Luss H, Li R K, Shapiro R A, Tzeng E, McGowan F X, Yoneyama T, Hatakeyama K, Geller D A, Mickle D A, Simmons R L, Billiar T R
Department of Surgery, University of Pittsburgh School of Medicine, PA 15261, USA.
J Mol Cell Cardiol. 1997 Apr;29(4):1153-65. doi: 10.1006/jmcc.1996.0349.
There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.
有证据表明，一氧化氮（NO）可能介导了由细菌脂多糖（LPS）和炎性细胞因子引起的一些心肌功能变化。然而，人类心肌细胞中炎性或诱导型一氧化氮合酶（iNOS）的表达尚未得到充分表征。因此，我们用肿瘤坏死因子-α（500 U/ml）、白细胞介素-1β（30U/ml）、干扰素-γ（100 U/ml）和脂多糖（大肠杆菌0111:B4，10 μg/ml）的组合处理培养的、去分化的人类心室心肌细胞。Northern印迹分析显示，在受刺激的人类心脏细胞中可检测到诱导型一氧化氮合酶（iNOS）约4.5 kb的转录本，而在未处理的细胞中未检测到。逆转录聚合酶链反应（RT-PCR）证实，iNOS mRNA仅存在于受刺激的细胞中。然而，用细胞因子和脂多糖处理心肌细胞长达96小时，并未导致培养基中亚硝酸盐+硝酸盐积累所测量的NO合成，并且在细胞裂解物中未检测到iNOS酶活性。蛋白质印迹分析未能检测到iNOS蛋白。因此，尽管细胞因子处理的细胞中iNOS mRNA水平高且持续存在，但在该实验模型中不存在iNOS蛋白。鸟苷三磷酸环水解酶I在mRNA和蛋白质水平均被诱导，导致生物蝶呤水平升高，表明存在足够量的辅因子四氢生物蝶呤（BH4），并且未能表达诱导型蛋白是iNOS特有的。为了确定iNOS蛋白的缺失是否是由于人类心肌细胞中存在新的心脏iNOS基因或修饰的iNOS转录本，我们从细胞因子处理的心肌细胞中克隆了iNOS cDNA。该克隆的测序和表达显示了一个功能性iNOS cDNA，与其他人iNOS cDNA克隆的同一性>99%。当用仅携带人类肝细胞iNOS cDNA编码区的逆转录病毒载体转导人类心脏细胞时，可检测到iNOS mRNA和蛋白。总之，这些源自培养的人类心肌细胞的细胞缺乏表达内源性iNOS蛋白的能力，其基础似乎是细胞特异性抑制或iNOS翻译失败。
