In endothelium, reoxygenation after
hypoxia (H/R) has been shown to induce production of
reactive oxygen species (ROS) by
complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of
ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of
hypoxia (PO2, approximately 20 mmHg) without
glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with
glucose. ROS production was measured by the fluorescent marker
2',7'-dichlorodihydrofluorescein diacetate, and cell death by
propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by
desipramine, an inhibitor of
sphingomyelinase, an
enzyme responsible for
ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of
ceramide (
N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by
complex III inhibitor:
antimycin A (24 +/- 3%, P < 0.0001), or
stigmatellin (31 +/- 2%, P < 0.0001); 3)
hypoxia/reoxygenation-induced ROS production was not affected by either
ceramide-activated
protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor
cyclosporin A but was significantly inhibited by the antiapoptotic
protein Bcl-2 (82 +/- 8%, P < 0.05); 4)
ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to
hypoxia and
glucose depletion followed by reoxygenation with
glucose, the pathway implicated in mitochondrial
complex III ROS production is
ceramide dependent and is decreased by the antiapoptotic
protein Bcl-2.