Karlsen A E, Andersen H U, Vissing H, Larsen P M, Fey S J, Cuartero B G, Madsen O D, Petersen J S, Mortensen S B, Mandrup-Poulsen T
Steno Diabetes Center, Gentofte, Denmark.
Diabetes. 1995 Jul;44(7):753-8. doi: 10.2337/diab.44.7.753.
An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.
诱导型一氧化氮（NO）合酶同工型（iNOS）在暴露于白细胞介素（IL）-1β的大鼠胰岛β细胞中被特异性诱导，提示NO在I型糖尿病发病机制中起作用。本研究的目的是从细胞因子暴露的胰岛中克隆并鉴定iNOS cDNA。在无细胞因子培养的大鼠胰岛或大鼠胰岛素瘤RIN-5AH β细胞中，既检测不到NO生成，也检测不到iNOS转录。单独添加IL-1β或与肿瘤坏死因子-α联合添加，可诱导iNOS基因的浓度和时间依赖性表达以及胰岛和RIN细胞中相关的NO生成（以亚硝酸盐测定）。通过逆转录聚合酶链反应从细胞因子暴露的大鼠胰岛和RIN细胞中克隆iNOS转录本，DNA序列分析显示其与最近发表的从细胞因子暴露的大鼠肝细胞和平滑肌细胞中克隆的iNOS cDNA有近100%的同一性。重组大鼠胰岛iNOS在人肾293成纤维细胞中瞬时和稳定表达，添加L-精氨酸类似物Nω-硝基-L-精氨酸甲酯和氨基胍可抑制其高酶活性。二维凝胶电泳显示重组iNOS为一系列斑点，预期分子量为131 kDa，pI值在6.8至7.0范围内。总之，从大鼠胰岛和RIN细胞中克隆并表达的IL-1β诱导的iNOS与在其他细胞类型中诱导的iNOS由相同的转录本编码。
