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Previous studies have demonstrated that the ceramide analog D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) inhibits glucosylceramide (GlcCer) synthase and thus leads to extensive depletion of glycosphingolipids (GSLs) biosynthesized from GlcCer [reviewed by Radin, N.S., Shayman, J.A., and Inokuchi, J. (1993) Adv. Lipid Res. 26, 183-213). In the present study, stereospecificity of PDMP activity was demonstrated with an enantiomeric pair, D-threo-PDMP and L-threo-PDMP. Treatment of B16 melanoma cells with the D-threo or L-threo isomer produced contrasting changes of GSL biosynthesis, as monitored by metabolic labeling with [3H]Gal. D-PDMP markedly inhibited incorporation of radioactivity into GlcCer, LacCer, and GM3 as expected, whereas the L-threo isomer significantly increased it. Homologs of L-PDMP having different N-acyl chains were synthesized and also tested for their effects. Among them, the compounds having C8-C14 acyl chains increased incorporation of the radioactivity into GSLs to different degrees, demonstrating that the stimulatory effect of the L-threo homologs depends on acyl chain length. In order to elucidate the biochemical mechanisms of these PDMP effects, the activities of GlcCer synthase, LacCer synthase, and GM3 synthase in B16 cell lysates were measured in the presence of PDMP. D-Threo-PDMP but not the L-threo isomer inhibited both LacCer and GM3 synthases as well as GlcCer synthase, suggesting that the ceramide-like structure of the D-PDMP molecule interacted stereospecifically with these GSL-synthesizing enzymes. On the other hand, L-PDMP had no effect in the in vitro assays.(ABSTRACT TRUNCATED AT 250 WORDS)

We reported previously that stereoisomers of 1 -phenyl-2-decanoylamino-3-morpholino-1 -propanol (PDMP), the D-threo and L-threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with L-or o-threo-PDMP. These isomers exhibited opposite effects on neurite outgrowth: D-PDMP was inhibitory at concentrations ranging from 5 to 20 1iM, whereas L-PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10-15 jiM. Rat neocortical explants were doubly labeled with [ 14C]serine and [3H]galactose at 15 jiM L-or D-PDMP. L-PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas D-PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of L-PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N-acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase(240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of L-PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides. Key Words: Lthreo-1 -Phenyl-2-decanoylamino-3-morpholino-1 -propanol -Neocortical neurons-Neurite outgrowth-Ganglioside biosynthesis-Glycosyltransferase.

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its enantiomer, L-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by D-PDMP and facilitation by L-PDMP. Up-regulation of synaptic activity by L-PDMP could be correlated with the slow but robust activation of p42 mitogen-activated protein kinase. Treatment with L-PDMP after transient forebrain ischemia in rats ameliorated the deficit of a well-learned spatial memory by an 8-arm maze task, suggesting a new potential therapeutic approach for neurodegenerative disorders.

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