title: Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor.
abstract: Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-alpha (PKC-alpha) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3-4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-alpha binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.
authors.affiliation: Department of Biochemistry, Signal Transduction Research Group, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada.
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
authors.forename: Tom C
authors.initials: TC
authors.lastname: Hobman
authors.forename: Jay
authors.initials: J
authors.lastname: Dewald
authors.forename: Michael
authors.initials: M
authors.lastname: Garbutt
authors.forename: David N
authors.initials: DN
authors.lastname: Brindley
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D015151:Immunoblotting; D010739:Phospholipase D / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D012412:Rubella virus / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
chemical_list: C064769:N-acetylsphingosine; D011493:Protein Kinase C; D010739:Phospholipase D; D020727:ADP-Ribosylation Factors; D013110:Sphingosine