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A. Li, C. T. Leung, K. Peterson-Yantorno, C. H. Mitchell, M. M. Civan; Knockdown of Pannexin-1 Hemichannel Inhibits ATP Release by Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):686.
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To test whether down-regulation of pannexin-1 (Px1) hemichannels in human trabecular meshwork (TM) cells reduces ATP release, the enabling step in purinergic regulation of aqueous humor dynamics. We previously identified Px1, connexin (Cx) hemichannels and ionotropic P2X7 receptor in TM cells by RT-PCR with DNA sequencing.
Cultured human TM (hTM5) cells were transduced with shRNA lentiviral particles and selected with puromycin (0.5µg/mL) for stable Px1 knockdown. Efficiency was determined by real-time PCR and semi-quantitative immunoblotting. ATP release was measured with a luminometer by the luciferin-luciferase reaction 24-48hrs after plating cells into 96-well plates. Cells were pre-incubated isotonically with/without drugs for 1hr, and stimulated by 50% hypotonicity or by raising cytosolic Ca2+ with 10µM ionomycin. ATP concentrations were monitored in isotonic and hypotonic solutions with/without drugs. Intracellular Ca2+ levels were similarly measured with fluorescent dye Fluo-4.
Lentivirus-mediated shRNA produced stable knockdown of Px1 mRNA by 67% (n=21) and protein by 39% (n=8) for >5 weeks. ATP concentration for the non-targeted control (NC) was 9.6+0.1nM (mean+SE, n=543 wells), increasing 3.6-fold to 31.3+0.5nM (n=746, P<0.01) after hypotonic exposure. Px1 knockdown (KD) did not change the isotonic baseline (n=147, P=0.71), but reduced the hypotonically-elicited ATP release by 27+2% (n=287, P<0.001). Relatively selective blockers of Px1 (100µM Probenecid), Cxs (1mM Heptanol) and P2X7 receptor (1µM KN-62) inhibited hypotonicity-induced release by 42+2% (n=246), 39+3% (n=61), and 33+3% (n=120) in NC, and by 12+2% (n=377), 63+2% (n=160), and 44+3% (n=120) in KD, respectively. KN-62 (1µM) abolished ionomycin-stimulated ATP release in both groups (NC: 100+1%, n=80; KD: 98+1%, n=120) without affecting Ca2+ influx.
Px1 knockdown decreases swelling-activated ATP release and largely nullifies the inhibitory effect of the Px1 blocker probenecid. Antagonists of other ATP conduits become correspondingly more efficacious. In contrast, the abolition of intracellular Ca2+-evoked ATP release by KN-62 suggests that a Px1-independent P2X7 pathway predominates after ionomycin stimulation. The results demonstrate that the conduits recruited by TM cells are stimulus-specific, and that Px1 contributes to swelling-initiated ATP release.
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