Ceramide interaction with the respiratory chain of heart mitochondria

A study is presented on the interaction of ceramide with the respiratory ch ain of rat heart mitochondria, and a comparison is made between the effects elicited by short- and long-chain ceramides, N-Acecylsphingosine (C-2-cera mide) and N-palmitoylsphingosine (C-16-ceramide) inhibited to the same exte nt the pyruvate+malate-dependent oxygen consumption. Succinate-supported re spiration was also inhibited by ceramides, but this activity was substantia lly restored upon the addition of cytochrome c, which, on the contrary, was ineffective toward the ceramide-inhibited NADH-linked substrate oxidation. Direct measurements showed that short- and long-chain ceramides caused a l arge release of cytochrome c from mitochondria. The ceramide-dependent inhi bition of pyruvate+malate and succinate oxidation caused reactive oxygen sp ecies to be produced at the level of either complex I or complex III. The a ctivity of the cytochrome c oxidase, measured as ascorbate/TMPD oxidase act ivity, was significantly stimulated and inhibited by C-2- and C-16-ceramide , respectively. Similar effects were observed on the activity of the indivi dual respiratory complexes isolated from bovine heart. Short- and long-chai n ceramides had definitely different effects on the mitochondrial membrane potential. C-2-ceramide caused an almost complete collapse of the respirati on-dependent membrane potential, whereas C-16-ceramide had a negligible eff ect. Similar results were obtained when the potential was generated in lipo some-reconstituted complex III respiring at the steady-state. Furthermore, C-2-ceramide caused a drop of the membrane potential generated by ATP hydro lysis instead of respiration, whereas C-16-ceramide did not. Finally, only short-chain ceramides inhibited markedly the reactive oxygen species genera tion associated with membrane potential-dependent reverse electron flow fro m succinate to complex I. The emerging indication is that the short-chain c eramide electron flow dependent collapse of membrane potential is a consequ ence of their ability to perturb the membrane structure, leading to an unsp ecific increase of its permeability.