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D. Reigada, W. Lu, C.H. Mitchell; Glutamate Acts at NMDA Receptors to Trigger ATP Release From the RPE . Invest. Ophthalmol. Vis. Sci. 2006;47(13):883.
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Glutamate is released by photoreceptors to signify changes in light levels to the proximal retina. This released glutamate could also contribute to the light–dependent changes in RPE physiology. As interactions between glutaminergic and purinergic systems are essential for neuron–glial communication throughout the nervous system, we asked whether glutamate altered ATP release from RPE cells and whether this altered cell signalling.
ATP release was detected with luciferase reaction assay from the apical face of fresh bovine RPE eyecups and from cultured human ARPE–19 cells grown in 96 well plates. Intracellular calcium was monitored with fura–2, while NMDA receptors (NMDARs) were detected with antibody to NR1 subunit.
Addition of glutamate produced a dose dependent release of ATP from ARPE–19 cells, with 300 µM glutamate doubling ATP levels. Receptors for NMDA were detected in the RPE plasma membrane, and the effect of glutamate was prevented by 30 µM NMDAR blocker MK801. NMDA produced a dose dependent release of ATP, with 300 µM producing a 2.1±0.1 fold increase in ATP levels. MK801 and a second NMDAR blocker AP5 (100 µM) inhibited the NMDA–induced ATP release. Blocking the glycine site of the NMDAR with 30 µM DCKA also prevented this release. NPPB (100 µM) prevented the increase, suggesting release was physiologic via Cl– channels. While levels of calcium in RPE cells were increased by NMDA, this rise was abolished by apyrase (1 unit/ml), implying ATP was an essential intermediary. The response was even more pronounced across the apical membrane of fresh bovine RPE cells, with 100 µM NMDA increasing ATP levels 3.4±1.0 fold.
These results suggest that glutamate acts at NMDA receptors on fresh and cultured RPE cells to released ATP. This secondary release amplifies the signal by increasing Ca2+ inside RPE cells, and could thus activate Ca2+–dependent conductances in the RPE. The interplay between glutaminergic and purinergic systems may be important for light–dependent interactions between photoreceptors and the RPE.
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