Chloride secretion by semicircular canal duct epithelium is
stimulated via
2-adrenergic receptors
Pierre G.
Milhaud1,
Satyanarayana R.
Pondugula2,
Jun Ho
Lee2,
Michael
Herzog2,
Jacques
Lehouelleur1,
Philine
Wangemann2,
Alain
Sans1, and
Daniel C.
Marcus2
1 Institut National de la Santé et de la
Recherche Médicale Unité 432 Vestibular Neurobiology,
Université Montpellier II, 34095 Montpellier,
France; and 2 Department of Anatomy and
Physiology, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
The ductal
epithelium of the semicircular canal forms much of the boundary between
the K+-rich luminal fluid and the Na+-rich
abluminal fluid. We sought to determine whether the net ion flux
producing the apical-to-basal short-circuit current
(Isc) in primary cultures was due to anion
secretion and/or cation absorption and under control of receptor
agonists. Net fluxes of 22Na, 86Rb, and
36Cl demonstrated a basal-to-apical Cl
secretion that was stimulated by isoproterenol. Isoproterenol and
norepinephrine increased Isc with an
EC50 of 3 and 15 nM, respectively, and isoproterenol
increased tissue cAMP of native canals with an EC50 of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had no
effect on Isc. Isoproterenol stimulation of
Isc and cAMP was inhibited by ICI-118551
(IC50 = 6 µM for Isc) but not
by CGP-20712A (1 µM) in primary cultures, and similar results were
found in native epithelium. Isc was partially inhibited by basolateral Ba2+ (IC50 = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, and
DIDS did not fully fit the profile for CFTR. Our findings show that the
canal epithelium contributes to endolymph homeostasis by secretion of
Cl
under
2-adrenergic control with cAMP as
second messenger, a process that parallels the adrenergic control of
K+ secretion by vestibular dark cells. The current work
points to one possible etiology of endolymphatic hydrops in Meniere's
disease and may provide a basis for intervention.
anion secretion; vestibular labyrinth; receptors; endolymph
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INTRODUCTION |
THE LUMEN OF THE VESTIBULAR
LABYRINTH is filled with endolymph, a fluid with a high
concentration of K+ (149 mM) and a low concentration of
Na+ (9 mM) (36). This composition is necessary
to support the transduction of acceleration by the vestibular sensory
cells into nerve signals to the brain. The epithelium forming the
boundary of the endolymphatic compartment is composed of many
epithelial cell types, including the neuroepithelial sensory hair
cells. Vestibular dark cells are known to be responsible for
K+ secretion (19) under adrenergic control
(31, 34), and transitional cells are known to be
responsible for cation reabsorption (15).
Net cation movements cannot occur in isolation and must be balanced by
transport of anions to maintain bulk electroneutrality. The
transcellular and/or paracellular routes of Cl
movements
in the inner ear have not previously been determined. It was of
interest to determine whether the canal ducts provide this function,
because a polarized primary culture of epithelial cells of the
semicircular canal duct from neonatal rats was recently developed that
produced an apical-negative transepithelial voltage (VT) and associated apical-to-basal
short-circuit current (Isc) (21).
This Isc could be due to anion secretion and/or
cation absorption.
The goals of the present study were to determine whether the
semicircular canal duct epithelium engages in anion secretion and/or
cation absorption, whether it is under adrenergic control, and whether
the primary culture has a phenotype that represents the native tissue.
Dysfunction of transport and its regulation by this epithelium may be
one basis of pathological states such as Meniere's disease.
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METHODS |
Temporal bones were removed after decapitation from neonatal
Wistar rats (3-5 days after birth) and adult gerbils (4- to
5-wk-old females), and the semicircular canal ducts were dissected from the vestibular labyrinth. Gerbils were anesthetized before euthanasia by injection of pentobarbital sodium (50 mg/kg, ip). All procedures conformed to protocols approved by the Institutional Animal Care and
Use Committees. Canals were dissected and prepared for primary culture,
transferred to a perfusion chamber on the stage of an inverted
microscope (Nikon TE-300) for measurement of Isc
density, or used for measurement of cAMP accumulation.
Epithelial cultures.
