Nitric oxide regulation of Na, K-ATPase activity in ocular ciliary epithelium involves Src family kinase

Mohammad Shahidullah, Amritlal Mandal, Guojun Wei, Nicholas A. Delamere

Research output: Contribution to journalArticle

12 Scopus citations

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 86Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100μM) or exogenously added cGMP (8-Br-cGMP) (100μM) and the SFK inhibitor PP2 (10μM) 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 86Rb 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; 10μM) and PKG (KT5823; 1μM). 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.

Original languageEnglish (US)
Pages (from-to)343-352
Number of pages10
JournalJournal of Cellular Physiology
Volume229
Issue number3
DOIs
StatePublished - Mar 1 2014

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

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