Department of Neurology, Saint Martin De Porres Hospital, Chiayi 600, Taiwan, ROC.
Life Sci. 2010 Dec 18;87(23-26):743-9. doi: 10.1016/j.lfs.2010.10.014. Epub 2010 Oct 27.
The generation of NO by endothelial nitric oxide synthase (eNOS) plays a major role in maintaining cardiovascular homeostasis. The objective of our present study was to investigate the effects of the flavone compound, apigenin, on eNOS activity and elucidate the molecular mechanisms underlying these effects in endothelial cells (ECs).
Bovine artery endothelial cells (BAECs) were exposed in a serum-free medium to apigenin. Cell viability was measured using an Alamar blue assay. The production of intracellular NO was determined using DAF-2/DA. The level of protein was examined by Western blotting. The intracellular Ca(2+) was measured using a fluorescent dye, Fura 2-AM.
Apigenin significantly induced NO production after 6h of treatment. This production was inhibited by pretreatment with the eNOS inhibitor, N(ω)-nitro l-arginine methyl ester (L-NAME). However, treatment with apigenin did not alter the eNOS protein levels but induced a sustained activation of eNOS Ser(1179) phosphorylation. Apigenin was further found to activate ERK1/2, JNK and Akt over various time courses in ECs. Treatment with specific PI3-kinase inhibitors significantly inhibited the increases in NO production and phosphorylation. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on NO production. In addition, apigenin stimulates an increase in the cytosolic Ca(2+) concentration. Apigenin-induced eNOS Ser(1179) phosphorylation and NO production are calcium-dependent, as pretreatment with extracellular or intracellular Ca(2+) chelators inhibits these processes.
Apigenin-induced calcium-dependent activation of eNOS is primarily mediated via phosphatidylinositol 3-kinase- and Akt pathways, and occurs independently of the eNOS protein content.
内皮型一氧化氮合酶（eNOS）产生的一氧化氮（NO）在维持心血管稳态中发挥主要作用。本研究旨在探讨黄酮类化合物芹菜素对 eNOS 活性的影响，并阐明其在血管内皮细胞（ECs）中的作用机制。
在无血清培养基中，将牛动脉内皮细胞（BAECs）暴露于芹菜素中。采用阿尔玛蓝法测定细胞活力。用 DAF-2/DA 测定细胞内 NO 的产生。通过 Western 印迹法检测蛋白水平。用荧光染料 Fura 2-AM 测定细胞内 Ca2+。
芹菜素处理 6 小时后可显著诱导 NO 的产生。这种产生可被 eNOS 抑制剂 N(ω)-硝基-l-精氨酸甲酯（L-NAME）预处理所抑制。然而，芹菜素处理并未改变 eNOS 蛋白水平，但诱导 eNOS Ser(1179)磷酸化的持续激活。在 ECs 中，芹菜素在不同时间内进一步激活 ERK1/2、JNK 和 Akt。用特异性 PI3-激酶抑制剂处理可显著抑制 NO 产生和磷酸化的增加。相反，ERK1/2、JNK 和 p38 的抑制对 NO 产生没有影响。此外，芹菜素刺激细胞浆 Ca2+浓度增加。芹菜素诱导的 eNOS Ser(1179)磷酸化和 NO 产生是钙离子依赖性的，因为细胞外或细胞内 Ca2+螯合剂预处理可抑制这些过程。
芹菜素诱导的 eNOS 钙离子依赖性激活主要通过磷脂酰肌醇 3-激酶和 Akt 途径介导，且与 eNOS 蛋白含量无关。
