Glutamate Catabolism Enzymes in Müller Cells of Monkeys with Experimental Glaucoma

Abstract

Abstract: : Purpose: Previous studies in our laboratory showed an accumulation of glutamine and of the glutamate aspartate transporter in Müller cells of monkeys with experimental glaucoma (Carter–Dawson et al., 1998; Zhang et al, 2003). These data indicate an increase in glutamate transport and metabolism. The purpose of the current study is to determine whether glutamate catabolism enzymes are changed in glaucomatous monkey retinas. Methods: Unilateral experimental glaucoma was induced by argon laser treatments to the trabecular meshwork in three rhesus monkeys and bilateral glaucoma induced in three monkeys. The intraocular pressure was measured weekly by hand–held applanation tonometry. Visual field defects were assessed by behavioral perimetry using a standard clinical field analyzer (Humphery Field Analyzer). Eyes were removed from deeply anesthetized monkeys and a retinal sample 5mm in diameter was collected from the inferior retina beginning at 20 degrees from the center of the fovea from each eye. The retina was detached from the pigment epithelium, placed in small conical tubes, quickly frozen in liquid nitrogen and stored at –80ºC. To determine glutamine synthetase (GS) and Gamma glutamylcysteine synthetase (GCS) content, samples were prepared for SDS–PAGE and proteins transferred for Western Blots. The blots were probed with anti–GS, GCS or ß–actin antibody and HRP conjugated secondary antibodies. Antibody binding was detected using chemiluminescense. Results: Our results show that expression of glutamine synthetase, which converts glutamate to glutamine, is reduced significantly. A reduction in GS is detected in eyes with mild and severe visual field defects. However, γ–glutamylcysteine synthetase, the enzyme which attaches glutamate to cysteine to form glutathione, shows no significant change in content in monkey eyes with experimental glaucoma compared to fellow control eyes. Conclusions: The abnormally low concentration of GS signifies a reduction in glutamate catabolism via the glutamate–glutamine cycle in monkeys with experimental glaucoma. This reduction in GS may be due to feedback inhibition by the product glutamine and/ or increased entry of glutamate into the TCA cycle and glutathione pathway which is consistent with increased extracellular glutamate and transport into Müller Cells.

Keywords: retina: neurochemistry • Muller cells • pathology: experimental