Santiago Ana R, Rosa Susana C, Santos Paulo F, Cristóvão Armando J, Barber Alistair J, Ambrósio António F
Center for Neuroscience and Cell Biology, Department of Zoology, Penn State College of Medicine, Hershey, PA, USA.
Invest Ophthalmol Vis Sci. 2006 Sep;47(9):4130-7. doi: 10.1167/iovs.06-0085.
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 alpha-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.
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.
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.
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.
谷氨酸能神经传递和钙稳态的改变可能导致糖尿病视网膜病变（DR）中视网膜神经细胞功能障碍和凋亡。本研究的目的是确定高糖对α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）和海人藻酸谷氨酸受体亚基蛋白质含量的影响，特别是GluR2亚基，因为它控制AMPA受体相关通道的Ca2+通透性。还研究了高糖对胞质游离钙浓度（[Ca2+]i）的影响。
通过蛋白质印迹法评估GluR1、GluR2、GluR6/7和KA2亚基的蛋白质含量。用钴染色法鉴定含有钙/钴可渗透AMPA受体的细胞。在加载了fluo-4的R28细胞和大鼠视网膜原代培养物中，通过活细胞共聚焦显微镜记录KCl或海人藻酸引起的[Ca2+]i变化。
在原代培养物中，高糖显著降低了GluR1和GluR6/7亚基的蛋白质含量，增加了GluR2和KA2亚基的蛋白质含量。高糖减少了钴阳性细胞的数量，表明通过AMPA受体相关通道的钙通透性降低。在高糖处理的细胞中，[Ca2+]i的变化大于对照细胞，且恢复到基础水平的时间延迟。然而，在不存在Na+的情况下，为防止电压敏感钙通道的激活，在存在抑制AMPA受体脱敏的环噻嗪时，海人藻酸引起的[Ca2+]i变化在高糖处理的细胞中显著低于对照培养物，进一步表明AMPA受体对钙的通透性较低。用作渗透对照的甘露醇与对照相比未引起显著变化。
结果表明，血糖升高可能改变视网膜中的谷氨酸神经传递和钙稳态，这可能与DR中视力丧失的机制有关。
