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.

中文翻译：

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TNF-α 激活的 eNOS 信号通过 S-亚硝基化途径增加白细胞粘附

一氧化氮 (NO) 是内皮中内皮一氧化氮合酶 (eNOS) 产生的炎症的关键因素，其活性在促炎细胞因子刺激后增加。NO 激活可溶性鸟苷酸环化酶-蛋白激酶 G 和 S-亚硝基化（蛋白质中游离硫醇半胱氨酸的 NO 修饰）途径。NO 经典地被描述为白细胞与内皮细胞粘附的负调节剂。然而，激活 NO 生成的激动剂会诱导快速白细胞粘附，表明 NO 可能正调节白细胞粘附。我们测试了 eNOS 诱导的 NO 通过 S-亚硝基化途径促进白细胞粘附的假设。我们使用肿瘤坏死因子 α (TNF-α) 作为促炎激动剂刺激白细胞粘附于内皮。通过生化和成像技术评估 ICAM-1 的变化。分别通过蛋白质印迹和生物素转换方法评估蛋白激酶 C zeta (PKCζ) 活性和 S-亚硝基化。TNF-α 在短时间刺激下激活 eNOS-S-亚硝基化途径，并在体内和体外引起白细胞与内皮细胞的粘附。TNF-α 诱导的 NO 导致细胞表面 ICAM-1 的变化，这是聚类的特征。TNF-α 诱导的 NO 还产生 PKCζ 的 S-亚硝基化和磷酸化，PKCζ 与 ICAM-1 和 ICAM-1 磷酸化的关联。PKCz 的抑制作用阻止了 TNF-α 诱导的白细胞粘附。PKCζ 的质谱分析将半胱氨酸 503 鉴定为蛋白质激酶结构域中的 S-亚硝基化残基。