Regulation of ENaC-mediated sodium transport by glucocorticoids in Reissner's membrane epithelium

Abstract

Reissner's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing Na+ from endolymph via amiloride-sensitive channels in gerbil inner ear. We used mouse Reissner's membrane to 1) identify candidate genes involved in the Na+ transport pathway, 2) determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone, and 3) obtain functional evidence for the physiological importance of these genes. Transcripts were present for α-, β-, and γ-subunits of epithelial Na+ channel (ENaC); corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); GR agonist regulator 11β-hydroxysteroid dehydrogenase (HSD) type 1 (11β-HSD1); Na+ transport control components SGK1, Nedd4-2, and WNKs; and K+ channels and Na+-K+-ATPase. Expression of the MR agonist regulator 11β-HSD2 was not detected. Dexamethasone upregulated transcripts for α- and β-subunits of ENaC (∼6- and ∼3-fold), KCNK1 (∼3-fold), 11β-HSD1 (∼2-fold), SGK1 (∼2-fold), and WNK4 (∼3-fold). Transepithelial currents from the apical to the basolateral side of Reissner's membrane were sensitive to amiloride (IC50 ∼0.7 μM) and benzamil (IC50 ∼0.1 μM), but not EIPA (IC50 ∼34 μM); amiloride-blocked transepithelial current was not immediately changed by forskolin/IBMX. Currents were reduced by ouabain, lowered bath Na+ concentration (from 150 to 120 mM), and K+ channel blockers (XE-991, Ba2+, and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone, but not spironolactone. These molecular, pharmacological, and functional observations are consistent with Na+ absorption by mouse Reissner's membrane, which is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral Na+-K+-ATPase, and K+-permeable channels and is under the control of glucocorticoids. These results provide an understanding and a molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph.