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 Ca2+ chelator) or depletion of extracellular Ca2+, whereas it was mimicked by stimulation of cells with the Ca2+ 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 Ca2+, which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca2+/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 Ca2+-CaMKKβ pathway. Activation of AMPK is a presently unrecognized important mechanism underlying the pharmacological effects of FFA.
Footnotes
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grants 17659255, 20590953 (to J.Y.); B21390324 (to H.M.)]; the Takeda Science Foundation; the Japan-China Medical Association; and a Strategic Project Grant from the University of Yamanashi.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.183020.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- AMPK
- AMP-activated protein kinase
- p-AMPK
- phosphorylated AMPK
- AICAR
- 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside
- CaMKK
- Ca2+/calmodulin-dependent kinase kinase
- FFA
- flufenamic acid
- LKB1
- liver kinase B1
- MPTP
- mitochondria permeability transition pore
- NSAID
- nonsteroidal anti-inflammatory drug
- TRP
- transient receptor potential
- IL
- interleukin
- TNF
- tumor necrosis factor
- siRNA
- short interfering RNA
- iNOS
- inducible nitric-oxide synthase
- ACC
- acetyl-coA carboxylase
- FBS
- fetal bovine serum
- NFκB
- nuclear factor κB
- SEAP
- secreted alkaline phosphatase
- NRK
- normal rat kidney
- MFA
- mefenamic acid
- TFA
- tolfenamic acid
- NFA
- niflumic acid
- MCFA
- meclofenamic acid
- PKC
- protein kinase C
- ERK
- extracellular signal-related kinase
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)
- A23187
- 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
- STO-609
- 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate
- SC-514
- 4-amino-[2′,3′-bithiophene]-5-carboxamide.
- Received April 14, 2011.
- Accepted July 13, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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