Ceramide induces release of pro-apoptotic proteins from mitochondria by either a Ca2+-dependent or a Ca2+-independent mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C-2- or C-16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+-independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C-2-ceramide causes cytochrome c release through a Ca2+-dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C-2-ceramide.