NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye

1. We developed a novel method to isolate nonpigmented epithelial (NPE) cells from porcine eyes in order to examine Na,K-ATPase responses to nitric oxide (NO) donors specifically in the epithelium. 2. Cells were treated with NO donors and other test compounds for 20 min prior to Na,K-ATPase activity measurement. 3. NO donors, sodium nitroprusside (SNP, 1 μM-1 mM), sodium azide (100 nM-1 μM) and S-nitroso-N-acetylpenicillamine (1 μM-1 mM) caused significant concentration-dependent inhibition of Na, K-ATPase activity. Detection of nitrite in the medium of L-arginine and SNP-treated NPE confirmed NO generation. 4. 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 (1-100 μM), a cell permeable analog of cGMP. The L-arginine effect was abolished when the NO synthesizing enzyme, NO-synthase, was inhibited by L-NAME (100 μM). 5. The inhibitory effect of SNP or sodium azide on Na,K-ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10 μM) or methylene blue (10 μM). 6. The inhibitory effect of 8p-CPT-cGMP on Na,K-ATPase was abolished by protein kinase G (PKG) inhibitors, H-8 (1 μM) and H-9 (20 μM), but not by the protein kinase A (PKA) inhibitor H-89 (100 nM). H-8 and H-9 partially suppressed the inhibitory effect of SNP on Na,K-ATPase. 7. Taken together the results indicate that Na,K-ATPase inhibition response to NO donors involves activation of sGC, generation of cGMP and activation of PKG. These findings suggest that Na, K-ATPase inhibition in NPE may contribute to the ability of NO donors to reduce aqueous humor secretion.