The protective effect of astrocyte-derived 14,15-epoxyeicosatrienoic acid on hydrogen peroxide-induced cell injury in astrocyte-dopaminergic neuronal cell line co-culture
Highlights
► 14,15-EET protects dopaminergic neurons from H2O2-induced cell death. ► Co-culture of these neurons with astrocytes and astrocyte-conditioned media also showed this protective effect. ► Increasing bioavailability of endogenous EETs confers cytoprotection. ► Blocking astrocytic EETs production obliterates the protective effect.
Introduction
Elevated production of hydrogen peroxide (H2O2) in the central nervous system has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson’s disease (PD). Superoxide dismutase (SOD) and catalase are important antioxidant enzymes that scavenge superoxide anion () and H2O2 to protect cells from oxidative damage. If abnormal formation of and H2O2 is over the capability of SOD/catalase defenses or the activities of SOD and catalase were to decrease abnormally, the production of reactive oxygen species (ROS) will induce cell death. However, the therapeutic strategy for neurodegenerative diseases using SOD/catalase has not been successful (Peng et al., 2005, Chen et al., 2008). Degeneration of dopaminergic neurons is thought to be the result of apoptosis induced by chronic oxidative stress. The source of increased oxidative stress is not completely known and therefore environmental factors, excitotoxins, dopamine homeostasis, and other factors have gained more attention (Sayre et al., 2008). Oxidative stress induces mitochondrial dysfunction, genetic mutation, and protein aggregation and ultimately causes cell death (Mattson et al., 2002).
Recognition of the importance of astrocytes in neurovascular regulation is increasing, specifically regarding the modulation of neural activity. Astrocytes actively participate in several aspects of neuronal growth and differentiation both by providing cell–cell interactions and by secreting neuronal growth-promoting factors. They take up and release several neurotransmitter substances and can modulate the concentration of a neurotransmitter substance at the synaptic cleft and thus monitor neuronal activity (Vernadakis, 1988). Certain neurotrophic factors like glutathione released from astrocytes have been shown to protect neurons against oxidative damage induced by neurotoxins (Spina et al., 1992). More recently several reports show that epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid (AA) by cytochrome P450 (CYP) epoxygenases, are released from neurons and astrocytes (Alkayed et al., 1996a, Iliff et al., 2010). In the brain, EETs play an important role in cerebral blood flow regulation (Alkayed et al., 1996b) and neurovascular coupling (Koehler et al., 2006). Furthermore, the expression of CYP epoxygenase in brain is increased by ischemic preconditioning, which was associated with protection from ischemic stroke induced in the rat by middle cerebral artery occlusion (Alkayed et al., 2002a). In addition, more recently the same group has demonstrated that EETs protect neurons (Koerner et al., 2007) and astrocytes (Liu and Alkayed, 2005) against ischemic cell death induced in vitro by oxygen–glucose deprivation (OGD), suggesting that EETs may exert a cytoprotective effect independent of its effect to dilate blood vessels and increase CBF. However the initial cellular mechanisms that mediate the action of EETs remain uncertain. One possibility is that EETs bind to a membrane receptor linked to an intracellular signal transduction pathway that initiates the functional response. The other is an intracellular mechanism in which EETs directly interact with and activate ion channels, signal transduction components, or transcription factors producing the functional response. It is likely that the actions of EETs are mediated by both mechanisms, thus accounting for their diverse effects. The mechanism involving a G protein-coupled receptor is provided by the observation that 11,12-EET induced activation of the BKCa channel and tissue plasminogen activator expression is mediated by the Gαs component of a heterotrimeric GTP-binding protein (Gebremedhin et al., 1992, Li and Campbell, 1997, Node et al., 2001). Angiogenesis initiated by 11,12-EET also involves a cAMP-PKA signaling pathway that induces cyclooxygenase-2 expression (Michaelis et al., 2005). In addition to the Gαs-cAMP-PKA pathway, a number of other signal transduction mechanisms have been found to be active in EET functional responses under various conditions. Activation of tyrosine kinase cascade, Src kinase, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt pathways mediate actions of EETs in endothelial cells, arterial smooth muscle cells, glomerular mesangial cells, renal tubular epithelial cells, and myocardium (reviewed in Spector and Norris, 2007). In addition, the anti-inflammatory effect produced by 11,12-EET in the endothelium is due to inhibition of cytokine-activated nuclear factor-B (NF-B)-mediated transcription. The fact that other agonists typically activate these pathways through membrane receptor mechanisms provides support for an EET receptor mechanism, but so far the putative EET receptor has not been conclusively identified.
There are four regioisomeric EETs: 5,6-, 8,9-, 11,12- and 14,15-EETs (Rifkind et al., 1995). Because the regioisomers have a number of similar metabolic and functional properties, EETs are generally considered as a single class of compounds. But there are quantitative and qualitative differences in the actions of various regioisomers, such as 14,15-EET is the good substrate for sEH (Spector and Norris, 2007) and 11,12-EET is the only regioisomer that inhibits basolateral K+ channels in the renal cortical collecting duct (Wang et al., 2008).
Nevertheless, no information is available regarding the effect of EETs against the oxidant-induced neuronal damage one of the hallmarks of pathogenesis of PD. Therefore, this study was designed to evaluate the neuroprotective effects of 14,15-EET against H2O2-induced dopaminergic neuronal damage.
Section snippets
Materials
H2O2 and miconazole were obtained from Sigma (St. Louis, MO, USA). EETs were kindly donated by Dr. John R. Falck (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA). 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA) was also kindly donated by Dr. John D. Imig (Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA). H2O2 was dissolved in a sterile saline solution (0.9% NaCl solution). Miconazole, EETs and AUDA
Toxic effect of H2O2 on co-cultured cells
Increased intracellular levels of ROS cause oxidative stress by activating a common pathway leading to neuronal cell death (Griendling and FitzGerald, 2003). In our study the effect of H2O2 on astrocytes, N27 cell lines were studied. Astrocytes, N27 cells were stimulated with two different doses of H2O2 for 1 h and then cell viability was measured by MTT assay. We found that neuronal cells stimulated with different doses of H2O2 (0.1 and 1 mM) for 1 h decreased cell viability to about 33–52%
Discussion
Oxidative stress has been considered as one of the major risk factors exacerbating neuronal damage in degenerative disorders of the central nervous system via different molecular pathways. (Chan, 2001, Jenner, 2003). Several components of ROS generated during neurodegeneration can cause damage to cardinal cellular components, such as lipids, proteins and DNA, initiating subsequent cell death via necrosis or apoptosis (Gorman et al., 1996). Based on these findings, there is increased interest in
Acknowledgments
We would like to thank Dr. Kalyanaraman B., PhD, Department of Biophysics, Medical college of Wisconsin for donating N27 dopaminergic neuronal cells, Dr. Imig J., PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin for providing sEH inhibitor-AUDA, Dr. Woodliff J., PhD, Department of Pediatrics, Medical College of Wisconsin for measurement of mitochondrial membrane potential and The Biochemical core, Department of Physiology for measuring the endogenous level of EETs.
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