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A. Li, W. Lu, G. C. T. Leung, K. Peterson-Yantorno, C. H. Mitchell, M. M. Civan; Potential Role of Pannexin Hemichannels in ATP Release From Native Bovine Ciliary Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3161.
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To determine whether the recently-identified, ubiquitous pannexin hemichannels serve as a conduit for ATP release, the first step in purinergic signaling and regulation of aqueous humor dynamics.
Mixed populations of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells were harvested from bovine ciliary processes. Cell ATP release was measured with a luminometer by the luciferin-luciferase reaction 24-48 hrs after plating into 96-well microplates. Cells were preincubated isotonically with/without drugs for 1 hr, and stimulated by 50% hypotonicity. ATP concentrations were calibrated in isotonic standard solutions and in hypotonic standard solutions with/without carbenoxolone (CBX), flufenamic acid (FFA) or NPPB.
ATP release by fresh bovine NPE and PE cells was readily measured. ATP concentration in the bath was 2.4 +0.1 nM (mean ±SE, n=124 wells) in control isotonic solution, increasing 3-fold to 8.6 +0.5 nM (n=114, P<0.001) after hypotonic exposure. CBX reduced hypotonicity-induced ATP release by 32 +6%, 45 +6%, and 52 +5% at 1 µM, 3 µM and 30 µM, respectively. The potency of CBX was very high, with a calculated IC50 of 0.7 +0.1 µM (n=15). FFA reduced the ATP release at 30 µM by 60 +4% (n=32). NPPB inhibited hypotonically-elicited release by 59 ±3% (n=57) at 100 µM, without affecting isotonic ATP release (n=30, P=0.469).
CBX reduced hypotonically-triggered ATP release of native NPE and PE cells with high potency, a signature characteristic of pannexin hemichannels. However, the maximum inhibition by CBX was only ~50%, raising the possibility of at least 2 conduits for ATP release. Since ATP release is the first step in purinergic regulation of aqueous dynamics, the current data suggest the possibility of a novel approach in selectively blocking ATP conduits in order to lower intraocular pressure.
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