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Background: The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids., Results: Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2'-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/- mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR-/- mice had no responses. Nonetheless, CFTR-/- mice showed no difference from CFTR+/- mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined., Conclusions: These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.

The lumen of the inner ear has an unusually low concentration of endolymphatic Na+, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na+ by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na+ transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC); the alpha1-, alpha3-, beta1-, and beta3-isoforms of Na+-K+-ATPase; inwardly rectifying potassium channels [IC50 of short circuit current (Isc) for Ba2+: 210 microM] Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.3, Kir4.1, Kir4.2, Kir5.1, and Kir7.1; sulfonyl urea receptor 1 (SUR1); GR; mineralocorticoid receptor (MR); 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2; serum- and glucocorticoid-regulated kinase 1 (Sgk1); and neural precursor cell-expressed developmentally downregulated 4-2 (Nedd4-2). On the other hand, transcripts for the alpha4-subunit of Na+-K+-ATPase, Kir1.1, Kir3.2, Kir3.4, Kir6.1, Kir6.2, and SUR2 were found to be absent, and Isc was not inhibited by glibenclamide. Dexamethasone (100 nM for 24 h) not only upregulated the transcript expression of alpha-ENaC (approximately 4-fold), beta2-subunit (approximately 2-fold) and beta3-subunit (approximately 8-fold) of Na+-K+-ATPase, Kir2.1 (approximately 5-fold), Kir2.2 (approximately 9-fold), Kir2.4 (approximately 3-fold), Kir3.1 (approximately 3- fold), Kir3.3 (approximately 2-fold), Kir4.2 (approximately 3-fold), Kir7.1 (approximately 2-fold), Sgk1 (approximately 4-fold), and Nedd4-2 (approximately 2-fold) but also downregulated GR (approximately 3-fold) and 11beta-HSD1 (approximately 2-fold). Expression of GR and 11beta-HSD1 was higher than MR and 11beta-HSD2 in the absence of dexamethasone. Dexamethasone altered transcript expression levels (alpha-ENaC and Sgk1) by activation of GR but not MR. Proteins were present for the alpha-, beta-, and gamma-subunits of ENaC and Sgk1, and expression of alpha- and gamma-ENaC was upregulated by dexamethasone. These findings are consistent with the genomic stimulation by glucocorticoids of Na+ absorption by SCCD and provide an understanding of the therapeutic action of glucocorticoids in the treatment of Meniere's disease.

Background: The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride. Findings: Transport function was assessed by measurements of short circuit current (Isc) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on Isc. By contrast, basolateral ATP transiently increased Isc and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. Isc was reduced by 42%, 50% and 63% after knockdown of α-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed. Conclusions: These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease. [ABSTRACT FROM AUTHOR]

The semicircular canal duct (SCCD) epithelium is a vestibular epithelial domain that was recently shown to actively contribute to endolymph homeostasis by Cl- secretion under control of β2-adrenergic stimulation. By analogy to other Cl- secretory epithelia, we hypothesized that SCCD also provides an active absorptive pathway for Na+ under corticosteroid control. Measurements of short-circuit current (Isc) demonstrated stimulation (7-24 h) by the glucocorticoids hydrocortisone (EC50 13 nM), corticosterone (33 nM), prednisolone (70 nM), and dexamethasone (13 nM) over physiologically and therapeutically relevant concentrations and its block by amiloride (IC50 470 nM) and benzamil (57 nM), inhibitors of the epithelial sodium channel (ENaC). Isc was also partially inhibited by basolateral ouabain and Ba2+, indicating the participation of Na+K+-ATPase and a K+ channel in Na+ transport. By contrast, aldosterone stimulated Isc only at unphysiologically high concentrations (EC50 102 nM). The action of all steroids was blocked by mifepristone (RU-486; Kd ∼0.3 nM) but not by spironolactone (Kd ∼0.7 µM). Expression of mRNA for the α-, β-, and γ-subunits of ENaC was demonstrated in the presence and absence of glucocorticoids. These findings are the first to identify SCCD in the vestibular labyrinth as a site of physiologically significant ENaC-mediated Na+ absorption and osmotically coupled water flux. They further demonstrate regulation of Na+ transport by natural and therapeutic glucocorticoids. The results provide for the first time an understanding of the therapeutic benefit of glucocorticoids in the treatment of Meniere's disease, a condition that is associated with increased luminal fluid volume. [ABSTRACT FROM AUTHOR]