Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism.

Andrea Guidarelli,Liana Cerioni,Mara Fiorani,Orazio Cantoni

doi:10.3390/ijms18081686

Andrea Guidarelli, Liana Cerioni + Show 2 more

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https://doi.org/10.3390/ijms18081686

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Abstract

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite.

Highlights

Peroxynitrite, the coupling product of nitric oxide and superoxide, promotes extensive damage on diverse biological molecules including lipids, proteins and DNA [1,2,3]
Relationships between Mitochondrial Superoxide Formation, with the Ensuing Downstream DNA Strand Scission, and Mitochondrial Ca2+ Accumulation in Cells Exposed to Ascorbic acid (AA) and Peroxynitrite
We demonstrate the mitochondrial fraction of vitamin C enhances the effects of peroxynitrite on complex III via a Ca2+-independent mechanism to promote mitochondrial superoxide formation, and downstream strand scission of genomic DNA, as well as mitochondrial dysfunction leading to mitochondrial permeability transition (MPT)-dependent cytotoxicity

Summary

Introduction

Peroxynitrite, the coupling product of nitric oxide and superoxide, promotes extensive damage on diverse biological molecules including lipids, proteins and DNA [1,2,3] While these effects can be directly mediated by peroxynitrite, it appears clear that the oxidant promotes secondary damage by triggering events resulting in the time-dependent formation of different damaging species. Our studies performed in U937 cells showed that peroxynitrite mobilizes Ca2+ from the ryanodine (Ry) receptor (RyR) and that the cation subsequently accumulates in the mitochondria, thereby enhancing the rate of superoxide/H2O2 formation. This response was named CRDM (Ca2+-/respiratory chain-dependent mechanism) [6]. Human monocytes or macrophages, while not expressing the RyR, displayed collateral resistance to CRDM [6,7]

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