Departments of Molecular Signaling, University of Yamanashi, Yamanashi, Japan.
J Pharmacol Exp Ther. 2011 Oct;339(1):257-66. doi: 10.1124/jpet.111.183020. Epub 2011 Jul 15.
Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway. Activation of AMPK is a presently unrecognized important mechanism underlying the pharmacological effects of FFA.
氟芬那酸（FFA）是一种非甾体抗炎药（NSAID）。它具有抗炎和退热作用。此外，它还调节多种通道活性。FFA 的药理作用机制目前尚不清楚。鉴于 AMP 激活蛋白激酶（AMPK）具有抗炎和调节通道的双重功能，我们研究了 FFA 是否诱导 AMPK 激活。1）FFA 暴露于几种不同类型的细胞中，导致 AMPKα 在 Thr172 处的磷酸化升高。这种 FFA 的作用可以通过功能和结构相似的甲芬那酸、托芬那酸、尼氟酸和甲氯芬那酸复制。2）FFA 诱导的 AMPK 激活在很大程度上被 1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四（乙酰氧甲基酯）（细胞内 Ca2+ 螯合剂）处理细胞或耗尽细胞外 Ca2+ 而被废除，而细胞内 Ca2+ 离子载体 A23187 或离子霉素刺激细胞则模拟了这一作用。3）FFA 触发细胞内 Ca2+ 升高，该升高被线粒体通透性转换孔阻滞剂环孢素废除。环孢素也废除了 FFA 诱导的 AMPK 激活。4）用 7-氧代-7H-苯并咪唑[2,1-a]苯并[de]异喹啉-3-羧酸乙酸盐（STO-609）抑制 Ca2+/钙调蛋白依赖性激酶激酶β（CaMKKβ）或用短干扰 RNA 下调 CaMKKβ 可显著废除 FFA 诱导的 AMPK 激活。5）FFA 显著抑制白细胞介素-1β和肿瘤坏死因子α触发的核因子-κB 活性和诱导型一氧化氮合酶表达。这种抑制作用也被 STO-609 大大废除。综上所述，我们得出结论，FFA 通过 Ca2+-CaMKKβ 途径诱导 AMPK 激活。AMPK 的激活是 FFA 药理作用的一个尚未被认识的重要机制。
