Mechanisms of ATP Release by Human Trabecular Meshwork Cells Initiating Purinergic Regulation of Aqueous Humor Outflow

Abstract

Purpose: : Ecto-enzymatic conversion of extracellular ATP to adenosine activates A₁ adenosine receptors, reducing aqueous humor outflow resistance and intraocular pressure (IOP). This study aimed at identifying stimulus-specific, ATP-release mechanisms in trabecular meshwork (TM) cells.

Methods: : ATP levels were measured with a luminometer by the luciferin-luciferase reaction 24-48 hrs after plating human transformed (TM5) or primary (HTM) TM cells into 96-well plates. Cells were pre-incubated isotonically with/without drugs for 1 hr, and stimulated by 50% hypotonicity or 10 µM ionomycin. ATP concentrations were calibrated in iso/hypotonic solutions with/without drugs. Cell viability was monitored by the LDH assay.

Results: : Hypotonicity (50%) increased the control ATP level of TM5 cells in the isotonic bath from 11.1±0.1 nM (n=1,878 wells) to 63.7±0.7 nM (n=1,904) with half-peak time (τ) of 8.5±0.1 min. Swelling-activated ATP release was reduced by the pannexin-1 (PX1)-blocker probenecid (PRO, 0.1 mM, 37±2%), the connexin (Cx)-blocker heptanol (HEP, 1 mM, 44±4%), and the P2X₇-receptor blocker KN-62 (1 µM, 33±3%), and was nearly abolished by adding the 3 drugs together (96±1%). The reducing agent dithiothreitol (10 mM) also lowered release by 26±1%, but didn't enhance inhibition by PRO alone (40±2%). Applying 10 µM ionomycin to TM5 cells bathed in isotonic solutions with 0.1 mM external Ca²⁺ triggered a much-delayed 11-fold larger ATP release (τ=74.9±0.7 min), which was unaffected by PRO or HEP, but abolished by 1 µM KN-62 (96±2%). Similar results were obtained in HTM cells. Further raising external Ca²⁺ to 2.5 mM elevated release 148-fold, and reduced both τ (23.1±0.2 min) and efficacy of KN-62 (3 µM, 90±1%). Cell viability was unchanged by 50% hypotonicity or ionomycin with 0.1 mM Ca²⁺, but fell 31% with 2.5 mM Ca²⁺ after ionomycin.

Conclusions: : Swelling-elicited ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels, and modulated by oxidation-reduction state. In contrast, P2X₇-dependent release is either physiologically through itself and/or hemichannels, or likely reflects apoptotic magnification of ATP release through membrane compromised by high cytosolic Ca²⁺. Differentially altering the ATP-release mechanisms in inflow and outflow pathways may permit novel modulation of the purinergic regulation of IOP.

Keywords: outflow: trabecular meshwork • ion channels • adenosine