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Flufenamic acid as an inducer of mitochondrial permeability transition
M C Jordani1, A C Santos, I M Prado
1Department of Physics and Chemistry, School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, Brazil.
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.
Insights
Flufenamic acid (FA) induces mitochondrial permeability transition (MPT) through a protonophoric mechanism, affecting respiration and membrane potential. Reactive oxygen species (ROS) are implicated in this FA-induced MPT process.
Area of Science:
- Mitochondrial Physiology
- Biochemistry
- Pharmacology
Background:
- Mitochondrial permeability transition (MPT) is a critical process.
- Nonpolar carboxylic acids are known to affect mitochondrial function.
- Understanding the precise mechanisms of MPT induction is essential.
Purpose of the Study:
- To elucidate the mechanism by which flufenamic acid (FA) induces mitochondrial permeability transition (MPT).
- To investigate the effects of FA on mitochondrial respiration, membrane potential, swelling, ion flux, and reactive oxygen species (ROS) generation.
Main Methods:
- Isolated rat liver mitochondria were energized and treated with varying concentrations of FA.
- Measurements included mitochondrial respiration, electrical transmembrane potential difference (delta psi), osmotic swelling, Ca2+ efflux, NAD(P)H oxidation, and ROS generation.
- Inhibitors like ruthenium red (RR) and cyclosporin A (CsA) were used to probe the MPT pathway.
Main Results:
- FA induced mitochondrial uncoupling and delta psi dissipation via a protonophoric mechanism with an IC50 of approximately 10 microM.
- FA triggered MPT in energized and deenergized mitochondria, leading to delta psi dissipation and Ca2+ efflux.
- FA markedly stimulated NAD(P)H oxidation and ROS generation, which were partly prevented by CsA.
- FA decreased protein thiol group content, suggesting interaction with inner mitochondrial membrane thiol groups.
Conclusions:
- Flufenamic acid induces MPT through a protonophoric mechanism.
- Reactive oxygen species (ROS) likely mediate FA-induced MPT by interacting with mitochondrial thiol groups.
- FA's effects on mitochondria involve a complex interplay between protonophoric action, MPT induction, and ROS generation.