Original ContributionKaempferol 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
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, O2− 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 O2− in synaptosomes [15], and that when inhibited by 4-hydroxyquinazoline, the lesser production of O2−, and consequently the decreased generation of O2−-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 O2− 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.
References (63)
- et al.
Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death
Neuron
(1995) - et al.
Cytochrome c is released from mitochondria in a reactive oxygen species (ROS)-dependent fashion and can operate as a ROS scavenger and as a respiratory substrate in cerebellar neurons undergoing excitotoxic death
J. Biol. Chem.
(2000) - et al.
Neuronal death signaling by beta-bungarotoxin through the activation of the N-methyl-d-aspartate (NMDA) receptor and L-type calcium channel
Biochem. Pharmacol.
(2003) - et al.
Superoxide in apoptosis: mitochondrial generation triggered by cytochrome c loss
J. Biol. Chem.
(1998) - et al.
Neuronal apoptosis in rats is accompanied by rapid impairment of cellular respiration and is prevented by scavengers of reactive oxygen species
Neurosci. Lett.
(1998) - et al.
Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes
Biochim. Biophys. Acta
(1995) - et al.
The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron–sulfur cluster N2
FEBS Lett.
(2001) - et al.
Nitric oxide and oxygen radicals: a question of balance
FEBS Lett.
(1995) - et al.
Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase
J. Biol. Chem.
(1992) - et al.
Growth factor-stimulated trans plasma membrane electron transport in HL-60 cells
FEBS Lett.
(1992)
The hormone-responsive NADH oxidase of the plant plasma membrane has properties of a NADH:protein disulfide reductase
J. Biol. Chem.
The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity
Biochim. Biophys. Acta
Flavonoids and isoflavonoids: a gold mine for metabolic engineering
Trends Plant Sci.
Interaction of flavonoids with ascorbate and determination of their univalent redox potentials: a pulse radiolysis study
Free Radic. Biol. Med.
Flavonoid antioxidants: rate constants for reactions with oxygen radicals
Methods Enzymol.
Inhibition of peroxynitrite-mediated tyrosine nitration by catechin polyphenols
Biochem. Biophys. Res. Commun.
Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein
Free Radic. Biol. Med.
Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms
Free Radic. Biol. Med.
N-Methyl-d-aspartate promotes the survival of cerebellar granule cells in culture
Neuroscience
The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+
J. Biol. Chem.
Inactivation of ecto-ATPase activity of rat brain synaptosomes
Biochim. Biophys. Acta
Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium
Trends Neurosci.
Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups
Toxicol. In Vitro
Synaptic signaling by nitric oxide
Curr. Opin. Neurobiol.
Behaviour of nitric oxide synthase in rat cerebellar granule cells differentiating in culture
FEBS Lett.
Accurate prediction of xanthine oxidase inhibition based on the structure of flavonoids
Eur. J. Pharmacol.
Preferential inhibition by (−)-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture
Biochem. Pharmacol.
Oxidation stress in cell culture: an under-appreciated problem?
FEBS Lett.
Involvement of free radicals in excitotoxicity in vivo
J. Neurochem.
Role of oxidative stress in the apoptotic cell death of cultured cerebellar granule neurons
J. Neurosci. Res.
Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells
J. Neurochem.
Cited by (98)
COVID-19 and retinal degenerative diseases: Promising link “Kaempferol”
2022, Current Opinion in PharmacologyHuman erythrocytes exposure to juglone leads to an increase of superoxide anion production associated with cytochrome b<inf>5</inf> reductase uncoupling
2020, Biochimica et Biophysica Acta - BioenergeticsCitation Excerpt :Noteworthy due to the higher contribution of the NADH-dependent reactions to the ROS production, we focused on this pathway. Based on our previous reports showing a contribution of Cb5R to the ROS production in other cell types [13,14,23], we quantified how much of the oxidative stress-induced by juglone in erythrocyte lysates was associated with Cb5R. For this purpose, we used an inhibitor of this enzyme named mersalyl to analyze the contribution of Cb5R to the NADH-independent and NADH-dependent ROS production which represent 30% and 46%, respectively and in comparison to the experiments assayed without inhibitor.
Kaempferol protects retinal ganglion ceils from high-glucose-induced injury by regulating vasohibin-1
2020, Neuroscience LettersThe role of flavonoids in autoimmune diseases: Therapeutic updates
2019, Pharmacology and TherapeuticsCitation Excerpt :Flavonoids such as epicatechin, 3′-O-methyl-epicatechin (Schroeter, Spencer, Rice-Evans, & Williams, 2001), kaempferol, quercetin (Ishikawa & Kitamura, 2000), hesperetin and its structural counterparts namely, isorhamnetin and isosakuranetin have reduced the neuronal apoptosis through inhibiting JNK pathway and activation of caspase-3 (Schroeter et al., 2001). Activation of JNK and nitric oxide synthase (NOS) in oxidative stress-related neuronal death are linked to cell calcium homeostasis and modulation of superoxide/calcium/NO signalling was implicated in kaempferol neuroprotective action (Marques-da-Silva & Gutierrez-Merino, 2014; Samhan-Samhan-Arias, & Martı́n-Romero, & Gutiérrez-Merino, 2004; Schroeter et al., 2001). Similar to other flavonoids such as genistein (Vallés et al., 2008), epicatechin (Vallés et al., 2008) protect the oxidative stress-induced neurodegeneration in diseased cases by activating the p38 MAPK pathway.
- 1
These authors contributed equally to this work.