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An inhibitor of glucosylceramide (GlcCer) synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), has been reported to deplete cells and mice of their glucosphingolipids. This inhibitor has proved useful for the elucidation of the many functions of this lipid family [reviewed by Radin, N.S.Inokuchi, J. (1991) Trends Glycosci. Glycotechnol. 3, 200-213]. In the present study, we have synthesized homologs of PDMP having different acyl chains (C6-C18) and compared their effectiveness for the inhibition of GlcCer synthase in vitro and their inhibition of GlcCer, protein, and DNA synthesis in cultured MDCK (Madin-Darby canine kidney) cells. Using MDCK homogenates and mouse brain and liver microsomes, we found that the C6 compound was relatively inactive and that the longer chain compounds did not differ much in inhibitory power. However, the use of intact MDCK cells showed that the longer chain homologs were much more effective in inhibiting GlcCer synthesis, cell growth, and incorporation of [3H]thymidine. Tests with two radioactive homologs showed that the inhibitor with a longer acyl chain was taken up much more effectively by MDCK cells and that this difference explains the much greater effectiveness of this homolog in intact cells. The inhibitors were effective when solubilized either with a nonionic detergent or with bovine serum albumin. The extent of decrease in DNA synthesis was not directly proportional to the decrease in cellular glucosylceramide, possibly because only a low level of the glycolipid is needed for DNA synthesis.

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.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.

An enzyme system consisting of washed acetone powder of pig brain, adenosine triphosphate, nicotinamide adenine dinucleotide, magnesium, fumaric acid, and ascorbic acid was found to catalyze the oxidative decarboxylation of 2-ketostearic acid. The products were carbon dioxide and heptadecanoic acid. Washing the enzyme powder with ethylene-diaminetetraacetate and other chelating agents destroyed the activity. Further properties of the enzyme system are described. It is believed that this enzyme system accounts in part for the 1-carbon degradation route for brain fatty acids.

Amides resembling ceramide (fatty acyl sphingosine) were synthesized and tested for their effects on rat brain cerebrosidase (galactosyl ceramide beta-galactosidase). The best inhibitor was N-decanoyl dl-erythro-3-phenyl-2-aminopro-panediol, which exhibited a K(i) of 0.4 mm. A Lineweaver-Burk plot indicated that the amide acted as a noncompetitive inhibitor, presumably by attachment to a site other than the substrate-active site. Preincubation did not affect the degree of inhibition, and inhibition was independent of incubation duration; these observations suggest that the inhibitor does not combine with the enzyme irreversibly. Structural variations produced decreased inhibitory activity: loss of one of the hydroxyl groups, replacement of the aromatic side chain with an aliphatic or substituted phenyl group, or isomeric inversion of the 3-hydroxyl group. It appears that the best activity is obtained with a substance most closely resembling natural ceramide. The cerebrosidases of rat spleen, kidney, and liver are also inhibited by the same amide.

The galactoside bond in cerebroside was found to be cleaved by an enzyme in rat and pig brain. Emulsified stearoyl-(14)C psychosine was used as the substrate and the extent of cleavage was studied by isolating and counting the stearoyl sphingosine (ceramide) formed. The reaction products, ceramide and galactose, were characterized by column and thin-layer chromatography. Cerebroside containing galactose-(3)H was also used to show liberation of galactose. Cholic acid was found to be required for activation of the enzyme, which has a pH optimum of 4.5. Similar cerebrosidase activity was found in spleen, kidney, and lung of rat; liver and heart showed very slight activity. The partially purified enzyme from pig brain also formed ceramide from ceramide lactoside, ceramide glucoside, and cerebronoyl psychosine. The enzyme was active toward o-nitrophenyl galactoside and could be fractionated by Sephadex chromatography into a fraction active toward the nitrophenyl galactoside only and a fraction active toward both this substrate and ceramide galactoside. Human spleen, normal and Gaucher, exhibited cerebrosidase activity.