Cytoskeletal dependence of adenosine triphosphate release by human trabecular meshwork cells

Ang Li, Chi Ting Leung, Kim Peterson-Yantorno, W. Daniel Stamer, Mortimer M. Civan

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

Purpose. To test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure. Methods. 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. Results. 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 A 1 adenosine receptors (A 1ARs) 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 A 1AR agonist CHA and the A 1AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively. Conclusions. 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 A 1ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.

Original languageEnglish (US)
Pages (from-to)7996-8005
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number11
DOIs
StatePublished - Oct 2011
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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