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Mortimer M. Civan, Juni Banerjee, Kim Peterson-Yantorno, Chi Ting Leung, Ang Li; Effects of Cardiotonic Steroids on Trabecular Meshwork Cells: Search for Mediator of Ouabain-Enhanced Outflow Facility. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5341.
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Ouabain administration (at least 30 nM, for 4 hrs or more) has been reported to reduce aqueous humor outflow resistance. We previously found that ATP release and ectoenzymatic conversion to adenosine may link cytoskeletal remodeling and outflow resistance modulation. We now tested whether altered ATP release might also be a mediator of ouabain's effect on outflow resistance.
ATP release from human TM5 trabecular meshwork cells was measured by the luciferin-luciferase reaction, matrix metalloproteinases (MMPs) by zymography, cell Na+ concentration by SBFI fluorometry, gene expression by real-time PCR, cell volume by electronic cell sorting, cell viability by LDH and MTT assays, and the actin cytoskeleton by confocal microscopy of phalloidin-stained cells.
Contrary to expectation, ouabain at concentrations 10 nM or higher inhibited swelling-triggered ATP release after 4 hrs or longer. Inhibition was enhanced by increasing ouabain concentration and exposure time. Similar effects were produced by the reversible cardiac aglycone strophanthidin. Ouabain (4 hrs, 30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and cell viability was unchanged by exposure to ouabain (30 nM to 1 μM). Preincubation with 30 nM ouabain for 4 hrs did not detectably change Na+ level, the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-D-glucose replaced Na+ in the extracellular fluid, ouabain still inhibited swelling-initiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP stimulated MMP secretion, which was largely blocked by inhibiting conversion of ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did not alter secretion of MMP-2 and MMP-9 from cells pretreated for 4 hrs or less.
The results suggest that: (1) ouabain can trigger enhancement of outflow facility independent of its transport and actin-restructuring effects exerted at higher concentration and longer duration; (2) ouabain exerts parallel independent effects on ATP release and outflow facility; and (3) these effects likely reflect ouabain-induced changes in the scaffolding and/or signaling functions of Na+, K+-activated ATPase.
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