CHANGES IN THE ACTIVITY AND MESSENGER-RNA LEVELS OF PHOSPHOLIPASE-D DURING CERAMIDE-INDUCED APOPTOSIS IN RAT C6 GLIAL-CELLS

N-Acetylsphingosine (C-2-ceramide), a membrane-permeable analogue, ind uced apoptosis in C6 glial cells. Phase-contrast micrographs showed th at the round cells appeared 3 h after exposure to 25 mu M C-2-ceramide and the number of floating cells increased time-dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel ele ctrophoresis at 12 h. To understand the mechanism underlying glia[ cel l death induced by C-2-ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTP gamma S-dependent PLD activity was down-regulated after ceramide treatment in a time-dependent manner. In the in vitro PLD assay, membrane-associated PLD activation in response to recombina nt ADP-ribosylation factor I was greatly suppressed. Furthermore, leve ls of rPLD1a and rPLD1b mRNAs were found to be down-regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription-polymerase c hain reaction. Decreases in GTP gamma S-dependent PLD activity were we ll correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken toge ther, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.