Cells from neonatal rat semicircular canal epithelium, exclusive of the
common crus, were dispersed and seeded on permeable culture dish
inserts and cultured as described previously (21). Cells
were seeded at a density of 5-18 canals/cm2 on inserts
with 0.4-µm pores in 15-µm-thick polyester membrane (1.6 × 106 pores/cm2). The inserts were either 6.5 (Transwell; Costar, Cambridge, MA) or 12 mm in diameter (Snapwell; Costar).
Confluent monolayers of primary cultures were mounted in an Ussing
chamber (catalog no. AH 66-0001; Harvard Apparatus, Holliston, MA)
maintained at 37°C. For most experiments, both sides of the epithelium were bathed in bicarbonate-buffered physiological saline, which was stirred by bubbling with a mixture of 95% O2 and
5% CO2. The composition of the solution was (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 5 glucose, pH 7.4. A HEPES-buffered solution
bubbled with air was used for the Ba2+ experimental series
to avoid potential problems with Ba2+ precipitation; its
composition was (in mM) 150 NaCl, 10 Na-HEPES, 3.6 KCl, 1 MgCl2, 0.7 CaCl2, and 5 glucose, pH 7.4. The
HEPES-buffered solution did not alter the response of
Isc to forskolin.
Experimental agents were added to the bath as 1,000× concentrates.
Histamine (catalog no. H-7375, Sigma, St. Louis, MO), vasopressin (catalog no. V-9879, Sigma), (
)-isoproterenol (catalog no. I-6504, Sigma), (
)-norepinephrine (catalog no. A-9512, Sigma), CGP-20712A (catalog no. C-231, Sigma), and ICI-118551 (catalog no. I-127, Sigma)
were dissolved in H2O, whereas forskolin (catalog no.
F-6886; Sigma), ouabain (catalog no. O-3125, Sigma), glibenclamide
(catalog no. G-0639, Sigma),
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; catalog no.
D-3514, Sigma), and 5'-(N-ethylcarboxamido)adenosine (NECA;
catalog no. E-2387, Sigma) were dissolved in dimethyl sulfoxide (DMSO).
DMSO never exceeded 0.6% final concentration.
Fluxes of Cl
, Na+,
and Rb+
(K+).
The following isotopes were used for measuring transepithelial ionic
fluxes from cultured neonatal rat semicircular canal epithelium:
22Na for sodium was used at 84 Bq/µl, 36Cl
for chloride was used at 38 Bq/µl, and 86Rb for potassium
was used at 260 Bq/µl. We assumed that the tracers moved in the same
ways as nonradioactive Na+, Cl
, and
K+. Cultures grown on 12-mm inserts were used for flux
measurements. Electrodes connecting the voltage-current clamp to the
Ussing chamber consisted of Ag-AgCl connected to the bath solutions via a bridge of 1 M KCl and 2% agarose (catalog no. Fluka 05066, Sigma).
The experimental protocol consisted of a 20-min initial period during
which VT reached a steady state. The
radioisotope was added to the apical or basal compartment, and the
epithelium was voltage clamped to zero and allowed to reach a steady
state for >20 min. Three samples of 100 µl were collected from each
compartment at this time and again 20 min later. Isoproterenol (10 µM) was added to the basal compartment, a steady-state current was
reached after 5-10 min, and three samples of 100 µl were again
collected from each compartment at this time and 20 min later. After
each withdrawal, fresh buffer was added to maintain a constant volume. Samplings were accompanied by measurement of current and open-circuit voltage.
36Cl and 86Rb were counted in 2 ml of liquid
scintillation fluid (Aquasafe 500 plus, Zinsser Analytic, Frankfurt,
Germany) for 5 min per sample. 22Na was counted in a gamma
counter for 10 min per sample, up to eight times because of high
background.
where C, expressed in counts per minute (cpm), is the quantity
of isotope arriving into the cold (unlabeled) compartment. C is
corrected for background and dilution due to samplings and refillings.
[B] (in µmol/ml) is the total concentration of ion under study.
S (in cm2) is the surface area of the
epithelium. T (in min) is the duration of the flux
measurement. [R] (in cpm/ml) is the concentration of radioactivity in
the hot compartment.
Net fluxes were obtained by subtraction of the mean
apical-to-basal flux from the mean basal-to-apical flux.
