Inhibition of phospholipase D activity by ceramide. PLD activity in serum-starved, [3H]-glycerol-labeled astrocytes was determined by the transphosphatidylation assay; in the presence of ethanol, PLD converts [3H]-phosphatidylcholine (PC) into [3H]-phosphatidylethanol (PEth) which reflects PLD activity. In (A), PLD activity was stimulated by addition of medium containing 10% fetal calf serum (FCS) for 5 min. In (B), PLD activity was stimulated by 4β-phorbol-12β,13α-dibutyrate (PDB; 1 μM) for 30 min. C2-ceramide ("C2") was added to the cultures in concentrations of 10 and 50 μM 45 min before the addition of FCS and PDB, respectively. Data are means ± S.E.M. of 7–8 experiments and are expressed as [%] PEth/PC. Statistics: ANOVA, (A) F4,36 = 14.0, p < 0.0001; (B) F4,37 = 75.2, p < 0.0001. **, p < 0.01 vs. controls. ##, p < 0.01 vs. stimulated PLD activity (Tukey-Kramer multiple comparisons test).