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Ang Li, Chi Ting Leung, Kim Peterson-Yantorno, W. Daniel Stamer, Mortimer M. Civan; Cytoskeletal Dependence of Adenosine Triphosphate Release by Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(11):7996-8005. doi: 10.1167/iovs.11-8170.
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To test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure.
ATP release was measured by luciferin-luciferase. Ecto-ATPases from transformed human trabecular meshwork (TM) cells (TM5) and explant-derived TM cells were identified by RT-PCR. Actin was visualized by phalloidin staining. Cell viability was assayed by lactate dehydrogenase and thiazolyl blue tetrazolium bromide methods and propidium iodide exclusion, gene expression by real-time PCR, and MMP release by zymography. Cell volume was monitored by electronic cell sorting.
Hypotonicity (50%) and mechanical stretch increased ATP release with similar pharmacologic profiles. TM cells expressed ecto-ATPases E-NPP1–3, E-NTPD2, E-NTPD8, and CD73. Prolonged dexamethasone (DEX) exposure (≥2 weeks), but not brief exposure (3 days), increased cross-linked actin networks and reduced swelling-triggered ATP release. Cytochalasin D (CCD) exerted opposite effects. Neither DEX nor CCD altered the cell viability, gene expression, or pharmacologic profile of ATP-release pathways. DEX accelerated, and CCD slowed, the regulatory volume decrease after hypotonic exposure. Activating A1 adenosine receptors (A1ARs) increased total MMP-2 and MMP-9 release. DEX reduced total A1AR-triggered MMP release, and CCD increased the active form of MMP-2 release. The A1AR agonist CHA and the A1AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively.
Cytoskeletal restructuring modulated swelling-activated ATP release, in part by changing the duration of cell swelling after hypotonic challenge. Modifying ATP release is expected to modulate MMP secretion by altering ecto-enzymatic delivery of adenosine to A1ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.
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