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M. M. Civan, C. T. Leung, K. Peterson-Yantorno, A. Li; Cytoskeletal Remodeling Regulates ATP Release by Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):687.
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© ARVO (1962-2015); The Authors (2016-present)
To test whether remodeling the cytoskeleton and shape of human trabecular meshwork (TM) cells modulates ATP release, the first step in purinergic regulation of aqueous humor dynamics.
Human TM (hTM5) cells were subcultivated and plated into 96-well microplates or stretching chambers 48hrs before experiments. ATP release was measured by the luciferin-luciferase reaction using a luminometer, and ATP concentrations were calibrated in isotonic and hypotonic solutions with/without drugs. After pre-incubation isotonically with/without drugs for 1hr, cells were stimulated either by 50% hypotonicity or graded mechanical stretch. Cytoskeletal-remodeling agents, including dexamethasone (DEX, 1µM for 14 days), H-7 (5µM and 50µM for 1hr) and cytochalasin D (CCD, 25µM for 1hr), were added and continuously maintained in corresponding cell baths at indicated time points prior to hypotonic exposure. Cell viability was estimated by the MTT method after hypotonicity or by propidium iodide staining after stretch.
ATP concentration in control isotonic solution was 10.4+0.2nM (mean+SE, n=200 wells), rising to 47.4+0.8nM (n=171, P<0.001) after 50% hypotonicity. Promoting actin-cytoskeleton polymerization with 1µM DEX reduced hypotonically-induced ATP release by 78.9+1.3% (n=28, P<0.01), whereas facilitating depolymerization with 25µM CCD, 5µM H-7 and 50µM H-7 increased swelling-activated release by 39.7+9.2% (n=54, P<0.01), 41.1+6.6% (n=36, P<0.01), and 50.4+6.1% (n=54, P<0.01), respectively. Altering cell shape by stretch also triggered ATP release. Compared with ATP levels before stretch, 10% and 30% extensions of the chamber surface area elicited 1.8±0.2 fold (N=17, P<0.01) and 6.5±1.0 fold (N=18, P<0.001) increments, respectively. Cell viability was unaffected by stretch or hypotonicity with/without drug applications.
The results indicate that ATP is released by physiologically-relevant stretch of TM cells. The data also provide the first evidence that actin polymerization inhibits, whereas depolymerization enhances hypotonically-triggered ATP release. Changes in ATP release will alter ecto-enzymatic formation of adenosine, thereby modulating release of metalloproteinase-2, mediated by A1 adenosine receptors in TM cells. This observation may be relevant to the dependence of aqueous humor outflow on the actin cytoskeleton, whose mechanism is not yet established.
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