Expression of epithelial calcium transport system in rat cochlea and vestibular labyrinth

<p>Abstract</p> <p>Background</p> <p>The low luminal Ca²⁺concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca²⁺-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium.</p> <p>Results</p> <p>We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca²⁺-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca²⁺channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)₂vitamin D₃(VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system.</p> <p>Conclusions</p> <p>These findings demonstrate for the first time the expression of a complete Ca²⁺absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.</p

Slc26a7 chloride channel activity and localization in mouse Reissner’s membrane epithelium

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner’s membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner’s membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO[subscript 3]ˉ over Clˉ and inhibited by Iˉ and NPPB. Elevated NO[subscript 3]ˉ currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Clˉ transport in Reissner’s membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Clˉ conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner’s membrane cells during local perturbations of pH

cAMP-stimulated Clˉ secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium

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 Clˉ 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 (I[subscript sc]) from rat SCCD epithelia demonstrated stimulation by forskolin (EC[subscript 50]: 0.8 μM), 8-Br-cAMP (EC[subscript 50]: 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 I[subscript sc]. Partial inhibition of stimulated I[subscript sc] 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 CFTR[subscript inh]-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 I[subscript sc] 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 I[subscript sc] 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 Clˉ secretion whereby Clˉ is taken up across the basolateral membrane by a Na+-K+-2Clˉ cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Clˉ channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids

Stria vascularis in <i>Slc26a7^+/+</i> and <i>Slc26a7^Δ/Δ</i> Mice.

<p>Expression of key functional transport proteins that are known to be in the stria vascularis and that mediate generation of the endocochlear potential and the high potassium concentration of the luminal fluid, endolymph. There were no differences in the expression between <i>Slc26a7^+/+</i> (<i>top panel; A, B, C</i>) and <i>Slc26a7^Δ/Δ</i> (<i>lower panel; D, E, F</i>) mice for the potassium channels <i>Kcnq1</i> and <i>Kcnj10</i> and for the Nkcc1 transporter <i>Slc12a2</i>.</p

Nitrate currents exceed chloride currents in wild-type but not knockout mice.

<p>Currents in NO₃^–-rich bath minus currents in Cl^–-rich bath are plotted for wild-type and for knockout mice. Excess NO₃⁻ currents (at +80 mV) are significantly greater in wild-type animals, consistent with the contribution of anion currents mediated by <i>Slc26a7</i> channels.</p