Ceramide-induced cell death in cultured rat retinal pigment epithelial cells

We investigated whether retinal pigment epithelial (RPE) responds to ceramide, a known second messenger of apoptosis. RPE cells were isolated by 6-8 day old Long Evans rat eye. We used MTS assay for viability test, and used Hoechst 33552 and propidium iodide for apoptic cell staining. In cultured rat RPE cells, the addition of membrane-permeable ceramide induced apoptosis-like cell death rapidly. RPE cell death was dependent on C2-ceramide concentration. The effective dose (ED,,) of Ca-ceramide was 33.64 mu M. Ceramide-induced RPE cell death was inhibited by zVAD-fmk, a CPP32-like protease inhibitor. Our findings indicated that ceramide in RPE cell death functions upstream of CPP32-like proteases.