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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Summary of expression of transcripts for isoforms of epithelial Ca²⁺ channels (TRPV5 and TRPV6), calbindin, NCX, PMCA and VDR with and without exposure to 1,25- (OH)₂vitamin D₃. Expression levels given as ratio of specific gene to 18S in primary cultures of semicircular canal duct (SCCD), native SCCD, native lateral wall (LW) and native stria vascularis (SV). All native tissues were incubated overnight after microdissection in order to reduce extracellular cross-contamination from RNA released from adjacent tissues (see Fig 1B). Open bars; control, hatched bars; 1,25-(OH)₂vitamin D₃ treated, Significant up- or down-regulation is indicated; *, P < 0.05; ND, not detected. For comparison, data in the left section are primary culture of SCCD, reproduced from [5].

Figure 2

TRPV5 protein detected by immunoblot. Incubation of primary cultures of SCCD for 24 h in the absence and presence of 100 nM 1,25- (OH)₂vitamin D₃. Protein expression of TRPV5 was compared with β-actin. A) Cultured SCCD cells with or without 1,25-(OH)₂vitamin D₃; kidney cortex and papilla were used for positive and negative controls. The blot was stripped of TRPV5 antibody and re-probed for beta-actin. B) Antigenic peptide blocked 80 kDa band. A band at 42 kDa is β-actin. The blot was directly probed for beta-actin without stripping since the TRPV5 antibody did not produce bands at the molecular weight of actin. C) PNGase F did not affect any TRPV5 bands. A band at 36 kDa is PNGase F itself. D) Intensity of 80 kDa TRPV5 bands standardized to β-actin. The mean intensity in 1,25-(OH)₂vitamin D₃ treated cells was larger but not statistically significant. Ctrl, control; Vit.D, 1,25-(OH)₂vitamin D₃ ; ns, P > 0.05; n = 14 each condition.

Figure 3

Epithelial calcium channel immunolocalization in the cochlea and vestibular system of rat. Double-staining with an antibody against TRPV5 or TRPV6 (green) and actin (red; phalloidin) (A-D) and single-staining with an antibody against TRPV5 (green) (E). A, B) Cross sections of SCCD were stained against TRPV5 and TRPV6. C) Cross section of cochlea was stained against TRPV5. Outer sulcus cells (OS), inner sulcus cells (IS) and Hensen's cells (left of OS) show significant staining for TRPV5. D) Cross section of cochlea was stained against TRPV6. Outer sulcus cells, inner sulcus cells and Hensen's cells show significant staining for TRPV6. E) Cross section of stria vascularis (SV). Marginal cells of stria vascularis (arrows) show staining for TRPV5 at the apical membrane. Sections exposed to primary antibodies preabsorbed with antigenic peptide were not stained (not shown). RM, Reissner's membrane; L, spiral limbus; SL, spiral ligament.