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Abstract #45446 Published in IGR 13-2
Cellular localization of glutamate and glutamine metabolism and transport pathways in the rat ciliary epithelium
Hu RG; Lim JC; Kalloniatis M; Donaldson PJInvestigative Ophthalmology and Visual Science 2011; 52: 3345-3353
Purpose. To investigate how glutamate and glutamine levels are established in the aqueous humor by identifying the transporters and metabolism pathways that contribute to the differential accumulation of glutamate and glutamine between the distinct epithelial cell layers that constitute the ciliary body. Methods. Postembedding immunohistochemistry and silver intensification were used to quantify the relative distributions of glutamate, glutamine, and related amino acids (aspartate, alanine, GABA, and glycine) in the pigmented (PE) and nonpigmented (NPE) epithelial cells of the ciliary body. Fluorescent immunocytochemistry was used to localize Na(+)-dependent glutamate transporters (EAAT1-5), glutamine transporters (LAT1, LAT2, and b(0,+)AT), and the enzyme glutamine synthetase (GS) in the ciliary epithelium. Intravitreal injection of the GS inhibitor methionine sulfoximine (MSO) or the EAAT functional probe D-aspartate was used to modulate GS activity and indirectly monitor glutamate uptake from the aqueous, respectively. Results. Although glutamate, glutamine, and alanine were preferentially accumulated in NPE relative to PE cells, no such differential distribution of aspartate, GABA, or glycine was observed. This differential distribution of amino acids was abolished by a single injection of MSO that caused a decrease in glutamine and an increase in glutamate levels in NPE compared with PE cells. This amino acid distribution plus an observed strong labeling of EAAT3 in the interface between the PE and the NPE cell layers indicate that EAAT3 mediates the uptake of glutamate from the blood. Weaker EAAT3 labeling of the basolateral membranes of NPE cells, coupled with the accumulation of injected D-aspartate by the ciliary epithelium, indicates that NPE cells also mediate glutamate uptake directly from the aqueous. In contrast, the basolateral localization of LAT1 and b(0,+)AT in NPE cells suggest that these transporters may mediate glutamine efflux from the NPE cells into the aqueous. Conclusions. The basolateral membrane localization of EAAT3 and LAT1/b(0,+)AT in NPE cells indicates that the low glutamate and high glutamine levels observed in the aqueous are determined by glutamate uptake and glutamine efflux, respectively. Furthermore, the concentration gradient for glutamine efflux appears to be generated by the active accumulation of glutamate by EAAT3, located in the apical membrane of NPE cells and the subsequent conversion of the accumulated glutamate to glutamine by GS in NPE cells. This suggests that in contrast to fluid transport, which uses both the PE and the NPE cell layers, the transepithelial transport of glutamine occurs primarily in the NPE cell layer.
Department of Optometry and Vision Science, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand.
Classification:
2.9 Ciliary body (Part of: 2 Anatomical structures in glaucoma)
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.3 Immunohistochemistry (Part of: 3 Laboratory methods)
