Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite.

DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) contribute to peroxynitrite-induced cellular injury. We investigated the role of PARS activation in the pathogenesis of endothelial dysfunction. In human umbilical vein endothelial cells (HUVEC), DNA strand breakage (alkaline unwinding assay), PARS activation (incorporation or radiolabeled NAD+ into proteins), mitochondrial respiration [conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan] and apoptotic index (cytoplasmatic release of histones) were measured. Endotoxin shock was induced in rats by bacterial lipopolysaccharide. Vascular reactivity of thoracic aortic rings were measured in organ chambers. In HUVEC, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, induced DNA strand breakage and caused an activation of PARS. Pharmacological inhibition of PARS reduced the acute and delayed suppression of mitochondrial respiration when cells were exposed to intermediate, but not high doses of peroxynitrite. Similarly, protection against the intermediate, but not high doses of peroxynitrite was seen in fibroblasts from the PARS-/- mice, when compared to wild-type controls. These data suggest that PARS plays a role in peroxynitrite-induced cytotoxicity, but at very high levels of oxidant exposure, PARS-independent cytotoxic mechanisms become predominant. Peroxynitrite-induced apoptosis was not affected by PARS inhibition. Vascular rings exposed to peroxynitrite and rings taken from rats subjected to endotoxic shock exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. The development of this endothelial dysfunction was ameliorated by the PARS inhibitor 3-aminobenzamide. Activation of PARS by peroxynitrite, therefore, may be involved in the development of endothelial dysfunction in endotoxemia.