Electrophysiological recordings.
VT, Isc, and resistance
(RT) were measured from cultured neonatal rat
canal with an epithelial voltage-current clamp amplifier (model VCC600,
Physiologic Instruments, San Diego, CA; or model DVC 1000p, World
Precision Instruments, Sarasota, FL). VT and RT were measured during current clamp, and the
equivalent Isc was calculated from
Isc = VT/RT. During flux
measurements, the epithelium was voltage-clamped to zero and
Isc was measured directly.
cAMP-assay.
Native canal ducts from neonatal rats were divided into several
approximately equal-sized samples. Samples were transferred into a NaCl
solution containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) and equilibrated for 6 min with
agitation at 37°C. Subsequently, samples were incubated for 12 min at
37°C with 0.1 nM-1 µM isoproterenol, 1-100 nM isoproterenol in
the presence of 1 µM CGP-20712A, or 10 nM-1 µM isoproterenol in the
presence of 10 µM ICI-118551, respectively. One sample of the canals
was not stimulated, serving as a control. The reaction was stopped by
addition of a lysis reagent containing 2.5% dodecyltrimethylammonium bromide and disruption of the tissue by sonication for 30 min at 4°C.
Tissue fragments were removed by centrifugation, and cAMP was measured
in the supernatant with a colorimetric immunoassay according to the
manufacturer's protocol (RPN 225, Amersham, Piscataway, NJ). The
sensitivity of the assay ranged from 12.5 to 3,200 fmol cAMP per well.
Results were normalized to the cAMP production induced by 1 µM isoproterenol.
Voltage-sensitive vibrating probe.
The vibrating probe technique was identical to that previously
described (15). Briefly, the current density (proportional to the Isc) was monitored from neonatal rat or
adult gerbil semicircular canal ducts by vibrating (200-400 Hz) a
Pt-Ir wire microelectrode with a Pt-black tip positioned 20-30
µm from the apical surface of the epithelium with
computer-controlled, stepper-motor manipulators (Applicable
Electronics, Forest Dale, MA) and probe software (ASET version 1.05, Science Wares, East Falmouth, MA). The bath references were 26-gauge
Pt-black electrodes. The signals from the phase-sensitive detectors
were digitized (0.5 Hz, 16 bit), and the output was expressed as
current density at the electrode. In this series of experiments, the
HEPES-buffered solution was used. The solution in the chamber was
exchanged 0.6 times per second and maintained at 37°C.
Pharmacology.
EC50 and KDB values were calculated
as described previously (26, 33, 34). The agonist
concentration that caused a half-maximal effect (EC50) was
obtained by fitting data to the Hill equation: E = Emax × Ch/(EC50h + Ch), where Emax is the maximal
effect, C is the concentration of the agonist, and h defines
the slope. The affinity of the antagonists to the receptor
(KDB) was obtained from cumulative dose-response curves in the absence and presence of antagonist.
KDB was obtained from the Schild equation:
p(KDB) = log (y)
log
(DR
1), where y is the concentration of the
antagonist and DR is the dose ratio. The DR was obtained according to
DR = EC50 antagonist/EC50 agonist, where
"EC50 antagonist" is the EC50 of
isoproterenol in the presence of antagonist and "EC50
agonist" is the EC50 in the absence of the antagonist.
All nonlinear curve fits were obtained by a least-squares algorithm
using a programmable spreadsheet and plotting software (Origin 6.1, OriginLab, Northampton, MA). The
1-,
2-,
and
3-adrenergic receptor subtypes can be distinguished
by the relative affinity of the antagonists ICI-118551 and CGP-20712A
(27, 33, 34).
Statistical analysis.
The Student's t-test was used to determine statistical
significance of paired samples. Variance homogeneity was verified with Fisher's or Bartlett's test before computing unpaired Student's t-test or ANOVA, respectively, for ion flux data
(30). A logarithmic transformation of data or the Aspin
Welch test (a modified Student's unpaired t-test) was used
when the variances were significantly different (30). Data
are expressed as means ± SE (n = no. of tissues).
Dose-response curves of agonists were normalized to the response to 10 µM forskolin or 10 µM isoproterenol. Increases or decreases were
considered significant for P < 0.05.
 |
RESULTS |
Confluent primary cultures of neonatal rat canal epithelium.
