Uc Aliye, Kooy Neil W, Conklin Jeffrey L, Bishop Warren P
Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Dig Dis Sci. 2003 Dec;48(12):2353-9. doi: 10.1023/b:ddas.0000007874.20403.1d.
Intestinal mucosa serves as an important barrier that may be disrupted by inflammation. A complex system of cellular and humoral factors, including epidermal growth factor (EGF), maintains the integrity of this barrier. We hypothesized that peroxynitrite, generated in inflamed intestinal epithelium, can alter the EGF receptor function by nitrating tyrosine residues and blocking ligand-activated tyrosine autophosphorylation. Caco-2 cells or A431 cell membranes were treated with peroxynitrite or its decomposed form. Cell proliferation was measured by [3H]thymidine uptake. Immunoblot and immunoprecipitation were used to assess the tyrosine phosphorylation and nitration. Binding of epidermal growth factor to its receptor was detected by affinity labeling with 125I-EGF. Peroxynitrite inhibited EGF-induced Caco-2 cell proliferation and binding of EGF to its receptor in a concentration-dependent manner. Peroxynitrite abolished EGF-stimulated receptor autophosphorylation and nitrated EGF receptor tyrosine residues. Peroxynitrite generated during inflammation may disrupt the EGF-induced signaling in intestinal epithelial cells.
肠黏膜是一道重要的屏障，可能会因炎症而遭到破坏。包括表皮生长因子（EGF）在内的细胞和体液因子组成的复杂系统维持着这一屏障的完整性。我们推测，在炎症肠道上皮中产生的过氧亚硝酸盐可通过硝化酪氨酸残基并阻断配体激活的酪氨酸自磷酸化来改变表皮生长因子受体的功能。用亚硝酸盐或其分解形式处理Caco-2细胞或A431细胞膜。通过[3H]胸苷摄取来测量细胞增殖。采用免疫印迹和免疫沉淀法评估酪氨酸磷酸化和硝化作用。通过用125I-EGF进行亲和标记来检测表皮生长因子与其受体的结合。过氧亚硝酸盐以浓度依赖的方式抑制表皮生长因子诱导的Caco-2细胞增殖以及表皮生长因子与其受体的结合。过氧亚硝酸盐消除了表皮生长因子刺激的受体自磷酸化，并使表皮生长因子受体酪氨酸残基硝化。炎症期间产生的过氧亚硝酸盐可能会破坏肠上皮细胞中表皮生长因子诱导的信号传导。
