Effects of phosphatidic acid on ceramide formation in astrocytes. Astrocytes were prelabeled with [3H]-serine for 72 hours, washed and treated with PA (200 μM) or ethanol (EtOH, 0.3 % v/v) during transient permeabilization with streptolysin-O (144 ng/ml) in calcium-free medium. After 15 min, the cultures were washed and re-exposed to calcium-containing medium to initiate pore repair. After (A) 1 hour and (B) 18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. During the experiments, PA was only present for 15 min during cell permeabilization whereas ethanol was present throughout the incubation period. Data (N = 9) are means ± S.E.M. and are expressed as [%] ceramide/sphingomyelin. Statistics: Repeated measures ANOVA, (A) F3,35 = 5.52, p = 0.005; (B) F3,35 = 14.5, p < 0.0001. **, p < 0.01 vs. controls ("Ctr"). #, p < 0.05; ##, p < 0.01 vs. effect of ethanol (Tukey-Kramer multiple comparisons test).