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. 2010 Dec;299(6):C1308-17.
doi: 10.1152/ajpcell.00333.2010. Epub 2010 Oct 6.

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

Ang Li  1 , Chi Ting Leung, Kim Peterson-Yantorno, Claire H Mitchell, Mortimer M Civan
Affiliations

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

Ang Li et al. Am J Physiol Cell Physiol. 2010 Dec.

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 P2X(7) receptor (P2RX(7)) 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 P2RX(7) 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 P2RX(7)-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.

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Figures

Fig. 1.
Fig. 1.
Purinergic regulation of aqueous humor formation. Fluid is secreted from the stroma through the bilayered ciliary epithelium into the aqueous humor. ATP release by pigmented ciliary epithelial (PE) cells leads to a cAMP-mediated, tamoxifen (TMX)-enhanced activation of maxi-Cl− channels (17, 19, 28), subserving Cl− recycling that would reduce net secretion. Ectoenzymatic degradation of ATP released by the nonpigmented ciliary epithelial (NPE) cells produces adenosine (ADO) that can activate A3 adenosine receptors, thereby stimulating Cl− channels and fluid secretion (7, 8, 27). jcn, Junction.
Fig. 2.
Fig. 2.
Effects of inhibitors on swelling-stimulated ATP release from native ciliary epithelial cells. Positive and negative values indicate inhibition and stimulation, respectively. Numbers refer to numbers of wells measured. The inhibitors are grouped according to their putative major targets: pannexin (PX) hemichannels, connexin (Cx) hemichannels, vesicular release (VR), and P2X7 ionoreceptors. Pro, probenecid, MFQ, mefloquine; CBX, carbenoxolone; HEP, heptanol; FFA, flufenamic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; BAF, bafilomycin.
Fig. 3.
Fig. 3.
Correlation between inhibition of ATP release from native cells (bCE) and cell lines (bPE and bNPE). Data plotted reflected measurements after exposure to 1 mM PRO, 10 mM DTT, 100 nM MFQ, 1 mM HEP, 30 μM CBX, 2 μM BAF, 50 μM Gd3+, 1 μM KN-62, 30 μM NPPB, or 1 mM PRO + 1 mM HEP. The line is generated from the linear regression relating inhibition of bPE and bNPE cell lines (y) to inhibition of bCE cells (x) by the same drug at the same concentration: y = (12.15 ± 6.84) + (0.83 ± 0.15)·x, with a correlation coefficient (R) of 0.78 (P < 0.0001).
Fig. 4.
Fig. 4.
Identification of bPX1–3 and P2RX7 in mRNA from the 3 cell preparations by RT-PCR. The results obtained with GAPDH (primer pair 1) confirm that the cDNA derived from total RNA is free of genomic DNA (gDNA) contamination, because the expected products (606 bp) are observed only in the presence of reverse transcriptase [RTase (+)], distinct from those (1,251 bp) generated by gDNA.
Fig. 5.
Fig. 5.
Long (PX1-L) and short (PX1-S) isoforms of the complete coding sequences of PX1 (PX1 CDS) in the 3 cell preparations, verified by RT-PCR (A). Biotinylated probes generated by random labeling of products amplified with gene-specific PCR primers, pair 2 for GAPDH and pair 3 for PX1, were used in later detection of GAPDH (B) and PX1 (C) by Northern blotting.
Fig. 6.
Fig. 6.
Relative gene expression in the 3 cell preparations measured by real-time PCR.
Fig. 7.
Fig. 7.
Heterologous expression of bPX1 in HEK293T cells. Western immunoblots confirm expression of bPX1 tagged with myc-6×His, using anti-myc and anti-6×His antibodies (A). Confocal images demonstrate the localization of the PX1 close to or within the plasma membrane (B).
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