May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Ouabain-Induced Increase in Outflow Facility in TM is Associated With Changes in Actin Cytoskeleton
Author Affiliations & Notes
  • D. Z. Ellis
    Pharmacodynamics, University of Florida, Gainesville, Florida
  • W. Dismuke
    Pharmacodynamics, University of Florida, Gainesville, Florida
  • C. Mbadugha
    Pharmacodynamics, University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  D.Z. Ellis, None; W. Dismuke, None; C. Mbadugha, None.
  • Footnotes
    Support  American Health Assistance Foundation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3710. doi:https://doi.org/
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      D. Z. Ellis, W. Dismuke, C. Mbadugha; Ouabain-Induced Increase in Outflow Facility in TM is Associated With Changes in Actin Cytoskeleton. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3710. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Inhibition of the Na,K-ATPase by the cardiac glycoside, ouabain results in decreased intraocular pressure, however the mechanisms of regulation are not completely understood. Intraocular pressure is determined by the rates of secretion of aqueous humor by the ciliary processes and rates of outflow of aqueous humor by the trabecular meshwork (TM) and the Schlemm’s canal. The purpose of the study was to examine the role of the ouabain and the Na,K-ATPase in regulating aqueous humor outflow facility and the effects of ouabain on TM cells.

Methods: : Porcine eye anterior segment organ culture using gravity driven constant pressure perfusion technique was used to measure outflow facility. A baseline flow rate of 8.0 µl /min at a pressure of 14 mmHg was maintained. Ouabain (30 nM) was used to inhibit Na,K-ATPase activity and its effect on outflow facility was measured. To assess changes in actin cytoskeleton in response to ouabain treatment, cells were stained with rhodamine-phalloidin and visualized using confocal microscopy.

Results: : Ouabain (30 nM), when added to the perfusate caused a 40 % increase in outflow facility above baseline levels (0.512-0.768 µl/min/mmHg). These increases were not immediate and occurred 4 hours following drug application and were sustained for up to 14 hrs after which outflow facility returned to baseline levels. Porcine TM cells exposed to ouabain (30 nM) for 4 hours resulted in changes in cell shape and an uneven distribution and less organization of F-actin filaments than control cells.

Conclusions: : We demonstrate a correlation between the time course for changes in cell shape and actin stress fibers in response to ouabain and the time course for the changes in outflow facility in response to ouabain. We do not know the biochemical mechanism by which ouabain increases outflow facility. However, studies in other tissues demonstrate that the α subunit of the Na,K-ATPase interacts with and contains a putative actin-binding and a cofilin-binding site. Exposure of cofilin, an actin binding protein, to ouabain leads to dephosphorylation of cofilin, and subsequent actin filament remodeling possibly through a Rho-mediated signaling pathway.

Keywords: cytoskeleton • outflow: trabecular meshwork • NaK ATPase 
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