EDITORIAL FOCUS
Osmotic pressure regulates 

-rENaC expressed in
Xenopus oocytes
Hong-Long
Ji,
Catherine M.
Fuller, and
Dale J.
Benos
Department of Physiology and Biophysics, University of Alabama,
Birmingham, Alabama, 35294-0005
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ABSTRACT |
The hypothesis
that amiloride-sensitive Na+
channels (ENaC) are involved in cell volume regulation was tested.
Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to
superfuse Xenopus oocytes expressing


-rat ENaC (

-rENaC). Whole cell currents were
reversibly dependent on external osmolarity. Under conditions of
swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased
and increased, respectively. In contrast, there was no change in
current amplitude of H2O-injected
oocytes to the above osmotic insults. Currents recorded from


-rENaC-injected oocytes were not sensitive to external
Cl
concentration or to the
K+ channel inhibitor
BaCl2. They were sensitive to
amiloride. The concentration of amiloride necessary to inhibit one-half
of the maximal rENaC current expressed in oocytes
(Ki; apparent
dissociation constant) decreased in swollen cells and increased in
shrunken oocytes. The osmotic pressure-induced
Na+ currents showed properties
similar to those of stretch-activated channels, including inhibition by
Gd3+ and
La3+, and decreased selectivity
for Na+.


-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first
demonstration that 

-rENaC heterologously expressed in
Xenopus oocytes may contribute to
oocyte volume regulation following shrinkage.
rat epithelial sodium channel; mechanosensation; cell volume; amiloride; voltage clamp
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INTRODUCTION |
EPITHELIA ARE ROUTINELY subjected to mechanical
stimuli, most commonly changes in osmotic and hydrostatic pressure.
Besides these, there are special forms of mechanical stress, such as
shear stress of the endothelial cells of the vasculature, peristalsis of the intestine and renal papilla, breathing in the lungs, and air
vibration on the hair cells of the inner ear, that impinge on
epithelia. It is well known that all epithelia are capable of adapting
to variations in osmotic microenvironments by regulating their
absorbing and secreting processes for water and electrolytes. At least
four classes of epithelial channels, namely, volume-sensitive Cl
channels (10, 38),
mechanoactivated K+ channels (6,
24), nonselective cation channels (13, 17), and amiloride-sensitive
Na+ channels (1), have been
described as sensitive to membrane distension or change in cell volume.
The amiloride-sensitive epithelial
Na+ channel (ENaC) is composed of
-,
-, and
-subunits. These ENaC subunits share significant homology to the degenerins of
Caenorhabditis
elegans, which are sensitive to mechanical stimuli. The same apparent membrane topology and structural motifs are found in ENaC and the
mec genes of C. elegans. These similarities suggest that ENaC may
possess inherent mechanosensitive properties. The mechanosensation of
ENaC was first reported for a cloned bovine renal ENaC (
-bENaC) by
incorporating channel proteins into planar lipid bilayers. A
hydrostatic pressure gradient across the bilayer increased channel
activity but produced a concomitant decrease in amiloride affinity and
monovalent cation selectivity (3). Similar observations were made in
planar lipid bilayers containing 

-rat ENaC (

-rENaC)
(14), although data obtained from whole cell patch-clamp studies were
less consistent (26).
Changes in cell volume mediated by changes in medium osmolarity also
place a mechanical stress on membrane proteins akin to what can be
induced by establishment of a hydrostatic pressure gradient across a
planar lipid bilayer. However, studies examining the effects of
osmolarity on Na+ channel activity
have reported variable findings. Activation of a
Na+ conductance has been reported
in hepatocytes (36), in rat fetal distal lung epithelium (23), and in
lymphocytes (1) under hypertonic stress. In contrast,
amiloride-sensitive Na+
conductances were reported to be both suppressed by cell shrinkage in
A6 cells (37) and increased in frog skin (7). Neither the molecular
identity of the channel involved in these responses nor the
intracellular signaling mechanisms involved are known.
The purposes of the present study were to characterize the responses of
a cloned amiloride-sensitive Na+
channel, 

