TNFalpha and oxLDL reduce protein S-nitrosylation in endothelial cells

Area of Science:

• Endothelial Cell Biology
• Molecular Signaling
• Biochemistry

Background:

• Nitric oxide (NO) is crucial for maintaining endothelial cell integrity.
• NO reacts with thiols to form S-nitrosothiols, important signaling molecules.
• Understanding S-nitrosothiol regulation in endothelial cells is key to vascular health.

Purpose of the Study:

• To characterize S-nitrosylated molecules in endothelial cells.
• To investigate the regulation of S-nitrosylation by inflammatory stimuli.
• To elucidate the role of S-nitrosylation/denitrosylation in endothelial cell function.

Main Methods:

• Gel filtration to determine molecular mass of S-nitrosothiols.
• Inhibition of glutathione synthesis to assess its role.
• Treatment with TNF-alpha and oxidized LDL (oxLDL).
• Saville-Griess assay, immunostaining, and Western blotting to assess protein S-nitrosylation.
• Analysis of caspase-3 subunit modification.

Main Results:

• Over 95% of S-nitrosothiols in endothelial cells are high molecular mass (>5000 Da), primarily proteins.
• Glutathione is a minor contributor to cellular S-nitrosylation.
• TNF-alpha and oxLDL induce widespread protein denitrosylation.
• Caspase-3 p17 subunit was denitrosylated by TNF-alpha.

Conclusions:

• Endothelial S-nitrosothiols are predominantly protein-bound.
• Pro-inflammatory factors regulate protein S-nitrosylation status in endothelial cells.
• S-nitrosylation and denitrosylation are critical regulatory mechanisms for endothelial cell monolayer integrity.