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Reactive oxygen species production by monoamine oxidases in intact cells
N Pizzinat1, N Copin, C Vindis
1INSERM U388, Pharmacologie Moléculaire et Physiopathologie Rénale, Toulouse, France.
Abstract:
Monoamine oxidase (MAO) A and B are mitochondrial enzymes involved in the oxidative deamination of endogenous and exogenous amines. At present, the production of H2O2 by MAO in intact cells and its functional consequences in cell function have not been extensively investigated. The aim of this study was to define whether, in intact cells, the metabolism of small amounts of MAO substrates was able to induce a detectable H2O2 production. Hydrogen peroxide production was measured using a luminol-amplified chemiluminescence assay in three cell types, rat mesangial cells, rabbit proximal tubule cells and Hep-G2 cells, containing different MAO A/MAO B ratios. Our results showed that cell incubation with tyramine (50 micromol/l) led to a time-dependent H2O2 generation which was fully inhibited by MAO A (clorgyline and RO 41-1049) and MAO B (selegiline and RO 19-6327) inhibitors. The extent of inhibition of H2O2 production by selective inhibitors was in agreement with the amount of MAO isoforms expressed in each cell type, as determined by Western blot analysis and enzyme assay. Altogether, these findings show that, in a normal cell environment, MAO can be a source of reactive oxygen species which could have a functional impact on cell functions. In addition, we propose the luminol-amplified chemiluminescence assay as a rapid and sensitive procedure to characterize the monoamine oxidase isoforms and their regulation in intact cells.
Insights
Monoamine oxidase (MAO) enzymes generate hydrogen peroxide (H2O2) in intact cells. This study demonstrates MAO as a source of reactive oxygen species impacting cell function, proposing a new assay for MAO isoform analysis.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Monoamine oxidase (MAO) A and B are mitochondrial enzymes crucial for amine metabolism.
- The role of MAO in producing hydrogen peroxide (H2O2) and its cellular impact remains under-investigated.
- Understanding MAO's contribution to reactive oxygen species (ROS) is vital for cellular function.
Purpose of the Study:
- To determine if MAO substrate metabolism in intact cells generates detectable H2O2.
- To investigate the functional consequences of MAO-derived H2O2 in cellular environments.
- To evaluate a chemiluminescence assay for characterizing MAO isoforms and their regulation.
Main Methods:
- Utilized a luminol-amplified chemiluminescence assay to measure H2O2 production.
- Employed three cell types (rat mesangial, rabbit proximal tubule, Hep-G2) with varying MAO A/B ratios.
- Applied selective MAO A and MAO B inhibitors (clorgyline, RO 41-1049, selegiline, RO 19-6327) and Western blot analysis.
Main Results:
- Tyramine incubation induced time-dependent H2O2 generation in all tested cell types.
- H2O2 production was fully inhibited by both MAO A and MAO B specific inhibitors.
- Inhibition levels correlated with MAO isoform expression, confirmed by Western blot and enzyme assays.
Conclusions:
- MAO enzymes are a significant source of ROS in normal cellular conditions.
- MAO-derived reactive oxygen species can influence cellular functions.
- The luminol-amplified chemiluminescence assay is a sensitive method for studying MAO isoforms in intact cells.