Nitric oxide (NO) is a key factor in inflammation produced by endothelial nitric oxide synthase (eNOS) in endothelium, whose activity increases after stimulation with pro-inflammatory cytokines. NO activates the soluble guanylate cyclase-protein kinase G and S-nitrosylation (NO modification of free-thiol cysteines in proteins) pathways. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce fast leukocyte adhesion suggesting that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium using tumor necrosis factor alpha (TNF-α) as pro-inflammatory agonist. ICAM-1 changes were evaluated by biochemical and imaging techniques. Protein kinase C zeta (PKCζ) activity and S-nitrosylation were evaluated by western-blot and biotin switch methods, respectively. TNF-α, at short times of stimulation, activated the eNOS- S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1 and ICAM-1 phosphorylation. The inhibition of PKCz blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of PKCζ identified cysteine 503 as the S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS-S-nitrosylation mechanism that induces leukocyte adhesion suggesting that PKCζ--S-nitrosylation might be an important regulatory step in early leukocyte adhesion.
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