High Glucose Decreases Ca2+ Permeability of AMPA Receptors and Alters Calcium Homeostasis in Retinal Neural Cells

Abstract

Abstract: : Purpose: Diabetes leads to abnormal retinal function and increases apoptosis of neurons in the retina. The purpose of this study was to test the hypothesis that elevated glucose alters calcium homeostasis and AMPA receptor permeability in retinal neural cells. Methods: Primary cultures of rat retina and R28 cells were grown in media with normal (5 mM) or high glucose (30 mM for primary cultures and 20 mM for R28 cells), or mannitol (with 5 mM glucose) for 7 days and 48–72h, respectively. Cobalt staining was used to identify cells with Ca²⁺–permeable AMPA receptors. MAP–2 immunocytochemistry was used to assess neuronal morphology. The [Ca²⁺]_i response to depolarization with KCl or kainate was quantified in individual cells by confocal microscopy with the calcium–sensitive dye Fluo–4. Results: There was no significant change in neuronal morphology due to high glucose. The number of cell bodies and the length of cell processes stained with Co²⁺ significantly decreased in high glucose–treated cells, compared to both normal glucose and mannitol groups, indicating that glucose reduces the Ca²⁺ permeability of AMPA receptors. The KCl–evoked increase in [Ca²⁺]_i was significantly higher in cells grown in high glucose compared to the controls, and did not recover to baseline during the course of the experiment. Similar results were obtained in primary cultures stimulated with kainate. In Na⁺ free medium (N–methyl–D–glucamine present) the [Ca²⁺]_i response to kainate, in the presence of cyclothiazide to prevent AMPA receptor desensitization, decreased in high–glucose treated cells compared to control, further indicating a decrease in Ca²⁺–permeable AMPA receptors. Conclusions: The results demonstrate that Ca²⁺ homeostasis and AMPA receptor regulation can be altered by high concentrations of extracellular glucose. These findings offer a potential mechanism for the loss of neural function and apoptosis in diabetes.

Keywords: excitatory neurotransmitters • diabetic retinopathy • apoptosis/cell death