Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.
It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.
Vos
J P
JP
Laboratory of Veterinary Biochemistry, Utrecht University, Netherlands.
Giudici
M L
ML
van Golde
L M
LM
Preti
A
A
Marchesini
S
S
Lopes-Cardozo
M
M
D000818:Animals; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D005260:Female; D005456:Fluorescent Dyes; D005699:Galactosylceramides; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000494:pharmacology*; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D012235:Rhodamines; D013433:Sulfoglycosphingolipids / Q000378:metabolism*
D005456:Fluorescent Dyes; D005699:Galactosylceramides; D012235:Rhodamines; D013433:Sulfoglycosphingolipids; C075857:N-lissamine rhodaminyl-(12-aminododecanoyl)cerebroside 3-sulfate; D008985:Monensin