Sodium transport across the human ocular non-pigmented ciliary epithelium (NPE) is fundamental to the production aqueous humour and maintenance of intraocular pressure. In sodium transporting tissues, serum and glucocorticoid regulated kinase isoform 1 (SGK1) has been identified as an early corticosteroid target gene in the activation of pre-existing epithelial sodium channels (ENaC). We previously demonstrated the presence of both the mineralocorticoid (MR) and glucocorticoid (GR) receptors within human NPE and have now analysed the expression and regulation of SGK1 and ENaC.
The expression of SGK1 and the 3 ENaC subunits (alpha, beta, gamma) were evaluated by in-situ hybridisation (ISH) studies using non-radioactively labelled digoxigenin antisense cRNA probes on paraffin-embedded sections of normal human anterior segments. Total RNA was extracted from a human NPE cell line, at 0, 30, 60, 120 and 240 minutes following incubation at 37degC with varying concentrations dexamethasone (DEX) or aldosterone (ALDO), and in the presence of either RU486 (GR antagonist), or RU26752 (MR antagonist) or both. Northern blot analyses were performed using 32P-labelled SGK1 cDNA. The affinity of the GR and MR for DEX and ALDO was assessed by ligand binding assays conducted with radio-labelled and unlabelled DEX or ALDO.
ISH studies confirmed expression of SGK, and all ENaC subunits within the NPE, whilst control sections were negative. 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 and 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 4x104 MR per ODM cell.
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 ocular ciliary epithelium.
08 - 11 Apr 2002
British Endocrine Societies