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Mohammad Shahidullah, Amritlal Mandal, Nicholas A Delamere; Insulin stimulates Src family kinases and causes a short term increase of Na,K-ATPase activity in the nonpigmented ciliary epithelium.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2464. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The metabolic hormone insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues including corneal endothelium (Hatou et al., (2010) IOVS; 51: 3935). Na,K-ATPase activity in the nonpigmented ciliary epithelium (NPE) plays a vital mechanistic role in aqueous humor secretion and changes of Na,K-ATPase activity are likely to impact the driving force for fluid production. Because we detect changes of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism.
Using primary cultured porcine NPE, ouabain-sensitive rubidium uptake by intact cells was measured as a functional index of the rate of Na,K-ATPase-mediated inward potassium transport. Na,K-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in cell homogenates. Protein phosphorylation was measured by western blot analysis. Data analysis used a two sample t-test and one way ANOVA.
NPE cells exposed to 100nM insulin for 5 min displayed an increase in ouabain-sensitive rubidium (Rb) uptake (54.4+4.6 vs 233.2 +7.9 µmoles/5 min/mg protein, mean+SE, n= 6, p=0.001) signifying Na,K-ATPase stimulation. Insulin caused an intrinsic increase of Na,K-ATPase activity as indicated by higher ouabain-sensitive ATP hydrolysis detected in homogenates obtained from cells exposed to insulin for 5 min (200.7±45.6 vs 508.9±44.0 nmoles ATP hydrolyzed/mg protein/30 min, p=0.002, n=4). Insulin-induced Src family kinase (SFK) phosphorylation (activation), the increases in Na,K-ATPase activity and the Rb uptake response all were abolished by a specific SFK inhibitor, PP2 (10 µM). AKT activation in insulin-treated cells was not suppressed by PP2 and the AKT inhibitor MK-2206 (2.5 µM) failed to prevent SFK activation. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 prevented SFK activation as well as the Na,K-ATPase responses suggesting the SFK activation step is downstream of PI3K.
Insulin activates a number of signaling pathways in the NPE. Insulin causes short term stimulation of Na,K-ATPase activity. The Na,K-ATPase response to insulin is dependent on SFK activation.
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