May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
NO Regulates the Ouabain–Induced Increases in Outflow Facility in TM
Author Affiliations & Notes
  • D.Z. Ellis
    Pharmacodynamics, University of Florida, Gainesville, FL
  • W.M. Dismuke
    Pharmacodynamics, University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships  D.Z. Ellis, None; W.M. Dismuke, None.
  • Footnotes
    Support  American Health Assistance Foundation/National Glaucoma Research
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 211. doi:
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      D.Z. Ellis, W.M. Dismuke; NO Regulates the Ouabain–Induced Increases in Outflow Facility in TM . Invest. Ophthalmol. Vis. Sci. 2006;47(13):211.

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

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Purpose: : Nitric Oxide (NO) decreases intraocular pressure (IOP) by increasing outflow facility but the mechanisms of regulation are not known. NO regulates the Na,K–ATPase via soluble guanylate cyclase and cGMP and thus plays a role in regulating physiological ion homeostasis. Additionally, the activity of the Na,K–ATPase is directly regulated by the cardiac glycoside, ouabain. The purposes of the study were to examine the role of the Na,K–ATPase in regulating outflow facility, and to determine if the ouabain– induced change in outflow facility is regulated by NO.

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. The ability of the NO donor, DETA–NO (100 µM) to regulate ouabain–induced changes in outflow facility was also determined. Cell viability assays were also performed.

Results: : DETA– NO caused an immediate increase in outflow facility (30%) (baseline outflow 0.57 µl/min/mmHg; n=6 for all experiments). This effect lasted for 30 minutes after which outflow facility was normalized. In other experiments, addition of ouabain to the perfusate increased outflow facility 40 % above baseline levels. Unlike NO, 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. While ouabain alone caused a 40% increase in outflow facility, the addition of DETA–NO 6 hours after the addition of ouabain caused an additional increase (30%) in outflow facility. Interestingly, in perfusates containing ouabain, the addition of DETA–NO altered the time course for outflow facility to return to baseline. Cell viability assays demonstrated that tissue contained living cells after medium/drug perfusion.

Conclusions: : Ouabain (30 nM) resulted in partial, nontoxic inhibition of the Na,K–ATPase and increases in outflow facility. The delayed activation of the ouabain response suggests that not only might the partial inhibition of the Na,K–ATPase by ouabain result in modest changes in TM ion flux, but also may involve the activation of signal transduction pathway(s). The additive effects of ouabain and DETA–NO on outflow facility, suggest that their mechanisms of regulation are different. However, the ability of NO to alter the normalization of outflow facility in response to ouabain would suggest that the NO/cGMP system may regulate the ouabain–induced increases in outflow.

Keywords: outflow: trabecular meshwork • NaK ATPase • nitric oxide 

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