Carol Wadham1, Angela Parker, Lijun Wang, Pu Xia. 1. Signal Transduction Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia.
Abstract
OBJECTIVE: Hyperglycemia-induced endothelial dysfunction, via a defect of nitric oxide (NO) bioactivity and overproduction of superoxide, is regarded as one of the most significant events contributing to the vascular lesions associated with diabetes. However, the mechanisms underlying such hyperglycemic injury remain undefined. We hypothesized that alterations in cellular protein S-nitrosylation may contribute to hyperglycemia-induced endothelial dysfunction. RESEARCH DESIGN AND METHODS: We exposed endothelial cells to high glucose in the presence and absence of reactive oxygen species inhibitors and used the biotin switch assay to analyze the alteration in the global pattern of protein S-nitrosylation compared with cells cultured under normal glucose conditions. We identified endogenous S-nitrosylated proteins by mass spectrometry and/or immunoblotting with specific antibodies. RESULTS: High-glucose treatment induced a significant reduction of endogenous S-nitrosylated proteins that include endothelial NO synthase, beta-actin, vinculin, diacylglycerol kinase-alpha, GRP78, extracellular signal-regulated kinase 1, and transcription factor nuclear factor-kappaB (NF-kappaB). Interestingly, these changes were completely reversed by inhibition of superoxide production, suggesting a key role for oxidative stress in the regulation of S-nitrosylation under hyperglycemic conditions. In addition, we found that in parallel with the restoration of decreased S-nitrosylation of NF-kappaB, high glucose-induced NF-kappaB activation was blocked by the superoxide inhibitors. CONCLUSIONS: The alterations in protein S-nitrosylation may underlie the adverse effect of hyperglycemia on the vasculature, such as endothelial dysfunction and the development of diabetic vascular complications.
OBJECTIVE:Hyperglycemia-induced endothelial dysfunction, via a defect of nitric oxide (NO) bioactivity and overproduction of superoxide, is regarded as one of the most significant events contributing to the vascular lesions associated with diabetes. However, the mechanisms underlying such hyperglycemic injury remain undefined. We hypothesized that alterations in cellular protein S-nitrosylation may contribute to hyperglycemia-induced endothelial dysfunction. RESEARCH DESIGN AND METHODS: We exposed endothelial cells to high glucose in the presence and absence of reactive oxygen species inhibitors and used the biotin switch assay to analyze the alteration in the global pattern of protein S-nitrosylation compared with cells cultured under normal glucose conditions. We identified endogenous S-nitrosylated proteins by mass spectrometry and/or immunoblotting with specific antibodies. RESULTS: High-glucose treatment induced a significant reduction of endogenous S-nitrosylated proteins that include endothelial NO synthase, beta-actin, vinculin, diacylglycerol kinase-alpha, GRP78, extracellular signal-regulated kinase 1, and transcription factor nuclear factor-kappaB (NF-kappaB). Interestingly, these changes were completely reversed by inhibition of superoxide production, suggesting a key role for oxidative stress in the regulation of S-nitrosylation under hyperglycemic conditions. In addition, we found that in parallel with the restoration of decreased S-nitrosylation of NF-kappaB, high glucose-induced NF-kappaB activation was blocked by the superoxide inhibitors. CONCLUSIONS: The alterations in protein S-nitrosylation may underlie the adverse effect of hyperglycemia on the vasculature, such as endothelial dysfunction and the development of diabetic vascular complications.