May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Glutamate secretion by retinal pigment epithelial cells is regulated by iron
Author Affiliations & Notes
  • M.C. McGahan
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • J. Harned
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • M. Mukunnemkeril
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • M. Goralska
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • L. Fleisher
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • J. Ferrell
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • Footnotes
    Commercial Relationships  M.C. McGahan, None; J. Harned, None; M. Mukunnemkeril, None; M. Goralska, None; L. Fleisher, None; J. Ferrell, None.
  • Footnotes
    Support  EY04900–21
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 641. doi:
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      M.C. McGahan, J. Harned, M. Mukunnemkeril, M. Goralska, L. Fleisher, J. Ferrell; Glutamate secretion by retinal pigment epithelial cells is regulated by iron . Invest. Ophthalmol. Vis. Sci. 2004;45(13):641.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Glutamate (glu) and iron have many essential functions in all cells. Although dysregulation of glu and iron metabolism has been shown in both neural and retinal degeneration, no link between these changes has been established. The presence of cytosolic forms of the mitochondrial enzymes aconitase and isocitrate dehydrogenase has led to questions about their function in this compartment. The coupled activity of these enzymes results in the formation of α–ketoglutarate, a substrate for glu dehydrogenase. Since the activity of c–aconitase, an enzyme with an iron–sulfur cluster, is regulated by iron availability we explored the novel hypothesis that glu production and secretion by retinal pigment epithelium (RPE) is regulated by iron. Methods: Primary cultures of canine RPE cells were used in these studies. RPE were incubated in glutamine and serum free medium for 24h and the cell conditioned medium (CCM) was then collected. After various treatments the glu content of the CCM was determined using an Amplex Red assay kit (Molecular Probes, OR). Aconitase activity was determined by measuring the disappearance of added aconitic acid from mitochondria free cytosolic extracts. Results: Glu accumulated in the CCM over time to a concentration of 96 µM (1.47 µmoles/mg protein) at 24 h. Iron supplementation with ferric ammonium citrate (FAC) increased aconitase activity by 15% and glu accumulation in the CCM over a 24h period by 50%. The iron chelator deferroxamine decreased glu accumulation by 33%. The aconitase inhibitor oxalomalate completely inhibited aconitase activity and decreased baseline glu accumulation (by 56%) and completely inhibited the FAC–induced increase in glu accumulation. Inhibition of glu transporters by L–trans–pyrrolidine–2,4–dicarboxylic acid resulted in a 57% increase in accumulation of glu in CCM, indicating that there is normally reuptake of glu secreted by RPE. Conclusions: While RPE cells have glu receptors and transporters, glu secretion by these cells has not, to our knowledge, been previously studied. Since RPE underlie and intimately interact with the retina, glu secretion by the RPE could have important physiological functions and its dysregulation could have pathological consequences. This study clearly demonstrates that iron regulates glu secretion by RPE cells via the cytosolic aconitase pathway. The effect of elevated iron concentration on glutamate secretion provides a possible link between increased iron deposition and glu–induced excitotoxic neural and retina degeneration.

Keywords: excitatory neurotransmitters • retina • retinal pigment epithelium 
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