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  μ M –1 m M ), sodium azide (100 n M –1  μ M ) and S ‐nitroso‐ N ‐acetylpenicillamine (1  μ M –1 m M ) 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 m M ), a physiological precursor of NO and 8p‐CPT‐cGMP (1–100  μ M ), 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  μ M ). 5 The inhibitory effect of SNP or sodium azide on Na,K‐ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10  μ M ) or methylene blue (10  μ M ). 6 The inhibitory effect of 8p‐CPT‐cGMP on Na,K‐ATPase was abolished by protein kinase G (PKG) inhibitors, H‐8 (1  μ M ) and H‐9 (20  μ M ), but not by the protein kinase A (PKA) inhibitor H‐89 (100 n M ). 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.British Journal of Pharmacology (2006) 148 , 871–880. doi: 10.1038/sj.bjp.0706795