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S Rauz, EA Walker, SV Hughes, M Coca-Prados, M Hewison, PI Murray, PM Stewart; Expression and Functional Role for the Serum and Glucocorticoid Regulated Kinase, and the Epithelial Sodium Channel in Human Ocular Ciliary Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2462.
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Purpose: Sodium transport across the human ocular non-pigmented ciliary epithelium (NPE) is fundamental to the production of aqueous humour and maintenance of intraocular pressure. In amiloride-sensitive sodium transporting tissues, such as the distal nephron, the serum and glucocorticoid regulated kinase isoform 1 (SGK1) has recently been identified as an early corticosteroid target gene in the activation of pre-existing epithelial sodium channels (ENaC). We previously demonstrated the presence of mineralocorticoid (MR) and glucocorticoid (GR) receptors within the human NPE and have now analyzed the expression and regulation of SGK1 and ENaC. Methods: The expression and distribution of SGK1 and the three ENaC subunits (α, ß, γ) was evaluated by in-situ hybridization using non-radioactively labelled digoxigenin antisense cRNA probes on paraffin-embedded sections of normal human anterior segments. Total RNA was extracted from the human NPE cell line, ODM2, at 0, 30, 60, 120 and 240 minutes following incubation at 37C with varying concentrations dexamethasone (DEX) or aldosterone (ALDO) (10-6M - 10-9M), and in the presence of 100 fold excess of either RU486 (GR antagonist), or RU26752 (MR antagonist) or both inhibitors. Northern blot analyses were performed using 32P-labelled SGK1 cDNA. The affinity of the GR and MR for the DEX and ALDO was assessed by ligand binding assays conducted with radiolabelled and unlabelled (200 fold excess) DEX or ALDO. Results: In-situ hybridization studies confirmed expression of SGK, α, ß, and γ ENaC subunits within the NPE, whilst control sections demonstrated minimal or no signal. There was marked dose-dependent induction of SGK1 with both DEX and ALDO maximal at 60 minutes. There was a moderate reduction in SGK induction by ALDO in the presence of RU26752 but induction was completely abolished in the presence of both inhibitors. There was no effect of RU26752 on SGK1 induction by DEX. Specific binding was detectable for both radiolabelled DEX (Kd 8x10-9) and ALDO (Kd 3.5x10-9), with 2x104 GR and 4x103 MR per ODM2 cell. Conclusion: Our results define expression of SGK and ENaCs within the human NPE. The induction of SGK1, even by ALDO involves both the GR and MR to stimulate sodium transport across the ciliary epithelium.
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