视网膜色素上皮细胞中诱导型一氧化氮合酶、精氨酸代谢相关酶的表达及一氧化氮对细胞紧密连接的影响

Expression of inducible nitric oxide (NO) synthase and arginine-metabolic relative enzymes in retinal pigment epithelial (RPE) cells and the effect of NO on tight junction of RPE cells

目的研究在免疫活化的视网膜色素上皮(RPE)细胞中诱导型一氧化氮合酶(iNOS)被诱导、产生NO时,其底物精氨酸的来源;产生的NO对RPE细胞紧密连接的影响。方法用干扰素γ(IFNγ)、肿瘤坏死因子α(TNFα)、脂多糖(LPS)刺激大鼠视网膜色素上皮细胞系(RPEJ)细胞,Northernblotting、Westernblotting方法研究RPEJ细胞内精氨酸再生系瓜氨酸NO循环的相关酶的表达及地塞米松和环磷酸腺苷(cAMP)对iNOS表达的影响。并通过免疫细胞化学和Westernblotting方法,研究产生的NO对RPEJ细胞紧密连接功能的影响。结果活化的RPEJ细胞中,iNOS和精氨酸的再生系瓜氨酸NO循环相关酶精氨酸琥珀酸合成酶(AS)在mRNA和蛋白质水平同时被诱导,精氨酸琥珀酸裂解酶(AL)没有被诱导,同时产生NO,cAMP可以增加NO的产量。NO不仅以精氨酸为底物产生,而且也可由瓜氨酸产生。产生的NO破坏RPEJ细胞的紧密连接功能,与紧密连接相关蛋白ZO1的合成量减少。结论在活化的RPEJ细胞中产生NO时,由瓜氨酸NO循环再生的精氨酸是RPEJ细胞中产生NO的底物精氨酸的主要来源;NO对RPEJ细胞的紧密连接起破坏作用。

Objective To detect the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in immunostimulated retinal pigment epithelial (RPE) cells to seek for the supplying of the arginine, a substrate for NOS; as well as the effects of produced NO on the tight junction of RPE-J cells. Methods Rat′s RPE-J cells were treated with interferon-γ(INF-γ), tumor necrosis factor-α(TNF-α) and lipopolysaccharide (LPS), and Northern and Western blotting were applied to analyze the expression of the citrulline-NO cycle enzymes and related enzymes and the effect of dexamethasone and cyclic adenosine monophosphate (camp) on the expression of iNOS. Immunocytochemistry reaction and Western blotting were used to evaluate the effect of produced NO on the tight junctions of RPE-J cells. Results iNOS and argininosuccinate synthetase (AS) were highly induced at both mRNA and protection levels in immunostimulated RPE cells while arginiosuccinate lyase (AL) was not induced. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. And the produced NO impaired the tight junction of RPE-J cells, decreased the production of tight junction related protein ZO-1. Conclusion In activated RPE-J cells, citrulline-arginine recycling is important for NO production, and the produced NO weakened the function of tight junction of RPE-J cells.