Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis |
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Authors: | Samhan-Arias Alejandro Khalil Martín-Romero Francisco Javier Gutiérrez-Merino Carlos |
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Affiliation: | Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías Agrarias, Universidad de Extremadura, Badajoz, Spain. |
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Abstract: | Micromolar concentrations of the flavonoid kaempferol were found to efficiently block cerebellar granule cell (CGC) death through low K+-induced apoptosis, as demonstrated by prevention of the activation of caspase-3, internucleosomal DNA fragmentation, and chromatin condensation, without a significant rise in intracellular free Ca2+ concentration. Half of the maximum protection against CGC apoptosis was attained with 8 +/- 2 microM kaempferol. Reactive oxygen species (ROS) were monitored with 2',7'-dichlorodihydrofluorescein diacetate. Quantitative analysis of intracellularly and extracellularly oriented ROS production up to 3 h from the onset of low K+-induced CGC apoptosis was carried out with acquired digital fluorescence microscopy images of CGC in culture plates using a CCD camera, and also with fluorescence measurements of resuspended CGCs. In both cases, nearly 90% of ROS production by CGCs during the early stages (up to 3 h) after induction of low-K+ apoptosis occurs at the plasma membrane. Kaempferol, at concentrations that blocked CGC apoptosis, has been found to be a particularly potent blocker of extracellularly oriented ROS production by CGCs, and to inhibit the ascorbate-dependent NADH oxidase and superoxide anion production activities of the neuronal plasma membrane redox chain. |
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