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J. Abrams, L. Srur, R. VanNorman, T.B. Redens, T.C. Welbourne, M.P. Langford; Localization of Immunoreactive Glutamate and Glutamate Transporter Activities in Human and Rabbit Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4100.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the intracellular distribution of immunoreactive glutamate (Glu) and to compare the Glu transport activities in SV40 transformed human (HLEC) and rabbit lens epithelial cells (RLEC).
Intracellular Glu concentrations were quantified by high performance liquid chromatography. Glu and excitatory amino acid transporter subtypes (EAAT1, 2, 3, 4 and 5) were detected by immunofluorescent antibody, enzyme–linked immunosorbent antibody and western analyses. Sodium dependency and substrate specificity were used to identify and characterize transport activity for L–Glu and L/D–aspartate (Asp).
The concentration of Glu was higher in RLEC (17±5 nanamoles/mg protein) than in HLEC (3±1 nanamoles/mg protein). The distribution of concentrated Glu varied within and between cells of a monolayer. The number of EAAT positive cells varied for each monolayer, but immunoreactive EAAT1–5 proteins were detected in HLEC while immunoreactive EAAT3, 4 & 5 proteins were readily detected in RLEC. HLEC uptake of radiolabeled L/D–Asp was dependent on sodium, but HLEC and RLEC uptake of [14C]–L–Glu occurred in absence of sodium. Uptake of [3H]–D–Asp and [14C]–L–Glu in HLEC was strongly inhibited by D–Asp, L–Glu, threo–ß–hydroxyaspartate, and serine–O–sulfate, but weakly by D–Glu and kainic acid consistent with predominant EAAT1 and 3 activities. RLEC uptake of [14C]–L–Glu was inhibited by L–Glu and serine–O–sulfate.
The results support concentration of free Glu in SV40–transformed HLEC and RLEC and suggest transport of Glu by RLEC is predominantly sodium–independent, while Glu/Asp transport by HLEC is 60% sodium–dependent EAAT1 & 3 and 40% sodium–independent. The results suggest polar distribution of intracellular Glu and differentiational expression of EAAT within cells of a monolayer.
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