The previously-found apical-side negative VT of
primary cultured epithelium from the semicircular canal ducts was
hypothesized to be due to Cl
secretion and/or
Na+ absorption. Preliminary experiments showed that the
responses to apical addition of the Na+ transport
inhibitors amiloride and ethylisopropyl amiloride (EIPA) were not
significant (data not shown). However, several
Cl
-secreting epithelia are known to be stimulated by
-adrenergic receptor activation (2, 6, 16, 23, 24).
Net fluxes of Cl
,
Na+, and
Rb+ across cultured neonatal rat canals.
To determine the ionic basis of electrogenic transport by this
epithelium, we measured unidirectional fluxes of Cl
,
Na+, and Rb+ (for K+) and
calculated the net fluxes. Inserts of high RT
(
5 k
-cm2) were selected to minimize the background of
passive paracellular fluxes. Net fluxes were also measured across
epithelia stimulated by the
-adrenergic receptor agonist isoproterenol.
In the absence of isoproterenol, a net Cl
secretion was
observed, but no net absorption of Na+ (Table
1). All of the Isc
could be accounted for by the net Cl
flux, because the
difference was not significantly different from zero. A small net
basolateral-to-apical Rb+ (K+) flux was seen
that amounted to only ~5% of the net Cl
flux. This
Rb+ flux was not due to the presence of
K+-secreting dark cells in the cultured epithelium because
cells from the common crus were assiduously excluded from the present series of experiments. We functionally tested for the presence of dark
cells by addition of DIDS (500 µM) to the apical bath and found no
response of VT (data not shown and Fig. 5). DIDS strongly increases the positive VT across dark
cells (29).
Addition of isoproterenol (10 µM) to the basolateral compartment led
to a strong increase in the net Cl
secretory flux but no
change in either the Na+ or Rb (K+) net fluxes
(Table 1). All of the Isc could be accounted for by the net Cl
flux (Fig. 1,
Table 1). Isc increased significantly and
RT decreased significantly after addition of
isoproterenol.

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Fig. 1.
Cl fluxes under stimulation by
isoproterenol. Open bars represent unidirectional 36Cl
tracer fluxes during stimulation with isoproterenol (10 µM). Hatched
bars represent net flux expressed in current and short-circuit current
(Isc), which are not significantly different;
net flux is significantly >0. BA, basolateral-apical flux; AB,
apical-basolateral flux. * P < 0.05; ns, not
significant.
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Increase in Isc by stimulation of
2-adrenergic receptors.
The synthetic and natural agonists for
-adrenergic receptors,
isoproterenol and norepinephrine, increased the magnitude of Isc of cultured neonatal rat canals with an
EC50 of 3 nM (pEC50 = 8.6 ± 0.1, n = 15) and 15 nM (pEC50 = 7.8 ± 0.3, n = 12; P < 0.05), respectively,
on the basal side (Figs. 2B
and 3, Table 2). Isoproterenol had no effect
when added to the apical solution (not shown).

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Fig. 2.
Representative recordings of transepithelial voltage
(VT) from primary cultures of semicircular canal
on permeable supports. A: response to forskolin
(10 5 M) on the basolateral side. B: response
to increasing concentrations of isoproterenol
(10 10-10 6 M) and to forskolin (FSK;
10 5 M) on the basolateral side. C: absence of
response to vasopressin (VP; 10 8 M) and histamine (Hist;
10 4 M). Pulses are the responses of
VT to current pulses (1 µA, 0.3-s duration,
repeated every 10 s).
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Fig. 3.
Concentration-response curves of the
Isc from primary cultured epithelia induced by
the adrenergic agonists isoproterenol and norepinephrine.
Isoproterenol: EC50 = 2.75 nM, Emax = 93%, and h = 0.72; norepinephrine:
EC50 = 15.0 nM, Emax = 87%, and
h = 0.56, where Emax is the maximal effect
and h represents the slope.
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Table 2.
