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

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)871-880
Number of pages10
JournalBritish Journal of Pharmacology
Volume148
Issue number6
DOIs
StatePublished - Jul 22 2006
Externally publishedYes

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Cyclic GMP-Dependent Protein Kinases
Nitric Oxide Donors
Swine
Epithelium
Single Nucleotide Polymorphism
Arginine
Sodium Azide
Nitric Oxide
S-Nitroso-N-Acetylpenicillamine
sodium-translocating ATPase
Aqueous Humor
Methylene Blue
NG-Nitroarginine Methyl Ester
Nitroprusside
Protein Kinase Inhibitors
Nitrites
Cyclic AMP-Dependent Protein Kinases
Nitric Oxide Synthase
Epithelial Cells

Keywords

  • Aqueous humor
  • Na,K-ATPase
  • Nitric oxide
  • Porcine eye
  • Protein kinase G

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye",
abstract = "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.",
keywords = "Aqueous humor, Na,K-ATPase, Nitric oxide, Porcine eye, Protein kinase G",
author = "Shahidullah, {Mohammad -} and Delamere, {Nicholas A}",
year = "2006",
month = "7",
day = "22",
doi = "10.1038/sj.bjp.0706795",
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volume = "148",
pages = "871--880",
journal = "British Journal of Pharmacology",
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TY - JOUR

T1 - NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye

AU - Shahidullah, Mohammad -

AU - Delamere, Nicholas A

PY - 2006/7/22

Y1 - 2006/7/22

N2 - 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.

AB - 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.

KW - Aqueous humor

KW - Na,K-ATPase

KW - Nitric oxide

KW - Porcine eye

KW - Protein kinase G

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M3 - Article

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JF - British Journal of Pharmacology

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