FIG. 5.

Ceramide mediates arachidonic acid-induced apoptosis. (A) Time course of arachidonic acid induced-ceramide generation in LLCPKcl4 cells. Quiescent cells were treated with or without arachidonic acid (10 μM) for the indicated times, the cells were then harvested, and ceramide levels were quantitated as described in Materials and Methods. (B) Arachidonic acid increased ceramide generation in Vector cells but not in BM3 cells. Vector cells and BM3 cells were rendered quiescent and treated with or without arachidonic acid (10 μM) for 48 h. Ceramide levels were determined as described in Materials and Methods. (C and D) Arachidonic acid increased ceramide levels and induced apoptosis in BM3 cells pretreated with a P450 inhibitor. Quiescent BM3 cells were pretreated with or without the P450 inhibitor 17-ODYA (20 μM) and treated with or without arachidonic acid (10 μM). The cells were then harvested and ceramide levels were measured (C), and apoptosis was determined (D). (E) 14,15-EET and arachidonic acid inhibited apoptosis induced by administration of ceramide in Vector cells and BM3 cells, respectively. Vector cells and BM3 cells were made quiescent and pretreated with or without 14,15-EET (Vector cells) or arachidonic acid (BM3 cells); they were then exposed to the cell-permeable C2-ceramide (25 μM), and apoptosis was assessed. (D and E) Leftmost lane, 100-bp DNA ladder; rightmost lane, 1-kb DNA ladder.