Pathways for ATP release by bovine ciliary epithelial cells, the initial step in purinergic regulation of aqueous humor inflow

Abstract

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X7 receptor (P2RX7) antagonist KN-62. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1–3, connexins Cx43 and Cx40, and P2RX7 were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX7-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.