Abstract
An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere’s disease.
Highlights
Materials and methodsHair cell function in the cochlea and the vestibular system is calcium (Ca2+)-dependent (Tanaka et al 1980; Ohmori 1985; Kozel et al 1998) and requires the endolymphatic Ca2+ concentration ([Ca2+]endolymph) to be tightly controlled at a level that is unusually low (0.017–0.133 mmol/l (Salt et al 1989)) for an extracellular fluid (e.g., [Ca2+]blood plasma = 1.2 mmol/l (Diem and Lentner 1970))
Based on the previously demonstrated similarities between the endolymphatic sac (ES) epithelium and the kidney distal tubular epithelium on the cellular and molecular levels, we mainly focused our immunohistochemical analysis on Ca2+ transport-associated proteins that are known to be crucial for the renal tubular handling of calcium
DAB immunolabeling of the calcium sensing receptor (CaSR), as well as of TRPV5, TRPV6, SERCA1, SERCA2, NCX2, PMCA1, and PMCA4 was visible in the intraosseous ES (iES) and extraosseous ES (eES) portions of the ES epithelium
Summary
(Takumida et al 2005; Wangemann et al 2007; Nakaya et al 2007; Ishibashi et al 2008)), ion exchangers (e.g., sodium-calcium exchangers [NCX] (Oshima et al 1997; Yamauchi et al 2010)), and Ca2+ ATPases (e.g., plasma membrane calcium ATPase [PMCA] (Crouch and Schulte 1995; Ågrup et al 1999)) Many of these transport proteins are directly regulated downstream targets of the calcium sensing receptor (CaSR), a G-coupled cell surface receptor that is prominently expressed in the primary tissues involved in whole-body Ca2+ homeostasis, including the kidney and the parathyroid and thyroid glands (Blankenship et al 2001; Topala et al 2009; Ranieri et al 2013). The sections were incubated with biotinylated antibodies raised in donkey and directed against mouse (1:400; 715-065-151, Milan Analytica AG, Rheinfelden, Switzerland) and Alexa Fluor 594-conjugated antibodies raised in goat and directed against rabbit (1:400; 111-585-144, Milan Analytica AG) for 1 h, followed by incubation with avidin-biotin-HRP complex (Vectastain ABC HRP Kit, Vector Laboratories). Mean values and standard deviations derived from six Spatial (proximal-to-distal) immunolabeling animals are shown
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