Electrophysiological response to isoproterenol of primary cultures of
semicircular canal duct epithelium
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-Adrenergic receptors are usually linked to adenylyl cyclase via the
heterotrimeric G protein of the Gs type. Activation of
adenylyl cyclase by forskolin (10 µM) caused a substantial increase
in Isc from 1.0 ± 0.3 to 2.4 ± 0.3 µA/cm2 (n = 20), although
RT in this experimental series did not change between control and forskolin conditions (1.8 ± 0.2 vs. 1.8 ± 0.2 k
· cm2) (Fig.
2A). Inserts were not selected for high resistance in this
series of experiments. At full stimulation with either isoproterenol or
norepinephrine, there was no further change in
VT or Isc with addition
of forskolin (Fig. 2B).
The
-adrenergic receptor antagonist CGP-20712A (1 µM) had no
effect (1.9 ± 1.3%, n = 10) after stimulation by
isoproterenol (100 nM), whereas the antagonist ICI-118551 inhibited
Isc with an IC50 of 6 ± 2 µM
(n = 15) and a KDB of 0.20 ± 0.06 µM, indicating that ion transport by this epithelium was
stimulated via
2-adrenergic receptors (Fig.
4; see DISCUSSION).

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Fig. 4.
Specific inhibition of the current from cultured
epithelia stimulated by -adrenergic receptors. The current
stimulated by isoproterenol (0.1 µM) was inhibited by CGP-20712A or
ICI-118551, specific antagonists of 1- or
2-adrenergic receptors, respectively.
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We tested agonists for several other Gs-linked receptors:
histamine for histamine receptors, arginine vasopressin for vasopressin receptors, and NECA for adenosine receptors. The agonists were added at
high concentration to the basolateral compartment of the epithelium,
and VT and RT were
continuously recorded. No significant changes in
VT or RT were observed
for histamine (10
4 M) (3) and vasopressin
(10
8 M) (34) (Fig. 2C) or for
NECA (10
5 M) (22).
Pharmacological test for apical CFTR.
Cl
secretion across the apical membrane in many epithelia
is mediated by the CFTR Cl
channel (28).
Although an unequivocal pharmacological criterion for the presence of
functional CFTR has not been developed, it is widely accepted that
stimulation of secretory current by apical genistein (30 µM),
inhibition by glibenclamide (50-300 µM), and no effect of the
broad-spectrum anion transport inhibitor DIDS (500 µM) indicate
mediation of the current by CFTR (1, 28).
Genistein (30 µM) significantly increased Isc
in primary cultures of neonatal rat canal epithelium in the absence of
forskolin and in the presence of submaximal (1 µM) forskolin (Fig. 5,
A and B),
consistent with CFTR. However, there was no effect of apical genistein
following stimulation of Isc with a higher
concentration of forskolin (10 µM) (Fig. 5C), and,
importantly, apical glibenclamide (300 µM) as well as DIDS (500 µM)
had no effect on Isc (Fig. 5, A and
C).

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Fig. 5.
Stimulation of Isc by genistein at
submaximal forskolin concentrations and lack of effect of glibenclamide
and DIDS. A: representative recording of
VT during addition of apical amiloride (A; 10 µM), apical genistein (G; 30 µM), basolateral forskolin (FSK; 10 µM), apical glibenclamide (Gb; 300 µM), and apical DIDS (D; 500 µM). B, left: summary of experiments showing stimulation
by genistein [after control conditions (C) and block of residual
Na+ absorption by apical amiloride (10 µM)] and further
stimulation by forskolin (F10; 10 µM) (n = 6);
right: stimulation by genistein (30 µM) after submaximal
forskolin (F1/G; 1 µM) (n = 5). C, left:
no further stimulation by genistein (30 µM) after forskolin (F10; 10 µM) (n = 6); right: no inhibition of
stimulated Isc by either glibenclamide (Gl; 300 µM) or DIDS (500 µM) (n = 5).
* P < 0.05.
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Decrease in Isc by blockers of
K+ channels and
Na+-K+-ATPase.
Basolateral addition of Ba2+ decreased the magnitude of
Isc of forskolin-stimulated cultured neonatal
rat canals with an IC50 of 0.27 mM (n = 3-6) (Fig. 6, A and
C).
