title: Flufenamic acid as an inducer of mitochondrial permeability transition.
abstract: To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3'-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (delta psi), osmotic swelling, Ca2+ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 microM Ca2+, 5 microM ruthenium red (RR) or 1 microM cyclosporin A (CsA) were used. The dose response-curves for both respiration release and delta psi dissipation were nearly linear, presenting an IC50 of approximately 10 microM and reaching saturation within 25-50 microM, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 microM Ca2+, followed by delta psi dissipation and Ca2+ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca2+. ADP, Mg2+, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H2O2 production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.
authors.affiliation: Department of Physics and Chemistry, School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, Brazil.
authors.forename: M C
authors.initials: MC
authors.lastname: Jordani
authors.forename: A C
authors.initials: AC
authors.lastname: Santos
authors.forename: I M
authors.initials: IM
authors.lastname: Prado
authors.forename: S A
authors.initials: SA
authors.lastname: Uyemura
authors.forename: C
authors.initials: C
authors.lastname: Curti
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005439:Flufenamic Acid / Q000494:pharmacology*; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling / Q000187:drug effects*; D010084:Oxidation-Reduction; D010539:Permeability / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D012430:Ruthenium Red / Q000494:pharmacology; D019802:Succinic Acid / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology*
chemical_list: D002614:Chelating Agents; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D012402:Rotenone; D012430:Ruthenium Red; D004533:Egtazic Acid; D005439:Flufenamic Acid; D019802:Succinic Acid; D002122:Calcium Chloride; D002118:Calcium