Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole.

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The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H-or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Fortyeight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H-and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of Hferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.Ferritins are the highly conserved iron storage proteins found in species from bacteria to mammals. Mammalian ferritins, located predominantly in cytoplasm, are heteropolymers of 24 subunits of two types, heavy (H 1 ; 21 kDa) and light (L; 19 kDa). Despite high (ϳ50%) sequence identity and similar three-dimensional structures, the subunits are genetically different and each has a distinct and complementary role in storing iron. The H subunit has a ferroxidase center responsible for uptake and oxidation of Fe 2ϩ into ferric ions. Translocation of these ions into the core of the ferritin shell and their mineralization for long term storage is facilitated by the L subunit. The intracellular level of ferritin correlates with the size of the pool of intracellular iron, which plays the main role in the regulation of ferritin expression. The iron-controlled expression of both chains is primarily translational and involves binding of iron-regulatory proteins IRP1 and IRP2 (iron sensors) to iron-response element (IRE), a stem-loop structure present in the 5Ј-untranslated region of the mRNA (1). The expression of H-chain gene is also regulated transcriptionally by cytokines (2), hormones, oncogenes (3), and inducers of cell differentiation (4).The H and L subunit ratio of mammalian ferritins varies and is highly tissue-specific; however, mechanisms by which cells maintain the chain-specific ratio are not fully understood. Although both chains complement each other in the process of sequestering and storing iron, H-chain has ...

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Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome. The formation of inclusion bodies in older lens epithelial cells, as demonstrated in the current investigation, is intriguing and could point to possible involvement of cytoplasmic L-chain-enriched ferritin aggregates in the formation of age-related cataract.