamine, and fucose. By subjecting the mucins to alkaline degradation, we determined that there are 19% fewer oligosaccharide chains per milligram of cancer-associated colonic mucin than there are in mucins from normal colons. We also found a reduction in mean oligosaccharide chain length in cancer-associated mucin (5.83 carbohydrate residues per chain) compared with those derived from normal colons (10.2 residues). Total and individual amino acid contents were greater in cancer-associated mucins, with the exception of three amino acids (threonine, serine, and proline), two of which represent the O-linked glycosylation sites for glycoproteins. Thus, mucins are aberrantly glycosylated in colon cancer, both in terms of the number and mean chain length of the oligosaccharide moiety. Because of their relative abundance in colonic tissue, mucins appear to be useful molecular species in the study of the derangements in protein glycosylation that occur during neoplasia.

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer.

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or 'species'. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P less than 0.0005), and there were significant increases in glycoconjugates eluted in fractions IV (P less than 0.008), III (P less than 0.0005), and II (P less than 0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or 'species', occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be of lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.

Formation of free fatty acid and ceramide during brain handling: lability of sphingomyelin.

Intact brain and brain homogenates readily form free fatty acids and ceramides, even in the cold during subcellular isolation procedures. The fatty acid formation is slightly stimulated by chelators and might be due to phospholipid hydrolysis by lysosomal phospholipases. The ceramide formation is accompanied by loss of sphingomyelin and is apparently due to the action of neutral, metal ion-activated sphingomyelinase. The latter reaction is inhibited by EDTA whereas both degradative processes are inhibited by mercuriphenylsulfonate, the thiol-reacting inhibitor. Cerebroside does not seem to be a source of accumulated ceramide.

Eicosapentaenoic acid: effect on brain prostaglandins, cerebral blood flow and edema in ischemic gerbils.

Eicosapentaenoic acid prevents platelet aggregation and inhibits arachidonate conversion into thromboxane A2 and prostaglandins. Consequently eicosapentaenoic acid might protect the brain from the ischemia that follows cerebral arterial occlusion. We studied the effect of eicosapentaenoic acid on cerebral ischemia in anesthetized gerbils. Ischemia was produced by bilateral carotid occlusion for 10 min, followed by reperfusion for 60 min, in gerbils fed either a standard diet (control) or a diet supplemented with menhaden fish oil for 2 months. The menhaden fish oil contained 17 mole % eicosapentaenoic acid. Regional cerebral blood flow was measured by the hydrogen clearance method and brain water by the specific gravity technique. In control animals cerebral blood flow was decreased 30 and 60 min after reperfusion (p less than .001) and brain water was increased (p less than .001). In the experimental group cerebral blood flow did not fall during reperfusion and edema did not appear. Brain prostaglandins and thromboxane were measured by radioimmunoassay. PGF2 alpha, PGE2, 6-keto PGF1 alpha and TXB2 increased after severe ischemia and reperfusion. The synthesis of brain diene prostaglandins was not altered by eicosapentaenoic acid. Our study indicates that eicosapentaenoic acid prevented post-ischemic cerebral edema and hypoperfusion, without affecting the levels of brain diene prostaglandin and thromboxane.

Free fatty acids in an animal model of Reye's syndrome.

Recent studies have indicated that viral infections, aspirin treatment and hyperammonemia are associated with Reye's syndrome. It has also been reported that free fatty acids in serum and total lipids in the liver of Reye's syndrome patients are elevated during illness. The role of the lipid changes in the development of the disorder cannot be optimally studied in human patients, because infection and aspirin ingestion occur prior to the earliest symptoms of Reye's syndrome. Effects of influenza B infection, aspirin treatment and hyperammonemia on the level of free fatty acids, total lipids and triacylglycerols in serum and liver of an animal model of Reye's syndrome are reported here. Hyperammonemia was produced in young, male ferrets either by feeding them small amounts of an arginine-deficient diet after overnight fasting or by an intraperitoneal injection of jackbean urease. The ferret model resembled Reye's syndrome in developing increased levels of individual and total serum free fatty acids, liver triacylglycerol and total lipids. The results also indicate that influenza infection or aspirin treatment, or both, while increasing the severity of encephalopathy in the deficient ferrets, did not cause a significant change in the level of serum free fatty acids. Other results suggest that elevation of serum ammonia, serum free fatty acid or liver lipids, either singly or in various combinations, does not provide conditions that can explain the rapidly developing encephalopathy in the arginine-deficient ferrets.

Determination of glucocerebroside, sphingomyelin, free fatty acid and total lipid by thin-layer chromatography and charring-scintillation quenching.

A previously described method has been extended to various specific lipids of liver and brain. The basic method involves thin-layer chromatography followed by charring to reveal the bands. The intensity of each band is determined by suspending the silica gel in a radioactive scintillation gel and measuring the optically quenched activities. The lipids are extracted with hexane/isopropanol and, in the case of total lipid determinations, the extract is simply applied to a silica gel plate and charred without use of a development step. For brain cerebroside, the extract is applied to the plate and developed in the usual way. For liver cerebroside, the dried lipid extract is fractionated with a silica gel column to purify the glycolipid, which is then purified further by development with a plate. For sphingomyelin the ester type lipids in the extract are cleaved by alkali for 1 min and the resultant lipids are applied directly to the thin-layer plate. Free fatty acids are chromatographed and measured after a preliminary solvent partitioning to remove most lipids. The method is useful for samples of 5-40 micrograms. Methods for quantitative application of samples to plates are described. A modification of the Camag sample streaker is described which yields precise 1-cm streaks.