Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells
Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells
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DOI：
10.1136/gut.52.2.231
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发表时间：
2003-02-01
期刊：
影响因子：
24.5
通讯作者：
Rask-Madsen, J
中科院分区：
文献类型：
--
作者：
Perner, A;Andresen, L;Rask-Madsen, J
背景：通过还原性烟酰胺二核苷酸（NADH）或还原性烟酰胺二核苷酸磷酸（NADPH）氧化酶的活性产生超氧化物（O-）已在多种细胞类型中被证实，但在人类结肠上皮细胞中未被证实。目的：测定人结肠上皮细胞培养中NAD(P)H氧化酶活性调节剂和潜在O-2（-）生成酶抑制剂的02生成及其影响。测定了NAD(P)H氧化酶、Nox1和gp91(phox) （phox，吞噬氧化酶）的催化亚基以及膜结合亚基p22（phox）的表达。方法：对转化后的DLD-1、HT-29和Caco-2结肠上皮细胞系进行亚融合、融合和分化后的研究。原代结肠上皮细胞从正常人结肠粘膜活检中分离出来。通过细胞色素c还原法或鲁米诺增强发光法测定细胞外O-2（-）的产生。采用逆转录-聚合酶链反应检测结肠上皮细胞和血液中性粒细胞中Nox1、gp91（phox）和p22(phox) mRNA的表达。结果：亚融合转化细胞的O-2（-）产率（平均（SEM） 35.8 (4.2) nmol/mg蛋白/h）和原代细胞（40.4(5.9)）高于融合转化细胞（6.0 (0.9)）；P < 0.01)。氧化还原酶抑制剂二苯碘显著抑制O-2（-）的生成，而NADPH和NADH则提高了O-2（-）的生成速率。相比之下，O-2（-）不受肉豆蔻酸佛波酯、n - g -硝基- l -精氨酸甲酯、吲哚美辛或别嘌呤醇的影响。Nox1 mRNA在所有结肠上皮细胞中均有表达，而gp91（phox）仅在HT-29细胞和中性粒细胞中检测到。P22 （phox）在所有细胞类型中均有表达。结论：人结肠转化上皮细胞和原代上皮细胞的培养可通过表达Nox1和p22（phox）的NAD(P)H氧化酶产生细胞外O-2（-）。
Background: Superoxide (O-) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells.Aims: To measure 02 production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O-2(-) generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91(phox) (phox, phagocytic oxidase), and the membrane bound subunit p22(phox) was assessed.Methods: The transformed colonic epithelial cell lines (DLD-1, HT-29, and Caco-2) were studied at subconfluence, confluence, and after differentiation. Primary colonic epithelial cells were isolated from mucosal biopsies from the normal human colon. Extracellular O-2(-) production was measured by the cytochrome c reduction assay or luminol enhanced luminescence. Nox1, gp91(phox), and p22(phox) mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptase-polymerase chain reaction.Results: Production rates of O-2(-) were higher in subconfluent transformed cells (mean (SEM) 35.8 (4.2) nmol/mg of protein/h) and primary cells (40.4 (5.9)) than in confluent transformed cells (6.0 (0.9); p < 0.01). The oxidoreductase inhibitor diphenylene iodonium significantly inhibited O-2(-) production whereas NADPH and NADH increased production rates. In contrast, O-2(-) was unaffected by phorbol myristate ester, N-G-nitro-L-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91(phox) was detected only in HT-29 cells and neutrophils. p22(phox) was expressed in all cell types.Conclusions: Cultures of transformed and primary epithelial cells from human colon may produce extracellular O-2(-) through an NAD(P)H oxidase expressing Nox1 and p22(phox).