Abstract
Purpose: :
Previously, nitric oxide (NO) donors have been shown to reduce the rate of aqueous humor formation (Shahidullah et al., Br. J. Pharmacol. 145: 84–92, 2005). Here, studies were conducted to examine the possible mechanism of action.
Methods: :
Aqueous humor flow (AHF) was measured in vitro by fluorescein dilution in the intact arterially perfused pig eye. The nonpigmented ciliary epithelium (NPE) cell layer was isolated from fresh pig eyes by a simple and novel method. NPE cells were incubated with test compounds then homogenized and Na,K–ATPase activity was measured using a colorimetric method. Nitrite was detected by the Griess assay.
Results: :
NO donors sodium nitroprusside (SNP, 1 µM–1mM), sodium azide (AZ, 100nM– 1µM) and S–nitroso–N–acetylpenicillamine (SNAP, 1µM–1mM) produced concentration–dependent inhibition of Na,K–ATPase activity in NPE cells. Detection of nitrite in the medium of SNP–treated cells confirmed NO generation by the NPE. Concentration–dependent inhibition of Na–K–ATPase was also obtained by L–arginine (1–3 mM), a physiological precursor of NO and 8p–CPT–cGMP (1–100µM), a cell permeable analog of cGMP. The L–arginine effect was abolished when the NO synthesizing enzyme, NO–synthase (NOS), was inhibited by L–NAME (100µM). The inhibitory effect of SNP or AZ on Na,K–ATPase activity was abolished by soluble guanylate cyclase inhibitors, ODQ or methylene blue (10 µM). The inhibitory effect of 8p–CPT–cGMP on Na–K–ATPase was abolished by selective protein kinase G (PKG) inhibitors, H–8 (1 µM) and H–9 (20 µM), but not by the specific protein kinase A (PKA) inhibitor H–89. H–8 or H–9 also partially suppressed the inhibitory effect of SNP on Na,K–ATPase. Since the results point to Na,K–ATPase inhibition by NO, studies were conducted to determine whether Na,K–ATPase inhibition changes AHF. In the intact eye, perfusion with ouabain (0.001–1.0 mM) was caused a dose–dependent inhibition of aqueous humor formation.
Conclusions: :
NO donors inhibit Na,K–ATPase activity in porcine NPE. The response involves NO generation by the NPE, the activation of soluble guanylate cyclase, generation of cGMP and activation of PKG. These findings suggest that the ability of NO donors to reduce AHF may be linked to inhibition Na–K–ATPase activity in the ciliary epithelium. Supported by NIH grant EY06915, RPB Inc. and the Kentucky Lions Eye Fndn.
Keywords: signal transduction: pharmacology/physiology • NaK ATPase • aqueous