Original Contribution
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

https://doi.org/10.1016/j.freeradbiomed.2004.04.002Get rights and content

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 μM 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.

Introduction

Reactive oxygen species (ROS) produced at the early phase of neuronal apoptosis have been recognized as mediators of intracellular apoptotic signaling cascades [1], [2], [3], [4], as ROS promote release of cytochrome c from mitochondria [5], [6], [7]. In glutamate neurotoxicity [5], [8], MPP+-induced apoptosis [9], or β-bungarotoxin [10] or glucocorticoid [11] neurotoxicity, there is a wide consensus on the thesis that most cell-damaging ROS are produced by mitochondria. However, the subcellular sources of the ROS burst produced 3–4 h after induction of cerebellar granule cell (CGC) apoptosis by lowering extracellular K+ concentration is a controversial matter [3], [4], [12]. Although an increase in ROS production by mitochondria during the early stages of low K+-induced CGC apoptosis has been observed [6], [13], [14], in recent years experimental evidence has pointed out that most of the ROS burst that potentiates the progress of this apoptosis is localized at the plasma membrane, as exogenous and extracellular SOD effectively blocks CGC apoptosis [3], [4]. Moreover, we have shown that the plasma membrane NADH oxidase activity of neuronal synaptic terminals releases superoxide anion as a co-product and, at maximal stimulation, is the major source of superoxide anion in neurons [15]. Superoxide anion production at the plasma membrane coupled to NADH and NADPH oxidase activities of the plasma membrane has also been noted for other mammalian cells [16], [17]. Therefore, there is a need for specific inhibitors of plasma membrane NADH oxidase activity to experimentally assess this point further in quantitative terms, as well as to critically evaluate the putative implication of the production of superoxide anion by the plasma membrane in apoptosis by other insults in different neurons.

In the nervous system, O2radical dot is produced as a side product of the mitochondrial electron transport chain [5], [13], [18], [19], [20], xanthine oxidase activity [5], [21], and plasma membrane NADH oxidase activity [15]. Recently our group described that the last system constitutes a major source of O2radical dot in synaptosomes [15], and that when inhibited by 4-hydroxyquinazoline, the lesser production of O2radical dot, and consequently the decreased generation of O2radical dot-derived species, led to blockade of low K+-induced apoptosis in CGCs [4]. However, 4-hydroxyquinazoline has also been reported to be an inhibitor of poly(adenosine-ribose) synthetase activity [22], with an inhibition constant close to that found for the plasma membrane NADH oxidase [4]. Therefore, more specific inhibitors of plasma membrane NADH oxidase are needed.

Plasma membrane NADH oxidase is one part of an electron transport chain [23], [24]. In addition to O2, natural electron acceptors for the trans-plasma membrane transport system include ascorbate free radical (AFR) [25] and protein disulfides [26]. This NADH oxidase activity may represent a terminal oxidase in the plasma membrane transport chain, where electrons would be transferred to molecular oxygen through different components, ubiquinone being an intermediate electron carrier of this redox chain [24], [27]. Flavonoids constitute a group of polyphenolic compounds widely distributed in various species of plants [28], with reduction potentials between 0.3 and 0.45 V [29], [30], i.e., higher than that of ubiquinone (0.06–0.1 V) [31]. As flavonoids are also lipophilic compounds with a quinonoid-like chemical structure, we tested the hypothesis that some flavonoids could be interfering with the generation of O2radical dot in apoptotic CGCs by plasma membrane NADH oxidase activity, thus acting as antiapoptotic agents. Their antioxidant activity has been related to their health-promoting functions by scavenging hydroxyl radicals, superoxide anion radicals, and lipid peroxyradicals in organisms [32], [33]. Among the flavonoids, flavonols and particularly kaempferol and kaempferol glycosides have been shown to afford significant neuroprotection against cell death induced by oxidized low-density lipoprotein in primary cultures of mouse striatal neurons [34], [35] or by glutamate in the mouse hippocampal cell line HT-22 and in primary cortical neurons [36], [37].

For this work we used primary cultures of CGCs, an experimental model for studying apoptosis of CNS neurons in vitro [12], [38], [39], [40], [41], [42], and we show that the flavonoid kaempferol efficiently blocks the oxidative stress associated with the early stages of low K+-induced CGC apoptosis and, as a consequence, completely blocks the execution of this apoptotic process.

Section snippets

Cerebellar granule cells primary cultures

Cultures of cerebellar granule neurons were obtained from dissociated cerebella of 7 day old Wistar rats as described previously [4], [41], [43]. Cells were plated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.1 mg/ml gentamicin, and 25 mM KCl on 35-mm dishes coated with poly-d-lysine, at a density of 2.75 × 106 cells/dish. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. Cytosine arabinofuranoside

Kaempferol blocks low potassium-induced apoptosis in CGCs

Initially, we did study the effects of prototype flavonols (kaempferol and quercetin) and flavones (apigenin) on cell survival after 24 h of incubation in K5 medium. Supplementation of K5 medium with micromolar kaempferol concentrations produced a dose-dependent blockade of cell death as monitored with the MTT reduction assay, and half-neuroprotection was observed with 8 ± 2 μM kaempferol (Fig. 1). Identical results were obtained with the trypan blue exclusion assay (not shown). Preincubation

Discussion

Kaempferol is a prototype member of the flavonols subclass of flavonoids, which had earlier been shown to afford efficient neuroprotection against several apoptosis and necrosis-inducing insults, such as oxidized low-density lipoproteins [34], [35] and l-glutamate [36], [37]. Ishige et al. [36] demonstrated that kaempferol efficiently blocked the increase in ROS associated with the oxidative stress caused by glutamate in the mouse hippocampal cell line HT-22. The results reported in this work

Acknowledgements

This work has been funded by Grants from the Spanish Ministerio de Ciencia y Tecnologı́a (SAF2003-08275) and from the Consejerı́a de Educación, Ciencia y Tecnologı́a (2PR03A060) of the Junta de Extremadura.

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