Abstract
Abstract: :
Purpose: Müller cells have specific functions in the retina, which are dependent on proteins expressed exclusively in these cells but not expressed in other retinal cells. Examples include enzymes such as glutamine synthetase (GS) and pyruvate carboxylase involved in neuronal glutamate homeostasis and glutamate detoxification. They also include specific transporters such as those that take up glutamate in a Na+–dependent way. We recently found that retinal Müller cells like brain astrocytes are remarkable in lacking a malate–aspartate shuttle (MAS) due to the absence of aspartate/glutamate carrier (AGC), which is a critical component of the shuttle. MAS is needed for mitochondrial oxidation of cytosolic NADH. Lack of MAS impairs the ability of Müller cells to oxidize lactic acid thereby reserving it for consumption by neurons. Not much is known about the regulation of expression of Müller cell specific proteins although an altered amount of GS is observed in retina of diabetic rats. The purpose of the present study was to investigate regulation of expression by hydrocortisone and insulin of GS and AGC in the newly developed conditionally immortalized rat Müller cell lines (TR–MUL). Methods: The cultured TR–MUL cells were grown in DMEM containing 5 mM glucose supplemented with 5% serum. Serum was then removed 2 hours prior to hyrdrocortisone or insulin (10 nM–1µM) treatments. The influence of time course and dose of both hormones were studied. To characterize the cultured TR–MUL cells, immunocytochemical staining was performed using antibodies to vimentin, AGC and GS. The GS and AGC protein contents were determined by Western immunoblotting. Results: The cultured TR–MUL cells were positive for vimentin, GS and AGC. Expression of GS and AGC protein in cultured TR–MUL were further confirmed by Western blotting. Exposure of these cells to 1µM hydrocortisone for 8, 16 and 72 hours resulted in 5, 8 and 25 fold induction respectively in GS and a decrease in AGC protein levels. Dose response indicated an increase in GS content with an increase in hydrocortisone concentrations (10nM–1µM). Time course and dose response of insulin resulted in a decrease in GS. Conclusions: Hydrocortisone induces an increase of GS expression and a decrease in AGC. Since GS is critical for lowering toxic interstitial glutamate levels, this suggests that hyrocortisone may be used for neuroprotective strategies.
Keywords: neuroprotection • Muller cells • retina: neurochemistry