Hatanaka Y, Fujii J, Fukutomi T, Watanabe T, Che W, Sanada Y, Igarashi Y, Taniguchi N
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochim Biophys Acta. 1998 Jul 31;1393(1):203-10. doi: 10.1016/s0005-2760(98)00066-6.
The sphingomyelin pathway, activated by stimuli, such as inflammatory cytokines, results in the formation of ceramide, a second messenger molecule. The purpose of the present study was to examine the mechanism by which macrophage-type nitric oxide synthase (NOS II) is induced by stimulation of the sphingomyelin pathway. When RAW264.7 cells were incubated with sphingomyelinase (SMase), nitrite production, NOS II activity, and NOS II mRNA were increased in a dose-dependent manner. Sphingosine, dihydrosphingosine, N-acetylsphingosine (C2-ceramide), and N-acylsphingosine (natural ceramide) had no effect on nitrite production, suggesting that signal molecules other than these were concomitantly produced by SMase treatment and required for NOS II induction. We then investigated the possible involvement of intracellular reactive oxygen species (ROS) in gene induction. SMase treatment increased the level of intracellular ROS, as assessed by flow cytometric analysis using a ROS-sensitive dye, dichlorofluorescin diacetate. Antioxidants, such as N-acetyl-l-cysteine and alpha-tocopherol, inhibited gene induction as well as nitrite production by SMase. These results suggest that activation of the sphingomyelin pathway induces gene expression and that the elevated ROS were somehow involved in this process.
鞘磷脂途径在诸如炎性细胞因子等刺激作用下被激活，导致第二信使分子神经酰胺的形成。本研究的目的是探讨鞘磷脂途径刺激诱导巨噬细胞型一氧化氮合酶（NOS II）的机制。当RAW264.7细胞与鞘磷脂酶（SMase）孵育时，亚硝酸盐生成、NOS II活性和NOS II mRNA以剂量依赖性方式增加。鞘氨醇、二氢鞘氨醇、N-乙酰鞘氨醇（C2-神经酰胺）和N-酰基鞘氨醇（天然神经酰胺）对亚硝酸盐生成无影响，这表明除这些分子外的信号分子是由SMase处理伴随产生的，并且是诱导NOS II所必需的。然后我们研究了细胞内活性氧（ROS）在基因诱导中的可能作用。通过使用对ROS敏感的染料二氯荧光素二乙酸酯进行流式细胞术分析评估，SMase处理增加了细胞内ROS水平。抗氧化剂，如N-乙酰-L-半胱氨酸和α-生育酚，抑制了SMase诱导的基因表达以及亚硝酸盐生成。这些结果表明鞘磷脂途径的激活诱导了基因表达，并且升高的ROS以某种方式参与了这一过程。