Isc was reduced 55 ± 4% (n = 6) by 1 mM Ba2+. Basolateral
ouabain (1 mM) decreased the magnitude of Isc by 30 ± 3% (n = 4) within 5 min (Fig. 6,
B and D). Preliminary results showed no effect of
either Ba2+ (1 mM) or ouabain (1 mM) on
Isc from the apical side. These findings are
consistent with the presence of K+ channels and the
Na+-K+-ATPase in the basolateral membrane of
these cells. The basis for the incomplete inhibition of
Isc by ouabain is not clear, but it could be due
to submaximal concentration, the presence of other ion pumps, or a
slower secondary phase of rundown.

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Fig. 6.
Inhibition of basolateral K+ channels and
Na+-K+-ATPase by barium and ouabain decreases
Isc. A: representative recording of
response of Isc to basolateral forskolin (10 µM) and basolateral barium (Ba; 1 µM). B: representative
recording of response of Isc to basolateral
forskolin (10 µM) and basolateral ouabain (1 mM). C:
summary concentration-response of Isc to barium
(n = 3-6) after stimulation by forskolin; initial
value after forskolin is 1.19 ± 0.12 µA/cm2
(n = 6); Hill plot with
Vmax = 0.68, Hill coefficient = 2.2, and IC50 = 0.27 mM. D: summary of response
of Isc to ouabain. Summary data are means ± SE. * P < 0.05.
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Native tissue.
The native tissue was used 1) to determine whether the
primary cultures had the same phenotype as the original tissue with respect to the adrenergic receptor and cAMP signal pathway and 2) to demonstrate that the assumed increase in cAMP during
exposure to agonists of the receptor or adenylyl cyclase did indeed
occur. The results showed that native canals had the same responses as the primary cultured epithelium.
The vibrating probe was used to measure current generated by the native
epithelium in neonatal rats and adult gerbils. The probe detected a
negative current (toward the epithelium) when the probe tip was
positioned near the apical cell surface of neonatal rat canals (Fig.
7A) and a positive current
(away from the epithelium) when the probe tip was positioned near the
basolateral cell surface of adult gerbil canals (Fig. 7B).

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Fig. 7.
Representative recordings of Isc
in native semicircular canals stimulated by increase in cAMP; current
density was recorded by vibrating probe (VP; see micrographs in
insets). A: Isc in
neonatal canals was reversibly stimulated by isoproterenol (ISO; 100 nM) and inhibited by ICI-118551 (ICI; 10 µM) but not by CGP-20712A
(CGP; 10 µM); forskolin (10 µM) was also reversibly active.
B: Isc in adult gerbil canals was
stimulated by cAMP via forskolin (10 µM) and isoproterenol (10 µM).
SCC, short-circuit current.
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The current from neonatal rat canals (n = 3) was
stimulated by isoproterenol (100 nM) and forskolin (10 µM), and the
isoproterenol-stimulated current was inhibited by ICI-118551 (10 µM)
but not CGP-20712A (10 µM) (Fig. 7A). Similarly, the
current from adult gerbil canals (n = 6) was stimulated
by isoproterenol (10 µM) and forskolin (10 µM) (Fig.
7B).
Isoproterenol caused a dose-dependent stimulation of cAMP production in
isolated native neonatal rat canals (Fig.
8). The EC50 for
isoproterenol-induced cAMP production was 5 nM (pEC50 = 8.3 ± 0.4, n = 7). The presence of 1 µM
CGP-20712A had no effect on the EC50 (3 nM,
pEC50 = 8.5 ± 0.8, n = 7). In
the presence of 10 µM ICI-118551, the dose-response curve was shifted
to the right and had an EC50 of ~10 µM. The data
provide evidence for the presence of
2- but not
1-receptors in semicircular canals of neonatal rats.

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Fig. 8.
Stimulation of cAMP by isoproterenol. The
concentration-response curve for cAMP stimulated by isoproterenol in
native neonatal rat canals was shifted to the right by ICI-118551 but
was not significantly affected by CGP-20712A.