-rENaC heterologously expressed in
Xenopus oocytes, to changes in cell
volume induced by altering external osmolarity. Xenopus oocytes provide an ideal model
system, since they promiscuously translate exogenous cRNAs. ENaC is a
widely distributed ion channel, being present in epithelial and some
endothelial tissues. Furthermore, its relationship to the
mechanosensitive degenerins of C. elegans is suggestive of a role for this channel in the
regulation of cell volume. The results show that expressed


-rENaC in oocytes is sensitive to hypotonicity (70 mosM) and
hypertonicity (450 mosM). Cell swelling decreased rENaC-associated
currents, whereas cell shrinkage increased the activity of rENaC. Under
anisosmotic conditions, the apparent affinity
(Ki) of rENaC
to amiloride and the ionic permeability ratios
(PNa/PX)
were shifted markedly. Measurements of relative oocyte volume in
hyperosmotic solutions by digital imaging were consistent with
recordings of amiloride-sensitive Na+ current changes produced by
hypertonic signals in shrunken rENaC-expressing oocytes, indicating
that expressed rENaC may participate in regulatory volume increase
(RVI) of oocytes.
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MATERIALS AND METHODS |
Expression of


-rENaC.
The vectors containing
-,
-, or
-rENaC subunit inserts were
linearized with Not I, and the insert
was in vitro transcribed with T7 polymerase (Ambion) to prepare cRNA.
The integrity of the cRNA was verified by denaturing gel
electrophoresis through 1% agarose-formaldehyde gels. The methods for
isolation of Xenopus oocytes and
expression of rENaC cRNA were as previously described (16). Briefly,
the ovarian tissue was removed from frogs under hypothermal anesthesia through a small incision in the lower abdomen. Oocytes at maturation stage V and VI were carefully defolliculated by
hand in Ca2+-free OR-2 solution
(in mM: 85.2 NaCl, 2.5 KCl, 10 HEPES, 1.0 Na2HPO4,
and 0.5% streptomycin, pH 7.5), and maintained in L-15 medium
(one-half strength L-15, with 15 mM HEPES, 5% heat-inactivated horse
serum, and 1% antibiotic-antimycotic, pH 7.5; GIBCO) at 18°C.
Oocytes were incubated in L-15 medium overnight before cRNA injection.
A Nanoject (Drummond Scientific, Broomall, PA) microinjector was used
for injection of 1:1:1 

