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Flufenamic acid as an inducer of mitochondrial permeability transition

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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 (ΔΨ), 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 μM Ca2+, 5 μM ruthenium red (RR) or 1 μM cyclosporin A (CsA) were used. The dose response-curves for both respiration release and ΔΨ dissipation were nearly linear, presenting an IC50 of approximately 10 μM and reaching saturation within 25-50 μM, 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 μM Ca2+, followed by ΔΨ 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.

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Authors and Affiliations

  1. Department of Physics and Chemistry, School of Pharmaceutical Sciences, University of São Paulo, Av. Café sn/n, 14040-903, Ribeirão Preto, São Paulo, Brazil

    Maria C. Jordani, Ieda M.R. Prado & Carlos Curti

  2. Department of Clinical, Toxicological and Bromatological Analysis, School of Pharmaceutical Sciences, University of São Paulo, Av. Café sn/n, 14040-903, Ribeirão Preto, São Paulo, Brazil

    Antonio C. Santos & Sérgio A. Uyemura

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  1. Maria C. Jordani
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  4. Sérgio A. Uyemura
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  5. Carlos Curti
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Jordani, M.C., Santos, A.C., Prado, I.M. et al. Flufenamic acid as an inducer of mitochondrial permeability transition. Mol Cell Biochem 210, 153–158 (2000). https://doi.org/10.1023/A:1007185825101

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