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DISCUSSION |
The vestibular labyrinth is comprised of the sensory hair
cells and many other epithelial cell types including transitional cells, dark cells, several "wall" cells, and the cells of the semicircular canal ducts. Sensory hair cell function depends on maintenance of endolymph ion composition and volume, which is the
function of the other epithelial cells of the vestibular labyrinth. The
contributions of vestibular dark cells (32) and
transitional cells (15, 35) have been investigated in much
detail, whereas relatively little is understood about canal duct
function. Our findings show for the first time that the semicircular
canal duct epithelium is capable of contributing to endolymph
homeostasis by secretion of Cl
, which complements the
secretion of K+ by vestibular dark cells. Both secretion of
K+ and of Cl
are controlled by
-adrenergic
receptors, leading to maintenance of bulk electroneutrality. Our
results also validate the primary culture model of semicircular canal
duct by demonstrating the homology of the salient results between the
cultured and native epithelia.
Anion transport.
The major anions in fluids of the inner ear are Cl
and
HCO
(36). It is likely that
HCO
secretion by semicircular canal duct epithelium
is small because 36Cl
flux accounted for the
basal and isoproterenol-stimulated Isc in the
presence of HCO
. Very recent evidence points to the
participation of vestibular transitional cells and cochlear outer
sulcus cells in the secretion of HCO
via an apical
pendrin transporter (T. Wu, E. White, P. Wangemann, and D.C. Marcus,
unpublished observations).
Ion transport by a variety of epithelia is controlled by
-adrenergic
receptors (4, 11, 24, 34). The classic view is that
stimulation of these receptors leads to an increase of intracellular
cAMP through coupling to heterotrimeric G proteins of the
Gs type and subsequent activation of adenylyl cyclase. Three
-adrenergic receptor subtypes (
1,
2, and
3) have been identified
(37), and the subtypes can be distinguished by the affinity of the antagonists ICI-118551 and CGP-20712A
(33). The Cl
secretion by semicircular canal
duct epithelium is clearly regulated by the
2-adrenergic
receptor acting via elevation of cAMP. The Isc
and cAMP level of canal epithelium from neonatal rats were stimulated
by agonists of
-adrenergic receptors. The affinity for ICI-118551 of
the receptor in the canal epithelium is distinctly greater than for
CGP-20712A, a constellation fitting only that for the
2-adrenergic receptor and not
1 or
3 (27, 33, 34).
Furthermore, this signal pathway is not restricted to early development
because isoproterenol and forskolin stimulated
Isc in adult canals. The finding that addition
of forskolin after maximal stimulation by isoproterenol had no
additional effect on Isc suggests that adenylyl
cyclase is mainly linked to
-adrenergic receptors rather than to
multiple receptors. This signal pathway is likely functional in vivo
and may be stimulated by agonists in the serum, because measured
concentrations of norepinephrine in human (14) and rat
(8) serum are in the nanomolar range (Fig. 3).
Cl
transport by several epithelia has been shown to be
under control of a cAMP signal pathway. Transporter proteins whose activities are modified by cAMP include Cl
channels
(10, 28), anion exchanger (25), and
Na+-K+-2Cl
cotransporter
(13). The constellation of transporters in semicircular canal duct epithelium that accounts for the observed Cl
secretion remains to be determined. The decrease in
RT during isoproterenol stimulation in the
radioisotope flux series is consistent with the activation of an apical
Cl
channel, such as CFTR.
Experiments were performed to test for electrophysiological responses
to genistein, glibenclamide, and DIDS; these agents are generally
accepted as pharmacologically defining the presence of functional CFTR
(1, 28). We found that the transepithelial current in the
cultured canal epithelium did not fully fit this profile, suggesting
that Cl
secretion may be carried by another
cAMP-dependent pathway.
Our current understanding of Cl
transport by the
semicircular canal duct epithelium is illustrated in Fig.
9. K+ is taken up into the
cell across the basolateral membrane by the
Na+-K+-ATPase, and the resulting high
intracellular K+ concentration is expected to develop a
negative basolateral membrane voltage via the
Ba2+-sensitive basolateral K+ channels. Because
the transepithelial voltage in the vestibular labyrinth is within a few
millivolts of zero (17), the apical membrane voltage would
also be negative and provide an electrical driving force for the exit
of Cl
into the lumen. This secretory pathway in the
apical membrane does not fully fit the pharmacological profile of CFTR.
Cl
secretion in this epithelium is regulated by cAMP via
2-adrenergic receptors. The molecular basis of this
control is not yet known.

