Inhibition of Glutamate Uptake Causes an Acute Increase in Aqueous Humor Protein

Abstract

Inhibition of glutamate transport has been shown to increase paracellular permeability of epithelial cell monolayersin vitro. To determine if blocking glutamate transport would affect tissue permeabilityin vivo,D-aspartate (D-Asp; 300 nmol 30 μl−1) (a non-toxic competitive inhibitor of glutamate transport) or a placebo was injected into the anterior chambers of the fellow eyes of 15 adult rabbits. [14C]-L-glucose and/or [125I]-rabbit albumin were included in the injection vehicle as aqueous humor (AH) outflow markers. The specific inhibition of glutamate uptake byD-Asp was indicated by a 15% increase in AH glutamate (174±9 nmol ml−1to 205±13 nmol ml−1;P=0.03) at 1–1.5 hr post injection. Also, the efflux of [14C]-L-glucose and [125I]-rabbit albumin from the AH of D-Asp injected eyes was increased 22% over the placebo-injected control eyes (P≤0.02). Concomitantly, the total protein concentration in the AH fromD-Asp injected eyes (517±35 μg ml−1) was 19% greater (P<0.02) than the protein concentration in AH from placebo-injected control eyes (420±36 μg ml−1). In additional studies, an irreversible inhibitor of glutamate transport, threo-β-hydroxyaspartate (THA; 30 nmol 30 μl−1), was shown to increase the efflux of [14C]-L-glucose (22%;P<0.05) from the anterior chamber and increase AH protein concentrations by 29% (484±112 μg ml−1in control AH versus 686±117 μg ml−1in THA AH,P=0.08) at 1 hr post intracameral injection. SDS-PAGE analysis of the AH associated the protein increase in theD-Asp and THA injected eyes but not placebo-injected control eyes with a detectable increase in a 66 kDa protein (aligns with serum albumin) and several lower molecular weight (23–35 kDa) AH proteins. The results found suggest that inhibition of glutamate transport from the AH acutely increases intraocular epithelial/endothelial paracellular permeability.