C₂-ceramide treatment of intact PMNs: inhibition of PLD activity in a reconstituted system. PMNs were [³H]-labeled, incubated with 10 μM C₂-ceramide, dihydro-C₂-ceramide, or no lipid (control cells) for 30 minutes at 22°C, then disrupted by probe sonication. Cytosol and plasma membrane fractions were separated by ultracentrifugation over sucrose gradients. Samples were incubated with 1% ethanol. Cytosol from control cells only was activated with GTPγS, then combined with equal amounts of membrane protein from control or ceramide-pretreated PMNs (10 minutes, 37°C). Samples were extracted, and TLC was performed as described in “PLD activity in cell-free system.” Data shown are the mean ± SEM of 5 experiments. ^*Significantly different from stimulated control; P < .05.