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Mohammad Shahidullah, Nicholas A. Delamere; DIDS Causes Src-Dependent Inhibition of Na,K-ATPase in the Nonpigmented Ciliary Epithelium and Slows Aqueous Humor Secretion by the Intact Porcine Eye. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1984.
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Aqueous humor (AH) secretion is the result of net ion transport that involves the coordinated operation of Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) with several other basolateral transporters and channels in the ciliary epithelium bilayer. Here, we report on the ability of DIDS to reduce AH secretion and present studies to examine the mechanism.
AH secretion rate in the intact eye was measured by fluorescein dilution technique. NPE cells were cultured to confluence on permeable supports, treated with drugs by adding to both sides of the membrane, and then harvested to measure Na,K-ATPase activity or to detect protein phosphorylation. Na,K-ATPase activity was measured either by rubidium uptake or by direct colorimetric estimation of ATP hydrolysis. Protein phosphorylation was detected by Western blot analysis.
In an intact pig eye preparation the rate of AH secretion was significantly reduced by DIDS added to the blood-side perfusate. In primary cultured NPE, DIDS inhibited ouabain-sensitive 86Rb uptake. Incubation of the cells with DIDS activated Src family kinase (SFK) and ERK1/2 as judged by Western blot analysis. DIDS and a second stilbene derivative, SITS, also inhibited Na,K-ATPase activity as measured by ATP hydrolysis. PP2, an SFK inhibitor, prevented the DIDS effect on 86Rb uptake, SFK and ERK1/2. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH. Parallel studies showed PP2 eliminates activation of SFK and ERK1/2 that occurs upon pH reduction.
The ability of DIDS to suppress AH secretion is consistent with inhibition of Na,K-ATPase in the NPE following a drop in cytoplasmic pH, SFK and ERK1/2 activation.
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