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A. Li, C. T. Leung, K. Peterson-Yantorno, C. H. Mitchell, M. M. Civan; ATP Release by Human Trabecular Meshwork Cells Through Various Pathways. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1484.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the relative functional contributions to ATP release by large channels we have identified in human trabecular meshwork (TM) cells.
ATP release was measured with a luminometer by the luciferin-luciferase reaction 24-48hrs after plating cells into 96-well plates. Cultured human TM (hTM5) cells were pre-incubated isotonically with/without drugs for 1hr, and stimulated by 50% hypotonicity or by raising intracellular Ca2+ with 10µM ionomycin. ATP concentrations were calibrated in isotonic and hypotonic solutions with/without drugs.
Control ATP concentration in the isotonic bath was 14.0+0.2nM (mean+SE, n=839 wells). Hypotonicity increased ATP concentration 4-fold to 60.9+0.5nM (n=751, P<0.01), and ionomycin 9-fold to 134.7+3.8nM (n=138, P<0.01). The selective pannexin-1 hemichannel (PANX1) blockers carbenoxolone (3µM, CBX), mefloquine (30nM) and probenecid (100µM) inhibited hypotonicity-triggered ATP release by 43+5% (n=24), 42+3% (n=36) and 21+3% (n=46), respectively. The relatively specific connexin (Cx) inhibitor heptanol (1mM) also reduced release by 43%+1% (n=53). Antagonists of the P2X7 ionotropic receptor, 1µM KN62 and 0.5µM brilliant blue G decreased hypotonicity-induced ATP release by 36+5% (n=36) and 33+2% (n=54), respectively. Blockers of the maxi-Cl- channel (1mM SITS or 50µM Gd3+), of CFTR (100µM glybenclamide), of CaMKII (1µM Lavendustin C), and of vesicular release (2µM bafilomycin or 100µM monensin) had no effect. Contrary to their effects on hypotonicity-elicited ATP release, KN62 totally abolished ionomycin-triggered release, while CBX had no effect.
PANX1, Cxs and P2X7, directly or via PANX1, contribute to swelling-activated ATP release, whereas the maxi-Cl- channel, CFTR and vesicular release are not involved. KN62, but not CBX, abolished cytosolic Ca2+-evoked ATP release, suggesting that PANX1-independent P2X7 pathways may dominate following ionomycin stimulation. The results reveal that homogenous populations of TM cells functionally express multiple ATP conduits, whose relative importance depends on the stimulus applied.
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