Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture

Abstract

We studied tbc metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with cithcr N-lissamina rhodaminyl-cerumidc (LRh-Cer) or N-(7-nitrobcnz-2-oxo-I,3-diazol-4-yl)-ccramide (NBD-Cer) to the cells cultured in a chemically-defined mcdium. With both probes the major fluorescent product turned out to be sphingamyelin (SM). Most of LRh-SM WPS not cell-associated but recovered from lhe culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefcldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA). galxtosyl-and sulfogalactosyl-ceramidcs (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylccramide (GlcCcr) and GalCcr but not to SGalCcr or NBD-FA. These data demonstrate that chemical mudiIicaliull> ofcclntilide alter its metabolism in oligcdcndrocytcs and that the mctsbo!itef of LRh-Cer reflect thc&~caligidcomposition of myclin more closely than those of NBD-Cer.

Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent‐ and radio‐labelled ceramide analogues

“…Thereafter, products are formed at a higher rate and after 24 h of labelling, the cellassociated fluorescence is approx. 8 nmol/106 cells, of which maximally 1 nmol cells can be attributed to C~2-LRh-metabolites [15]. Therefore, we feel confident that the intracellular CI2-LRhCer concentration is not limiting C~2-LRh-SM synthesis.…”

“…This could result from: (i) a lack of substrate(s); or (ii) limited access of the residual fluorescent ceramide to SM synthase. Option (i) is unlikely, because we have demonstrated previously that only a small fraction of the cellassociated C~2-LRh-Cer is metabolized [15] and the intracellular level of CI2-LRh-Cer decreased but slightly (<20%) during the chase period (results not shown). Moreover, inspection by confocal scanning laser microscopy showed that the perinuclear region where the Golgi apparatus is situated was labelled intensely already after 3 h of incubation with C~2-LRh-Cer SUV (cf.…”

“…Lipid extraction from the cells and medium, separation by HPTLC, lipid extraction from the silica and fluorometry of the fluorescent lipids were performed as described [15]. Incubations were carried out in triplicate.…”

“…Then 1.6 ml of chloroform/methanol (1:2, v/v) was added to the mixture and lipids were extracted by adding 0.5 ml chloroform and 0.5 ml 0.88% (w/v) KCI/10 mM acetic acid. The NBD-labelled sphingolipids were separated by HPTLC and quantified [15]. Fig.…”

“…The suggestion of a high capacity for SM synthesis at the plasma membrane of myelin producing cells [8,9] and the potential importance of SM in signalling events during myelination, prompted us to investigate the assembly of SM at the plasma membrane of rat spinal cord oligodendrocytes in culture. C~2-Lrh-Cer or C6-NBD-Cer were incorporated into small unilamellar egg-PC vesicles (SUV) [15]. Alternatively, labelled ceramides were complexed with BSA immediately before use [16].…”