Open Access

Jörg Hoffmann, Judith Haendeler, Andreas M. Zeiher, Stefanie Dimmeler

• Nitric oxide (NO) plays an important role in the regulation of the functional integrity of the endothelium. The intracellular reaction of NO with reactive cysteine groups leads to the formation of S-nitrosothiols. To investigate the regulation of S-nitrosothiols in endothelial cells, we first analyzed the composition of the S-nitrosylated molecules in endothelial cells. Gel filtration revealed that more than 95% of the detected S-nitrosothiols had a molecular mass of more than 5000 Da. Moreover, inhibition of de novosynthesis of glutathione using N-butyl-sulfoximine did not diminish the overall cellular S-NO content suggesting that S-nitrosylated glutathione quantitatively plays only a minor role in endothelial cells. Having demonstrated that most of the S-nitrosothiols are proteins, we determined the regulation of the S-nitrosylation by pro-inflammatory and pro-atherogenic factors, such as TNFα and mildly oxidized low density lipoprotein (oxLDL). TNFα and oxLDL induced denitrosylation of various proteins as assessed by Saville-Griess assay, by immunostaining with an anti-S-nitrosocysteine antibody, and by a Western blot approach. Furthermore, the caspase-3 p17 subunit, which has previously been shown to be S-nitrosylated and thereby inhibited, was denitrosylated by TNFα treatment suggesting thatS-nitrosylation and denitrosylation are important regulatory mechanisms in endothelial cells contributing to the integrity of the endothelial cell monolayer.