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 microM-1 mM), sodium azide (100 nM-1 microM) and S-nitroso-N-acetylpenicillamine (1 microM-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 microM), 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 microM). 5. The inhibitory effect of SNP or sodium azide on Na,K-ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10 microM) or methylene blue (10 microM). 6. The inhibitory effect of 8p-CPT-cGMP on Na,K-ATPase was abolished by protein kinase G (PKG) inhibitors, H-8 (1 microM) and H-9 (20 microM), 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.
Shahidullah
Mohammad
M
Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky 40202, USA. m0shah01@louisville.edu
Delamere
Nicholas A
NA
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D001120:Arginine / Q000494:pharmacology; D002924:Ciliary Body / Q000166:cytology / Q000201:enzymology*; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009573:Nitrites / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013552:Swine
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009573:Nitrites; D030421:Peroxynitrous Acid; D001120:Arginine; D017869:Cyclic GMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester