Abstract
Purpose: :
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).
Methods: :
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).
Results: :
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.
Conclusions: :
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.
Keywords: excitatory neurotransmitters • immunohistochemistry • metabolism