-rENaC cRNA (5 ng/50 nl) or the same
volume of RNase-free water (control).
Electrophysiological recording.
Whole cell currents were sampled from the oocytes at room temperature
24 h after cRNA injection, using pCLAMP version 5.1, and analyzed using
pCLAMP 6.0 software (Axon Instruments). Voltage-clamp potentials were
evoked using a TEA-200 voltage clamp (Dagan) controlled by a personal
computer connected via a TL-1 interface (Axon Instruments). The
injected oocyte was placed in a small chamber (1 ml) and initially perfused with ND-96 medium (in mM: 96 NaCl, 1 MgCl2, 2 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.4),
followed by Cl
-free ND-20
(in mM: 20 sodium gluconate, 1 MgCl2, 2.0 KCl, 1.8 CaCl2, and 5.0 HEPES, pH 7.4) at a
flow rate of 1.5-2 ml/min for at least 5 min before
recording. Microelectrodes filled with 3 M KCl had a
resistance of 0.5-3.0 M
. The bath was clamped by two chlorided
silver wires through 3% agar bridges in 3 M KCl. The oocytes were
clamped at a holding potential of 0 mV. Data were filtered at
0.5-1 kHz, digitized, and stored on hardware for offline analysis.
For step current-voltage
(I-V)
recordings, test voltages were stepped from the holding potential to
100 mV through +100 mV in 20-mV increments for 500 ms. The
currents at
80 mV and +80 mV were monitored at 20-s intervals.
Samples at 200 and 450 ms of each episode were used as the steady-state currents to average data, and the sample at 490 ms was plotted in the
I-V
curve. Linear components of capacitance and leak currents were not
subtracted. The resting membrane potentials
(Vm) were read
directly from the monitor window of the voltage clamp before and after
application of amiloride.
Measurement of oocyte volume.
The methods for digital imaging of oocytes have been described
elsewhere (40). Briefly, osmotic cell volume changes at room temperature (22°C) were monitored with an Olympus phase-contrast microscope equipped with a video camera connected to a Macintosh computer. Oocytes were transferred from 200 to 450 mosM ND-20 media
with or without 10 µM amiloride. Oocytes were equilibrated in
isotonic ND-20 medium for >10 min before being transferred to
anisotonic ND-20 media. An oocyte image was digitized by IP-Lab Spectrum software (Signal Analytics) and stored at 30-s intervals for a
total of 5 min. The imaging at time
zero was taken as control. Imaging data were stored on
a Zip disk (Iomega). The surface area of the sequential images was
measured using the same software, assuming that the oocytes were
spheres without microvilli. The oocyte volume was calculated from
surface area (A) using the formula V = (4/3) × A × (A/
)0.5,
and the relative cell volume
(V/Vo, where
Vo is volume at
time zero and V is volume at time
t) was calculated from the oocyte surface area at time zero
(Ao) and at
time t
(A):
V/Vo = (A/Ao)1.5.
Statistics.
Student's t-test was used to analyze
the differences among groups. The results are presented as means ± SE; n is the number of oocytes. Data
from rENaC-expressing oocytes for quantification of the inhibitory
effect of amiloride were normalized to the maximum current in the
absence of amiloride. Plots of normalized currents as a function of
amiloride were fitted with the following multisite inhibition
equation, using a nonlinear least-squares method:
I = Imax/{1 + ([Amil]/Ki)n},
where I is the steady-state current
for a given amiloride concentration ([Amil]),
Imax is the
current amplitude in the absence of amiloride (normalized to 1.0), and
n is the slope of the fitting curve.
PNa/PX
were computed from a modified Goldman-Hodgkin-Katz equation (12):
PNa/PX = exp [F(Er,Na
Er,X)/RT],
where Er,X and Er,Na are the
reversal potentials for the tested cation (X) and Na+, respectively, and
F, R,
and T are Faraday's constant, the gas constant, and absolute temperature in kelvin, respectively.
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RESULTS |
Basal current of expressed rENaC.
The resting Vm of


-rENaC-expressing oocytes were depolarized to varying degrees,
from
20 to +20 mV, and were generally around 0 mV (
5.5 ± 0.3 mV, n = 20), indicating that
expression of rENaC increased Na+
permeability and depolarized the
Vm. In the
presence of 10 µM amiloride or in
Na+-free superfusate (equimolar
N-methyl-D-glucamine substituted for
Na+), the resting
Vm in oocytes
expressing rENaC were hyperpolarized compared with those of
H2O-injected oocytes
(
38.1 ± 1.6 mV, n = 11). The resting
Vm of
H2O-injected oocytes or uninjected
oocytes was always less than
30 mV (
31.8 ± 0.9 mV,
n = 16). The average current at +80 mV
was 4,545.2 ± 205 nA (n = 9) for


