Progressive hypoxia inhibits the de novo synthesis of galactosylceramide in cultured oligodendrocytes.
Neonatal rat oligodendrocyte (OLG) cultures exposed to 6 h of gradual, progressive hypoxia in a GasPak (BBL, Becton Dickinson) apparatus were not injured or metabolically impaired, but instead showed a specific inhibition of de novo synthesis (measured by [3H]palmitic acid labeling) of the major myelin component galactosylceramide (GalCer). De novo synthesis of the 2-hydroxy fatty acid GalCer (HFA-GalCer) species, which requires O2 for its synthesis, was most severely inhibited (by 65%), while non-hydroxy GalCer species (NFA-GalCer) were less affected. The synthesis of membrane glycerophospholipids and sphingomyelin was unaffected by hypoxia. Treatment of OLG with 12 nM oligomycin, an inhibitor of mitochondrial ATP synthesis, resulted in an inhibition (by 50-60%) of synthesis of all GalCer species. [3H]Palmitate labeling of NFA-ceramide, the ungalactosylated precursor of NFA-GalCer species, increased in both hypoxia and oligomycin treatments, suggesting that the conversion of newly synthesized ceramide to GalCer was blocked. Newly synthesized HFA-ceramide did not accumulate in OLG, but the small labeled HFA-ceramide pool present during hypoxia was not converted into HFA-GalCer. Pulse-chase studies indicated that NFA- and HFA-ceramides labeled during these treatments were available for galactosylation and could be converted into GalCer upon reoxygenation. [3H]Galactose labeling of NFA-GalCer species was enhanced 2-fold in hypoxia, in contrast to the inhibition seen with [3H]palmitic acid labeling. Thus, while de novo GalCer synthesis was blocked in hypoxia, galactosylation of pre-existing ceramide pools was actually enhanced. Our evidence suggests that hypoxia results in a reversible inhibition of transport of newly synthesized ceramide from its site of synthesis to its site of galactosylation, but causes an increase in galactosylation of subcellular pools of pre-existing ceramide.
Kendler
A
A
Joseph P. Kennedy, Jr. Mental Retardation Research Center, Department of Pediatrics, University of Chicago, Illinois 60637.
Dawson
G
G
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D002554:Cerebrosides / Q000096:biosynthesis*; D005699:Galactosylceramides / Q000096:biosynthesis*; D005700:Galactosyltransferases / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D009186:Myelin Sheath / Q000378:metabolism*; D009836:Oligodendroglia / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D051381:Rats; D013997:Time Factors
D002554:Cerebrosides; D005699:Galactosylceramides; D009840:Oligomycins; D005700:Galactosyltransferases