title: Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase.
abstract: 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.
authors.affiliation: Departments of Molecular Signaling, University of Yamanashi, Yamanashi, Japan.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chi
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.forename: Qiaojing
authors.initials: Q
authors.lastname: Yan
authors.forename: Schuichi
authors.initials: S
authors.lastname: Koizumi
authors.forename: Liye
authors.initials: L
authors.lastname: Shi
authors.forename: Shuhei
authors.initials: S
authors.lastname: Takahashi
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsue
authors.forename: Masayuki
authors.initials: M
authors.lastname: Takeda
authors.forename: Masanori
authors.initials: M
authors.lastname: Kitamura
authors.forename: Jian
authors.initials: J
authors.lastname: Yao
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D005439:Flufenamic Acid / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013045:Species Specificity; D013552:Swine; D014162:Transfection
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D005439:Flufenamic Acid; D052247:Nitric Oxide Synthase Type II; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D000469:Alkaline Phosphatase; D002118:Calcium