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|
Fig. 9.
Cell model of Cl transport by semicircular
canal duct epithelium. Cl is taken up across the
basolateral membrane by an unidentified mechanism. A
Na+-K+-ATPase and K+ conductance in
the basolateral membrane creates a negative cell voltage that drives
Cl exit across the apical membrane. Cl
secretion is stimulated by activation of a basolateral
2-adrenergic receptor coupled to a cAMP second messenger
pathway. The apical pathway is responsive to genistein but not to
glibenclamide and is therefore only partially consistent with the
profile for the CFTR Cl channel.
|
|
Cation transport.
Previous investigations of the physiological function of the
semicircular canal ducts have focused on transport of monovalent cations, because K+ and Na+ concentrations are
maintained farthest from equilibrium between endolymph and
perilymph (36). Cellular mechanisms of K+
secretion have been well characterized in vestibular dark cells of the
utricle and ampulla (18-20). Na+ was
shown to be absorbed by the frog ampulla, and the absorptive flux was
partially inhibited by amiloride (9). More recently, it
was shown that mammalian transitional cells of the ampulla are
responsible for Na+ absorption and that this occurs through
amiloride-sensitive nonselective cation channels in the apical cell
membrane (7, 15).
We found a small K+ secretion by semicircular canal ducts,
but there was no evidence for Na+ absorption. The
relatively small flux of K+ under basal conditions and the
absence of K+ flux in the presence of isoproterenol suggest
that K+ is of little or no physiological significance. The
previous report of a small K+ secretion by this epithelium
(21) may have been the result of a minor presence of dark
cells in the culture from inadvertent inclusion of parts of the common
crus. The common crus is the confluence of the anterior and posterior
canal ducts that is partially composed of dark cells (12).
Cells from the common crus were assiduously excluded from the present
series of experiments, and a functional test for dark cells with DIDS
confirmed their absence. Furthermore, isoproterenol would have caused
an increase in K+ secretion (31, 34), contrary
to our observations.
Physiological significance.
The present study demonstrated for the first time that semicircular
canal duct epithelium contributes to the homeostasis of vestibular
endolymph by secretion of Cl
under adrenergic regulation.
K+ secretion by vestibular dark cells has recently been
shown to be stimulated by
1-adrenergic receptors and by
downstream events in the signal pathway, including activation of
adenylyl cyclase and increase of intracellular cAMP levels (31,
34).
The vestibular labyrinth, therefore, has the means to control
vestibular endolymph composition not only by cation secretion (dark
cells) and absorption (transitional cells) but also by the primary
anion, Cl
.
-Adrenergic receptor agonists carried to
both the dark cells and semicircular canal duct cells would increase
secretion of both K+ and Cl
. These two
processes would be physiologically linked, because both cells respond
to a similar range of agonist. Pathological dysfunctions of the
vestibular labyrinth include vertigo associated with endolymphatic
hydrops (Meniere's disease). The possible involvement of adrenergic
receptors in Meniere's disease has been discussed (5,
33). The current work points to one possible etiology of
endolymphatic hydrops in Meniere's disease and may provide a basis for intervention.
 |
ACKNOWLEDGEMENTS |
We thank Prof. M. Rossi for providing P. G. Milhaud with
excellent working conditions in the Department of Nuclear Medicine and
L. Cambon for helpful discussions. We thank Bambi Harlow for excellent
technical assistance. We thank Dr. Robert Bridges for the design
modification of the Harvard/Navicyte Ussing chamber to accommodate
Transwell inserts.
 |
FOOTNOTES |
This work was supported by National Institute on Deafness and Other
Communication Disorders Grants R01-DC-00212 (to D. C. Marcus) and
R01-DC-01098 (to P. Wangemann) and by Centre National d'Etude Spatiale
Grant 793/01/8529/00.
Address for reprint requests and other correspondence:
D. C. Marcus, Dept. of Anatomy and Physiology, Kansas State
Univ., 1600 Denison Ave., Manhattan, KS 66506 (E-mail:
marcus{at}ksu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 28, 2002;10.1152/ajpcell.00283.2002
Received 20 June 2002; accepted in final form 19 August 2002.
 |
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