-rENaC-expressing oocytes perfused with ND-96 medium, whereas
that for the water-injected oocytes was 124.2 ± 33.7 nA
(n = 15). Amiloride had an effect on
the whole cell current in rENaC-expressing oocytes that was similar to
that of lower external Na+
concentration (>80% of the current was inhibited in 30 s).
Additionally, 5 mM BaCl2 (a
K+ channel inhibitor) or 200 µM
DIDS (an anion channel inhibitor) had no effect on the current in
oocytes expressing rENaC. The I-V
curve was linear in the range
100 to +100 mV, consistent with
the electrophysiological properties of the native and cloned ENaCs.
Cloned rENaC responds to osmotic pressure.
To inhibit the endogenous, swelling-activated
Cl
channel in
Xenopus oocytes,
Cl
-free hypotonic ND-20 (70 mosM medium; see MATERIALS AND
METHODS) was used in the extracellular solution, and
isotonic ND-20 (200 mosM) or hyperosmotic ND-20 (450 mosM) solutions
were produced by adding mannitol. The
Na+ concentration in each medium
was constant at 20 mM (pH 7.5). Under these experimental conditions,
endogenous swelling-activated Cl
channels were not
observed (2, 39). Basal currents in rENaC-expressing oocytes were
activated by switching the superfusion of isotonic ND-20 to hypertonic
ND-20 medium, whereas the rENaC-induced basal currents were depressed
in swollen oocytes perfused with hypotonic ND-20 (Fig.
1). As shown in Fig.
2, these osmotic effects on rENaC currents
were observed in most oocytes exposed to hypotonic ND-20 (22 of 24 oocytes) or hypertonic ND-20 (18 of 20 oocytes).

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Fig. 1.
Regulation of osmotic pressures on   -rat amiloride-sensitive
epithelial Na+ channels
(  -rENaC) expressed in Xenopus
oocytes. A: 1 representative recording (of 7) demonstrates that changes in current
evoked by changes in external osmolarity are reversible. Differing
osmolarity was produced by addition of mannitol to hypotonic ND-20
medium (20 mM Na+, 70 mosM).
Arrows, beginning of application of 10 µM amiloride (up arrows) and
of washout (down arrows). B: stepping
currents of rENaC treated with 200 mosM (isotonicity) and 450 mosM
(hypertonicity). C: rENaC-associated
currents recorded from an oocyte under unstretched (200 mosM) or
swollen (70 mosM) conditions.
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Fig. 2.
Summarized data comparing expressed rENaC currents with those of
H2O-injected
oocytes. A: currents
recorded at holding potential of 80 mV in swollen oocytes
(n = 18) are decreased significantly.
B: currents recorded at 80 mV
in shrunken cells are stimulated markedly
(n = 24). For comparison of
effects of anisosmolarity on rENaC, scale of y-axis is same
in A and B.
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The sensitivity of the cloned rENaC expressed in oocytes to changes in
external osmolarity was reversible. Figure
1A shows a typical recording
(repeated in 7 oocytes) for osmolarity-induced changes in total rENaC
currents. Currents recorded in both hypotonic and hypertonic ND-20 were
sensitive to nanomolar concentrations of amiloride. Application of
amiloride directly to the chamber or to the chamber perfusate
immediately blocked the currents in rENaC-expressing oocytes (>90%
of the total current). Both the increase and decrease in current
induced by changes in solution osmolarity were reversible. The current
was restored after washing out the applied amiloride, and application
of amiloride could be repeated several times on the same oocytes with
identical results.
The osmolarity dependence of expressed rENaC currents was investigated
by superfusing oocytes with ND-20 media of variable osmolarities (from
70 to 490 mosM; Fig. 3). In
H2O-injected oocytes, no
significant changes in the current magnitude were found (Fig. 3A). However, in rENaC-expressing
oocytes, osmolarities as high as 490 mosM increased current, whereas
perfusing the cells with hypotonic ND-20 decreased currents (Fig.
3B). Figure
3C shows that the relationship between
current and osmolarity was almost linear over the range of osmolarities
studied. Osmolarities >500 mosM caused an irreversible increase in
current, and the electrodes were easily dislodged from the oocytes due
to the large decrease in cell volume.

