Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells.

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.

Measurement of intracellular biomolecular oxidation in liver ischemia-reperfusion injury via immuno-spin trapping.

Hepatic ischemia-reperfusion (I/R) can lead to liver failure in association with remote organ damage, both of which have significant rates of morbidity and mortality. In this study, novel spin trapping and histopathological techniques have been used to investigate in vivo free radical formation in a rat model of warm liver I/R injury. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was administered to rats via intraperitoneal injection at a single dose of 1.5g of pure DMPO/kg body wt 2h before the initiation of liver ischemia. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60min, followed by 60min reperfusion. The effects of DMPO on I/R injury were evaluated by assessing the hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Immunoelectron microscopy was performed to determine the cellular localization of DMPO nitrone adducts. Levels of nitrone adducts were also measured to determine in situ scavenging of protein and DNA radicals. Total histopathological scoring of cellular damage was significantly decreased in hepatic I/R injury after DMPO treatment. DMPO treatment significantly decreased the hepatic conversion of xanthine oxidase and 4-hydroxynonenal formation in I/R injury compared to the untreated I/R group. The distribution of gold-nanoparticle-labeled DMPO nitrone adducts was observed in mitochondria, cytoplasm, and nucleus of hepatocytes. The formation of protein- and DNA-nitrone adducts was increased in DMPO-treated I/R livers compared to DMPO controls, indicating increased in situ protein and DNA radical formation and scavenging by DMPO. These results suggest that DMPO reduces I/R damage via protection against oxidative injury.