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R.G. Hu, P.J. Donaldson, M. Kalloniatis; Amino Acid Localization and Transport in the Ciliary Epithelium of the Rat Eye . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3322.
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Purpose: A continuous supply of nutrients from the ciliary epithelium is critical for the maintenance of normal lens physiology. Our aim was to investigate the amino acid metabolism in the ciliary epithelium. Methods: Rats (post–natal age 25–30 days) were sacrificed, the eyes dissected, hemi–sectioned and placed in aldehyde fixatives for either post–embedding silver intensified immunogold detection of amino acids IgGs, or for cryosectioning. We applied a range of IgG antibodies (gift of Dr RE Marc, Uni of Utah), directed against the amino acids: glutamate, glycine, glutamine, aspartate, alanine, arginine, and taurine, or used a range of commercially available antibodies directed against amino acid transporters or enzymes associated with amino acid metabolism. To verify the functional integrity of glutamate transporter, D–Aspartate (2.5 mM) was injected into the vitreous and detected by an antibody targeting D–Aspartate and visualized using immunofluorescence. Results:The amino acids glutamate, glutamine, alanine, arginine, and taurine were localized within the ciliary epithelium. A key feature of the labelling pattern was the differential distribution of glutamate and glutamine. Both amino acids showed higher levels of immunoreactivity in the non–pigmented epithelial (NPE) layer than in the pigmented epithelial (PE) layer. In addition, the enzyme that catalyzes the conversion between glutamate and glutamine, glutamine synthetase, was exclusively localized in the NPE layer. Screening for glutamate transporters (EAAT1–5) revealed the presence of EAAT3 in the ciliary epithelium. The labelling was predominantly punctate in the cytoplasm of NPE cells, intense in the apical–apical interface of NPE and PE cells, and between adjoining NPE cells. A functional amino acid transporter was confirmed by the transport of D–Aspartate secondary to intra–vitreal injection. Conclusions:We have characterized the amino acid distribution in the ciliary epithelium and shown the existence of unique localization patterns within the NPE and PE cells. The expression of glutamine synthetase and EAAT3 suggests the existence of a glutamate–glutamine cycle in the ciliary epithelium. The glutamate–glutamine cycle in the ciliary epithelium would provide glutamate/glutamine required by the lens and cornea for metabolic purposes.
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