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Fig. 3.
Osmotic pressure dependence of rENaC. Oocytes were perfused with ND-20
media, ranging from 70 to 490 mosM. A:
overlapped current traces from a water-injected oocyte evoked at
clamping potential of 80 mV. B:
inward currents of rENaC-expressing oocyte at 80 mV are
sensitive to external osmolarity. C:
currents in B normalized to that
obtained in isotonic ND-20 medium (1.0) and replotted as a function of
extracellular osmolarity. Data in A
and B are representative of at least 3 similar experiments.
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Inhibition by amiloride.
Amiloride is not only a specific inhibitor of ENaC (at the nanomolar
level) but also an inhibitor (at the micromolar level) of
stretch-activated channels in hair cells and in
Xenopus oocytes (for review, see Ref.
11). Observations from lipid bilayer experiments incorporating
-bENaC or 

-rENaC (3, 14) and whole cell patch-clamp studies
on human B lymphocytes (1) showed that the amiloride sensitivity of
ENaC tended to decrease with stretch. Our results using
Xenopus oocytes expressing rENaC
showed that hypertonic solutions (450 mosM) caused a leftward shift in
the dose-inhibition curve of amiloride, and that hypotonic solutions (70 mosM) shifted the dose-response curve of amiloride to the right
(Figs. 4 and
5). In rENaC-expressing oocytes exposed to hypotonic ND-48 medium (containing 48 mM
Na+; 100 mosM), the
Ki of amiloride
increased to 800 nM from 176 nM (recorded in isotonic ND-48 medium),
consistent with the observations for the shift in amiloride
Ki recorded in
ND-20 medium and the results of planar lipid bilayer experiments (14).

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Fig. 4.
Inhibition of current by amiloride in rENaC-expressing
Xenopus oocytes.
Top: currents in ND-20 perfusing media
(control). Middle: currents following
application of amiloride (100 nM).
Bottom: amiloride-sensitive
currents.
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Fig. 5.
Osmotic pressures shift amiloride affinity of rENaC. Apparent
dissociation constant
(Ki) of
amiloride for rENaC-expressing oocytes in 200 mosM ( ), 450 mosM
( ), and 70 mosM ( ) superfusates are 114 ± 0.03 nM
(n = 5), 55 ± 0.001 nM
(n = 3-5), and 4,199 ± 1.1 nM
(n = 4 or 5), respectively. Remaining
percentages of total current (100%) are plotted against concentration
of applied amiloride.
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To determine whether the osmotic pressure sensitivity of the currents
recorded from rENaC-expressing oocytes was a specific characteristic of
Na+ channel expression, amiloride
(10 µM) was added to the chamber containing isotonic ND-20 medium
before the chamber was switched to hypotonic or hypertonic ND-20 medium
with an equal concentration of amiloride. Amiloride inhibited >90%
of the total current in oocytes injected with rENaC cRNA. As expected,
application of osmotic pressure (70 or 450 mosM) did not modify the
amiloride-insensitive currents in rENaC-expressing oocytes, identical
to the results obtained from uninjected or
H2O-injected oocytes studied under the same conditions (Fig. 6).

