ATP was found to be released by RPE cells
10,16,45 –47 and by neural retina
48,49 and to be capable of acting on P2X receptors in the RPE cells in an autocrine or a paracrine manner. The study by Sullivan et al.
12 supports the presence of functional P2X receptors in the RPE cells. Sullivan et al.
12 applied 100 μM ATP to cultured human RPE cells and found that ATP induced an initial Ca
2+ peak and sustained rise in [Ca
2+]
i . In the absence of extracellular Ca
2+, the ATP-induced Ca
2+ peak was reduced, and the sustained [Ca
2+]
i increase was abolished.
12 Ryan et al.
13 presented evidence that cultured rat RPE cells exhibited functional P2Y and P2X receptors and showed that ATP-induced increases in [Ca
2+]
i did not depend on extracellular Ca
2+ influx. The discrepancies between studies could be explained by cell strains or cell lines, cell sensitivity, cell proliferation state, ATP concentration, and exposure time to ATP. Dutot et al.
19 showed that YO-PRO-1 dye uptake was increased in ATP- and BzATP-stimulated ARPE-19 cells and P2X
7 receptor protein was detected in ARPE-19 cells. In this study, we detected not only P2X
7 receptor protein but also P2X
7 receptor mRNA in human RPE cells. Our functional data indicate that P2X receptors contribute to both ATP- and BzATP-induced Ca
2+ increases and apoptosis in human RPE cells because the BzATP- and ATP-triggered Ca
2+ responses were abolished or largely blocked after the removal of extracellular Ca
2+. Our findings that the reduction or removal of extracellular Ca
2+ or the buffering of intracellular Ca
2+ with BAPTA-AM significantly inhibited or blocked ATP-induced apoptosis suggest that RPE apoptosis is triggered by the ATP-induced rise in [Ca
2+]
i (
Fig. 4). However, ATP-induced apoptosis seems to be lower at low extracellular Ca
2+ than in the nominal absence of extracellular Ca
2+ (
Fig. 7), indicating that some extracellular Ca
2+ may be required for RPE cell survival. Our pharmacologic data further support the notion that the P2X
7 receptors contribute to the responses of RPE cells to ATP because ATP-induced Ca
2+ influx and apoptosis were blocked by oATP, and the selective P2X
7 agonist BzATP induced a Ca
2+ influx that was significantly inhibited by oATP. Furthermore, BzATP-induced RPE apoptosis was blocked or significantly inhibited by P2X
7 receptor antagonists BBG, KN-62, and oATP. However, Ca
2+ influx evoked by ATP was higher than that by equimolar BzATP, indicating that in addition to P2X
7, other P2X receptors might be present because BzATP is known as a much better P2X
7 agonist than ATP.
43 Both ATP and BzATP-triggered Ca
2+ influx were insensitive to suramin, suggesting that P2X
4 and P2X
7 receptors may contribute to the induced Ca
2+ influx given that P2X
1, P2X
2, P2X
3, and P2X
5 receptors, but not P2X
4 and P2X
7 receptors, were found to be sensitive to suramin,
35,50 and the P2X
6 receptor cannot form a homomeric channel by itself.
35,44 Further studies are needed to test whether P2X
4 homomeric channels, P2X
4/P2X
7 heteromeric channels, or both are expressed in the RPE. This is important because P2X
4 and P2X
7 receptors are coexpressed in immune cells and epithelial cells, and heteromerization can change both the functional and pharmacological properties of P2X receptors.
51,52 Future studies to determine whether knockdown of P2X
7 reduces ATP-induced RPE Ca
2+ responses and apoptosis may further support our findings. Taken together, our results support the idea that P2X, especially P2X
7, receptors mediate ATP-induced Ca
2+ signaling and apoptosis in human RPE.