Abstract
Purpose:
We reported earlier that the nitric oxide (NO) donor SNP reduces aqueous humor secretion in isolated porcine eyes and inhibits Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE) through a cGMP/PKG-mediated pathway. Here we examine the mechanism that links SNP-induced PKG activation to inhibition of Na,K-ATPase activity.
Methods:
Porcine NPE was established in primary culture. Na,K-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in NPE lysates. Na,K-ATPase-mediated transport was measured as ouabain-sensitive potassium (86Rb) uptake by intact NPE monolayers grown on permeable supports. Protein phosphorylation was studied by western blot analysis.
Results:
SNP and 8-Br-cGMP both inhibited Na,K-ATPase activity and 86Rb uptake and the Src family kinase (SFK) inhibitor PP2 blocked the responses. SNP and 8-Br-cGMP caused activation of SFK, ERK1/2 and p-38 MAPK as indicated by increased phosphorylation. Although it is selective for SFK, PP2 prevented SNP-mediated activation of ERK1/2 and p-38 MAPK as well as SFK. The selective ERK1/2 inhibitor U0126 prevented SNP-induced ERK1/2 and p-38 MAPK activation but not SNP-induced SFK phosphorylation or Na,K-ATPase inhibition. SNP did not detectably change Na,K-ATPase α1 abundance on the plasma membrane or Ser16 phosphorylation of Na,K-ATPase α1. On the other hand, SNP-treated NPE displayed significant phosphorylation of Na,K-ATPase α1 at Tyr10.
Conclusions:
NO elicits phosphorylation of SFK through a cGMP/PKG pathway and Na,K-ATPase activity reduction appears to be SFK-mediated. The Na,K-ATPase response is associated with Tyr10 phosphorylation of Na,K-ATPase α1 protein. NO-elicited activation of ERK1/2 and p-38 MAPK does not play a role in the regulation of Na,K-ATPase activity.
Keywords: 606 NaK ATPase •
427 aqueous •
570 ion transporters