Elevated glucose changes the expression of ionotropic glutamate receptor subunits and impairs calcium homeostasis in retinal neural cells

Abstract

PURPOSE. Altered glutamatergic neurotransmission and calcium homeostasis may contribute to retinal neural cell dysfunction and apoptosis in diabetic retinopathy (DR). The purpose of this study was to determine the effect of high glucose on the protein content of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptor subunits, particularly the GluR2 subunit, because it controls Ca2 permeability of AMPA receptor-associated channels. The effect of high glucose on the concentration of cytosolic free calcium ([Ca2 ]i) was also investigated. METHODS. The protein content of GluR1, GluR2, GluR6/7, and KA2 subunits was assessed by Western blot. Cobalt staining was used to identify cells containing calcium/cobalt-permeable AMPA receptors. The [Ca2 ]i changes evoked by KCl or kainate were recorded by live-cell confocal microscopy in R28 cells and in primary cultures of rat retina, loaded with fluo-4. RESULTS. In primary cultures, high glucose significantly decreased the protein content of GluR1 and GluR6/7 subunits and increased the protein content of GluR2 and KA2 subunits. High glucose decreased the number of cobalt-positive cells, suggesting a decrease in calcium permeability through AMPA receptor-associated channels. In high-glucose–treated cells, changes in [Ca2 ]i were greater than in control cells, and the recovery to basal levels was delayed. However, in the absence of Na , to prevent the activation of voltage-sensitive calcium channels, the [Ca2 ]i changes evoked by kainate in the presence of cyclothiazide, which inhibits AMPA receptor desensitization, were significantly lower in high-glucose–treated cells than in control cultures, further indicating that AMPA receptors were less permeable to calcium. Mannitol, used as an osmotic control, did not cause significant changes compared with the control. CONCLUSIONS. The results suggest that elevated glucose may alter glutamate neurotransmission and calcium homeostasis in the retina, which may have implications for the mechanisms of vision loss in DR.