Endolymphatic sodium homeostasis by extramacular epithelium of the saccule

Abstract

The saccule is a vestibular sensory organ that depends upon regulation of its luminal fluid, endolymph, for normal transduction of linear acceleration into afferent neural transmission. Previous studies suggested that endolymph in the saccule was merely derived from cochlear endolymph. We developed and used a preparation of isolated mouse saccule to measure transepithelial currents from the extramacular epithelium with a current density probe. The direction and pharmacology of transepithelial current was consistent with Na(+) absorption by the epithelial Na(+) channel (ENaC) and was blocked by the ENaC-specific inhibitors benzamil and amiloride. Involvement of Na(+),K(+)-ATPase and K(+) channels was demonstrated by reduction of the current by ouabain and the K(+) channel blockers Ba(2+), XE991, and 4-AP. Glucocorticoids upregulated the current via glucocorticoid receptors. Dexamethasone stimulated the current after 24 h and the stimulation was blocked by mifepristone but not spironolactone. No acute response was observed to elevated cAMP in the presence of amiloride nor to bumetanide, a blocker of Na(+),K(+),2Cl(-) cotransporter. The results are consistent with a canonical model of corticosteroid-regulated Na(+) absorption that includes entry of luminal Na(+) through apical membrane Na(+) channels and active basolateral exit of Na(+) via a Na(+) pump, with recycling of K(+) at the basolateral membrane via K(+)-permeable channels. These observations provide our first understanding of the active role played by saccular epithelium in the local regulation of the [Na(+)] of endolymph for maintenance of our sense of balance.

Figure 1.

Structure of the inner ear. The inner ear comprises the endolymphatic space and perilymphatic spaces, which are filled with endolymph ([K⁺] = ∼150 mm; [Na⁺] = ∼1–10 mm) and perilymph ([K⁺] = ∼5 mm; [Na⁺] = ∼150 mm), respectively. The saccule (S) is situated in the endolymphatic system between the base of the cochlea and the rest of the vestibular labyrinth [utricle (Ut), ampulla (A), and semicircular canals (SCC)]. ES, Endolymphatic sac; ED, endolymphatic duct; OW, oval window; RW, round window; ME, middle ear. Endolymph composition of the saccule was previously thought to be completely derived from cochlear endolymph by longitudinal diffusion; the present study demonstrates that at least Na⁺ composition of the saccule is regulated locally by absorptive transport. The drawing was adapted with permission from Lim (2002) (their Fig. 2–4).

Figure 2.

Preparation of saccular extramacular membrane for the measurement of current density. A, Separated membranous labyrinth of vestibule. The saccule is separated with microscissors from the utricle (red dotted line). B, Isolated saccule and separation of extramacular membrane from saccular macula. The extramacular membrane is separated along red dotted line with one side attached to the longest edge of the macula. C, Tissue folding. The tissue is folded along the yellow dotted line with the apical side of the epithelium facing out. D, Prepared tissue before mounting on stage of microscope. E, Photo of mounted tissue for current measurement. S, Saccule; Am, ampulla; Ut, utricle; CC, common crus; SCC, semicircular canal; *otoconia of saccule; VP, vibrating probe.

Figure 3.

Effect of Na⁺ transport inhibitors on current from saccular extramacular epithelium. A, Amiloride (n = 5); bars show duration of perfusion at concentrations (molar) expressed as the negative log of the number in the bar. B, Benzamil (n = 5). C, EIPA (n = 5). D, Concentration–response relationships. E, pH 6, 5, and 4 and 1 μm amiloride (n = 4); bars show the duration of perfusion at the pH values in the bar. Amiloride (Amil; 1 μm) was perfused at pH 7.4. Graphs in A–C and E are derived from averages at each time point at 500 ms intervals of all experiments; the SE bars are drawn only at larger intervals for clarity.

Figure 4.

Effect of stimulators of cAMP-dependent chloride transport on current from saccular extramacular epithelium. Recording is digital averages of all experiments; SE is drawn only at intervals for clarity; n = 5. The following concentrations were used: cAMP (bromo-cAMP), 500 μm; forskolin (Forsk), 10 μm; IBMX, 125 μm.

Figure 5.

Effect of Na⁺,K⁺-ATPase and Na⁺,K⁺,2Cl⁻ cotransporter inhibitors on current from saccular extramacular epithelium. Current is shown in the absence and presence of ouabain (A) and bumetanide (Bumet) (B). Recordings are digital averages of all experiments; SE is drawn only at intervals for clarity; n = 8, each.

Figure 6.

Effect of K⁺ channel blockers on current from saccular extramacular epithelium. Current changes after perfusion of Ba²⁺ (A; n = 10), 4-aminopyridine (4-AP) (B; n = 6), XE991 (C; n = 11), tetra-ethyl-ammonium (TEA; 30 mm), and NMDG (30 mm) (D; n = 5) are shown.

Figure 7.

Dexamethasone acts at GR to regulate current density in saccular extramacular epithelial cell. A, Test for nongenomic (acute) effect of dexamethasone (Dex; 100 nm). B, Changes of current density under the conditions of control, dexamethasone (Dex; 100 nm), dexamethasone + GR antagonist mifepristone (Mif; 100 nm), and dexamethasone + MR antagonist spironolactone (Spi; 100 nm). Values are means ± SE; n = 5. NS, Not significant, dexamethasone versus dexamethasone + spironolactone. *p < 0.05, control versus dexamethasone, dexamethasone versus dexamethasone + mifepristone, and before amiloride versus during amiloride (1 μm) in all conditions.

Figure 8.

Model of Na⁺ absorption by saccule extramacular epithelium. The saccule is bounded by an epithelial monolayer with one tubular connection that bifurcates to the endolymphatic sac (es) and utricle (u) and another tubular connection to the cochlea via the ductus reuniens (dr). Na⁺ moves into and out of the saccule by at least four routes (red arrows). 1, Na⁺ is removed from the lumen by active transepithelial transport as illustrated in the expanded cell (upper left). 2, Na⁺ diffuses in from the utricle and sac, which have [Na⁺] higher than the saccule. 3, Na⁺ diffuses into the saccule from the cochlea, which has a slightly lower [Na⁺] than the saccule, but also a large positive voltage with respect to the saccule. The net electrochemical driving force on Na⁺ is in the direction cochlea to saccule, but the magnitude of the flux is expected to be small due to the low [Na⁺]. 4, Na⁺ diffuses between the epithelial cells from the perilymph to the saccular endolymph. Na⁺ from endolymph moves into the cells through Na⁺-permeable channels in the apical membrane. This pathway has the pharmacological fingerprint of ENaC, but may include other channels. Na⁺ exits the cell at the basolateral membrane by active extrusion via the Na⁺-pump, Na⁺,K⁺-ATPase. K⁺ brought into the cell on the Na⁺-pump leaves the cell across the same membrane by electrodiffusion via K⁺-permeable channels. The rate of absorption is stimulated by activation of glucocorticoid receptors (GR). Candidate isoforms of the transporters are discussed in the text.