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Fig. 6.
Amiloride pretreatment blocks response to mechanical signals of rENaC.
Left: currents.
Right: corresponding current-voltage
curves. A: control;
basal current recorded from an rENaC-expressing oocyte in isotonic
ND-20 medium. Vm,
membrane potential. B: inhibition of
current in isotonic ND-20 medium in presence of 10 µM amiloride.
C: in continual presence of 10 µM
amiloride, there is no change in current following superfusion with
hypertonic medium (450 mosM ND-20). Data are representative recordings
of 3 oocytes.
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Effect of osmotic pressure on ion selectivity.
ENaC are highly selective for Na+
and Li+ and much less permeable to
K+ and other monovalent cations
(4, 8, 9). The application of hydrostatic or osmotic pressure on human
B lymphocytes (1) and hydrostatic pressure across planar lipid bilayers
containing ENaC (3, 14) showed that mechanical stress on ENaCs may
modify the ionic selectivity of the pore. The ionic selectivity of
rENaC was calculated according to the reversal potentials of
amiloride-sensitive currents (Table 1)
recorded from the same oocyte with different extracellular monovalent
cations.
Oocytes were superfused in ND-20 with variable osmolarity or media in
which Na+ was replaced with the
equimolar test cation (K+,
Li+, or
Cs+). An
I-V
curve was then run from
100 to +80 mV in the presence or absence
of 10 µM amiloride. As summarized in Table
2, the relative permeability of
Na+ vs.
K+ or
Cs+ was higher in ND-20 media than
in ND-96; the
PNa/PK
was 20.0 ± 0.9 (n = 7) in ND-20
vs. 5.0 ± 0.8 in ND-96, and
PNa/PCs
was 33.3 ± 1.1 (n = 8) in ND-20
vs. 8.3 ± 1.8 in ND-96. However, under conditions
of identical ionic strength, osmotic pressure resulted in changes in
ion selectivity. The
PNa/PK
decreased to 6.3 and 11.0 for rENaC stressed by hypotonicity and
hypertonicity, respectively, compared with a selectivity of 20 in
isotonic ND-20 medium. The selectivity of the channel for
Cs+
(PNa/PCs)
similarly changed to 5.0 and 12.5 in 70 and 450 mosM ND-20 solutions,
respectively, from a ratio of 33 in isotonic ND-20.
Changes in the external osmotic pressure did not alter the relative
permeability sequence for monovalent cations. The sequence of ionic
permeability was the same in isotonic ND-96, isotonic ND-20, hypotonic
ND-20, and hypertonic ND-20 media, namely,
Li+ > Na+ > K+ > Cs+. The
PNa/PLi
was also less sensitive to changes in osmotic pressure.
Mechanogated channel inhibitors.
Although La3+ and
Gd3+ inhibit several ion channels
(19, 35), they have been applied to stretch-activated and
volume-sensitive cation or anion channels in a variety of cell species
(11). To test the hypothesis that the response of expressed rENaC to osmolarity represents its potential mechanosensitivity, the effects of
GdCl3 (50 µM) and
LaCl3 (2 mM) were studied on the
volume-sensitive currents in shrunken or swollen oocytes expressing
rENaC (Fig. 7). The currents of rENaC under
isotonic conditions were
1,560.2 ± 201 and
1,511.3 ± 115 nA in the absence and presence of 2 mM La3+, respectively. Similarly, no
significant change in current amplitude was found with 50 µM
Gd3+ (
1,747.8 ± 201 nA
for control vs.
1,750.1 ± 58 nA for
Gd3+). However, in both shrunken
and swollen cells, the rENaC-associated currents became sensitive to 2 mM La3+. The currents decreased to
45.9 ± 7.3% (n = 4, P < 0.01) and 80.3 ± 14.2%
(n = 4, P < 0.05) of control in
shrunken and swollen oocytes, respectively. Inhibition
of volume-sensitive currents by 50 µM
Gd3+ was similar to that by
La3+. Under conditions of
hypertonicity and hypotonicity, currents reduced to 75.3 ± 3.0%
(n = 4, P < 0.05) and 61.9 ± 10%
(n = 3, P < 0.05) of controls, respectively.
Pretreatment of rENaC cRNA-injected oocytes with
Gd3+ or
La3+ prevented the response of
rENaC-injected oocytes to changes in bath osmolarity (data not shown)
without affecting the basal currents. The inhibitory effects of these
mechanogated channel inhibitors were concentration dependent (data not
shown). In H2O-injected oocytes,
neither Gd3+ nor
La3+ had any effect on basal
current under conditions of swelling or shrinkage, consistent with the
response of uninjected oocytes to changes in solution osmolarity.

