Figure 2. Regulation of the recycling pathway for the generation of ceramide by ROS.

(A) Cells were pre-treated with 15 mM GSH (lane 5) or 100 μM H₂O₂ (lane 6) for 2 h, which was followed by treatment with 20 μM C₆-cer for an additional 2 h (lane 2), and their effects on the generation of long-chain ceramide formation was measured by palmitate labelling, compared with controls (lane 1). Lanes 3 and 4 contain samples treated with GSH and H₂O₂ alone. The quantification of ceramide bands are shown beneath the lanes. (B) Cells were treated with GSH and H₂O₂ as in (A) in the presence or absence of C₆-cer, and endogenous ROS levels were measured using DCFH-DA as indicated in the Experimental section. (C) The levels of endogenous ceramides were determined using HPLC/MS after treatment of cells with C₆-cer (2 h) following 15 mM GSH or 100 μM H₂O₂ for 2 h. The results shown are representative of three independent experiments performed in duplicate. (D) Cells were treated with or without 5 μM D-e-Sph in the presence of 1 μCi/ml of [³H]palmitate for 2 h, without (lane 2), or with (lane 3) 15 mM GSH, 100 μM H₂O₂ (lane 4), 2 nM TNF-α (lane 5), 50 μM FB1 (lane 6) or 50 nM MYR (lane 7). Lane 1 contains untreated control. Results are means±S.D. * and #, P<0.05 compared with controls or ceramide alone respectively.