Hartell Nicholas A, Archer Helen E, Bailey Clifford J
Department of Pharmacology, The School of Pharmacy, University of London, WC1N 1AX, UK.
Biochem Pharmacol. 2005 Mar 1;69(5):781-90. doi: 10.1016/j.bcp.2004.11.022. Epub 2005 Jan 18.
Insulin exerts a vasodilator effect by stimulating endothelial nitric oxide (NO) production. Studies in cultured cells suggest that insulin might activate endothelial nitric oxide synthase (eNOS) by an atypical, calcium-independent mechanism. This study investigates the mechanism of insulin-stimulated endothelial NO production in intact aortic wall. Real time fluorescence imaging with 4,5-diaminofluorescin diacetate (DAF-2 DA) or 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and FURA 2-AM was used to simultaneously visualise NO and intracellular calcium concentrations at multiple locations in the endothelium and vascular smooth muscle of isolated rat and mouse aorta after exposure to insulin. Inhibitors of intracellular insulin signalling were used to determine the pathway for insulin-stimulated NO production. Unlike acetylcholine, which stimulated endothelial NO production with a typical increase in free intracellular calcium, insulin (10(-8) to 10(-6)M) stimulated endothelial NO production without elevating intracellular calcium levels. Insulin-stimulated NO production was concentration dependent and detected within 30s of application. Peak increases in NO occurred between 60 and 120 s and declined slowly thereafter. Separate measurements of NO production by fluorescence of 2,3-diaminonaphthalene (DAN) noted that selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) inhibited insulin-stimulated NO production, whereas these inhibitors alone did not alter NO production or acetylcholine-stimulated NO production. Insulin-stimulated NO production by endothelium is an acute calcium-independent effect mediated via the PI3K-PKB signalling pathway.
胰岛素通过刺激内皮细胞一氧化氮（NO）生成发挥血管舒张作用。在培养细胞中的研究表明，胰岛素可能通过一种非典型的、不依赖钙的机制激活内皮型一氧化氮合酶（eNOS）。本研究探讨完整主动脉壁中胰岛素刺激内皮细胞产生NO的机制。使用4,5-二氨基荧光素二乙酸酯（DAF-2 DA）或4-氨基-5-甲基氨基-2',7'-二氟荧光素二乙酸酯（DAF-FM DA）以及FURA 2-AM进行实时荧光成像，以在离体大鼠和小鼠主动脉的内皮细胞和血管平滑肌的多个位置同时观察NO和细胞内钙浓度，观察胰岛素作用后的变化。使用细胞内胰岛素信号抑制剂来确定胰岛素刺激NO生成的途径。与乙酰胆碱不同（乙酰胆碱通过典型的细胞内游离钙增加来刺激内皮细胞产生NO），胰岛素（10⁻⁸至10⁻⁶M）刺激内皮细胞产生NO时不会升高细胞内钙水平。胰岛素刺激的NO生成具有浓度依赖性，在应用后30秒内即可检测到。NO的峰值增加发生在60至120秒之间，此后缓慢下降。通过2,3-二氨基萘（DAN）荧光单独测量NO生成发现，磷脂酰肌醇3-激酶（PI3K）和蛋白激酶B（PKB）的选择性抑制剂可抑制胰岛素刺激的NO生成，而这些抑制剂单独使用时不会改变基础NO生成或乙酰胆碱刺激的NO生成。内皮细胞中胰岛素刺激的NO生成是一种通过PI3K-PKB信号通路介导的急性、不依赖钙的效应。
