Kuemmerle J F
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0711, USA.
Am J Physiol. 1998 Jan;274(1):G178-85. doi: 10.1152/ajpgi.1998.274.1.G178.
Interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured as L-[3H]citrulline production, was increased 24.3 +/- 6.9 pmol.min-1.mg protein-1 by 1 nM IL-1 beta and 3.2 +/- 1.1 pmol.min-1.mg protein-1 by 1 nM TNF-alpha, and increased synergistically by a combination of the two (51.8 +/- 7.3 pmol.min-1.mg protein-1). Measurement of NOS II activity as nitrite production yielded similar results: IL-1 beta, 27.2 +/- 1.2; TNF-alpha, 1.6 +/- 0.1; and IL-1 beta + TNF-alpha, 46.8 +/- 3.2 pmol.min-1.mg protein-1 above basal. LPS (10 micrograms/ml) had a small but significant effect at 48 h that was only additive with that of IL-1 beta. The increase in NOS II activity induced by IL-1 beta and TNF-alpha was inhibited 73-86% by transforming growth factor-beta 1 (TFG-beta 1). The NOS isoform induced by IL-1 beta and TNF-alpha was identified as NOS II by Western immunoblot analysis and confirmed by its 66-97% inhibition by 100 microM S-methylisothiourea, a selective NOS II inhibitor, and its Ca(2+)-independent activity. We conclude that the cytokines IL-1 beta and TNF-alpha act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-beta 1.
研究了白细胞介素 -1β（IL -1β）、肿瘤坏死因子 -α（TNF -α）和脂多糖（LPS）调节培养的大鼠结肠平滑肌细胞中诱导型一氧化氮合酶（NOS II）活性和蛋白水平的能力。用这些试剂处理导致 NOS II 活性呈时间依赖性增加。48 小时后，以 L -[3H]瓜氨酸生成量衡量的 NOS II 活性，1 nM IL -1β使其增加 24.3±6.9 pmol·min⁻¹·mg 蛋白⁻¹，1 nM TNF -α使其增加 3.2±1.1 pmol·min⁻¹·mg 蛋白⁻¹，二者联合使用则协同增加（51.8±7.3 pmol·min⁻¹·mg 蛋白⁻¹）。以亚硝酸盐生成量衡量 NOS II 活性得到类似结果：IL -1β为 27.2±1.2；TNF -α为 1.6±0.1；IL -1β + TNF -α比基础值高 46.8±3.2 pmol·min⁻¹·mg 蛋白⁻¹。LPS（10 微克/毫升）在 48 小时时有微小但显著的作用，且仅与 IL -1β的作用具有相加性。IL -1β和 TNF -α诱导的 NOS II 活性增加被转化生长因子 -β1（TFG -β1）抑制了 73 - 86%。通过 Western 免疫印迹分析鉴定出由 IL -1β和 TNF -α诱导的 NOS 同工型为 NOS II，并用选择性 NOS II 抑制剂 100 μM S -甲基异硫脲对其 66 - 97%的抑制作用及其不依赖 Ca²⁺的活性进行了证实。我们得出结论，细胞因子 IL -1β和 TNF -α通过一种对 TGF -β1 抑制敏感的机制独立且协同地刺激大鼠结肠平滑肌中 NOS II 的表达和酶活性。
