title: Nitric oxide regulation of Na, K-ATPase activity in ocular ciliary epithelium involves Src family kinase.
abstract: The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation.
authors.affiliation: Department of Physiology, University of Arizona, Tucson, Arizona.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Shahidullah
authors.forename: Amritlal
authors.initials: A
authors.lastname: Mandal
authors.forename: Guojun
authors.initials: G
authors.lastname: Wei
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Delamere
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects / Q000201:enzymology*; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D006162:Guanylate Cyclase / Q000378:metabolism; D006868:Hydrolysis; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D014443:Tyrosine; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D014443:Tyrosine; D000255:Adenosine Triphosphate; D019061:src-Family Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000254:Sodium-Potassium-Exchanging ATPase; D006152:Cyclic GMP
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