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Mary C. McGahan, Malgorzata Goralska, Lloyd Fleisher, Marilyn Lall, Steven Nagar, Philip Mzyk, Jill Harned; Hypoxia Increases Glutamate Secretion by Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):890.
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Hypoxia-inducible factor-1 (HIF-1) is a transcription factor regulating the synthesis of dozens of proteins, including most iron regulatory proteins. Conversely, iron regulates the activity of HIF-1 by affecting stability and nuclear translocation of the HIF-1α subunit. Even under normoxic conditions, iron depletion can increase HIF-1 activity by increasing HIF-1α translocation to the nucleus where it combines with the constitutively expressed HIF-1β subunit to form active HIF-1. We have shown that iron regulates the synthesis and secretion of the excitatory amino acid neurotransmitter glutamate via effects on the cytosolic aconitase pathway. The purpose of this study was to determine if hypoxia could also induce changes in iron-regulated glutamate secretion.
Cultured canine RPE cells were used. Cells were exposed to either hypoxic (0.5% oxygen in a Ruskinn hypoxia chamber) or normoxic conditions in a standard incubator. During treatment, samples of the cell-conditioned medium (CCM) were taken for determination of glutamate (Amplex Red Assay Kit) and vascular endothelial cell growth factor (VEGF; ELISA), which is known to be induced by HIF-1. At the end of the treatment time cells were lysed and nuclear extracts prepared for analysis of HIF-1α levels.
Hypoxic conditions (0.5% oxygen) increased nuclear HIF-1α levels as well as VEGF(by 341%) accumulation in the CCM, which is consistent with activation of HIF-1. Hypoxia also increased glutamate secretion by 226%, which was significantly inhibited by the iron chelator Dp44mT and by the aconitase inhibitor oxalomalate.
The increase of HIF-1α levels in RPE nuclear extracts as well as the increase in VEGF secretion in cells exposed to 0.5% oxygen confirm that hypoxic conditions were attained in the Ruskinn chamber. Hypoxia induced a very large increase in the secretion of glutamate. The ability of iron chelation and inhibition of aconitase activity to decrease the hypoxia-induced increase in glutamate secretion indicate that hypoxia increases the availability of intracellular iron. The downstream effect of this was activation of the aconitase pathway resulting in increased glutamate production and secretion. These findings have important implications for neural and retinal ischemic injury caused by release of excitotoxic levels of glutamate. While oxidative damage is considered the major pathological consequence of dysregulated iron metabolism, iron's effects on glutamate secretion could cause additional significant damage to the brain and retina in ischemia.
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