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Fig. 7.
Inhibition by La3+ and
Gd3+ of osmotic pressure-sensitive
rENaC currents. Left:
LaCl3 (2 mM) inhibition.
rENaC-associated currents ( 80 mV) in swollen
(n = 4) or shrunken
(n = 4) oocytes were sensitive to
LaCl3. Currents were normalized to
maximal amplitudes without LaCl3.
Right:
GdCl3 (50 µM) decreased currents
in swollen (n = 9) or shrunken
(n = 4) oocytes injected with rENaC
cRNA.
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Relative oocyte volume.
It has been proposed that stretch-activated epithelial ion channels can
function as a sensor of cell volume changes (30). We therefore
hypothesized that rENaC may be involved in regulation of cell volume.
As shown in Fig.
8A,
oocytes expressing rENaC kept the cell volume constant following a
slight decrease in the first 1 min of exposure to hypertonic ND-20
medium; in contrast, the volume of the
H2O-injected oocyte was much
smaller after 5-min incubation in hypertonic ND-20 medium. The ability
of rENaC-expressing oocytes to regulate their volume in hypertonic
ND-20 medium was diminished with amiloride (10 µM). The absolute
oocyte volumes at time zero and 5 min
in 450 mosM ND-20 medium are summarized in Table
3. The normal absolute volume of
Xenopus oocytes
(H2O-injected oocytes) was 9.4 × 10
4
cm3
(n = 12), similar to that reported
previously (40). In oocytes incubated for 2 h in hypertonic media, 63.1 ± 7.0% of the eggs injected with
H2O shrank. In contrast,
rENaC-expressing oocytes were able to regulate their volume such that
only 18.4 ± 3.7% of the eggs shrank, consistent with activation of
rENaC conductance by an increase in external osmotic pressure (Fig.
8B). Digital imaging of both
water-injected and 

-rENaC-injected oocytes showed that,
although water-injected eggs shrank on exposure to hypertonic ND-20
medium, rENaC-expressing oocytes maintained their volume over a 5-min
time course (Fig. 8C)

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Fig. 8.
rENaC expression mediated cell volume regulation.
A: time course of relative cell volume
(RCV) of treated oocytes in hypertonic ND-20 (450 mosM). RCV of oocytes
expressing rENaC was kept approximately constant, and 10 µM amiloride
inhibited ~60% of regulatory volume increase, while RCV of
uninjected oocytes decreased markedly. Data are representative of 8 measurements. B: average percentages
of shrunken oocytes following 2-h incubation in hypertonic ND-20 medium
(450 mosM) of H2O-injected oocytes
and   -rENaC-injected oocytes
(n = 3).
C: digital images of
H2O-injected oocyte
(top) and   -rENaC-expressing
oocyte (bottom) treated in
hypertonic ND-20 (450 mosM). Images were taken at
time 0 (control), and then 1 image/min
was taken. Left to
right: images from 0, 1, 2, 3, 4, and
5 min. H2O-injected oocytes shrank
under these conditions, whereas rENaC-expressing oocytes maintained
their volume.
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DISCUSSION |
The purpose of the present study was to test whether wild type


-rENaC expressed in Xenopus
oocytes could respond to changes in osmotic pressure using the
two-electrode voltage-clamp technique. The main finding of our study is
that wild type 

-rENaC expressed in oocytes behaved as a
mechanoactivated cation channel sensitive to changes in external
osmotic pressure. Extracellular hypotonicity inhibited the oocyte whole
cell currents, whereas extracellular hypertonicity stimulated
rENaC-associated currents in a reversible fashion. Changes in osmotic
pressure also modified the sensitivity of rENaC to amiloride and the
ionic selectivity of the channel for monovalent cations. Under
conditions of hypertonic stress, rENaC-associated currents were
sensitive to the mechanogated channel antagonists
La3+ and
Gd3+. Moreover, rENaC in
heterologously expressed oocytes was involved in regulatory cell volume
restoration of oocytes.
Although Xenopus oocytes have been
used as an expression model to translate a number of volume-sensitive
Cl
channels (20) and ENaCs
(3, 8, 9, 14, 15), an important concern of the present study was
whether the current component activated by changes in external osmotic
pressure was due to activation of endogenous channels. Expressed
currents were not sensitive to 200 µM DIDS or 100 µM niflumic acid,
whereas endogenous Cl
channels were activated by oocyte swelling rather than shrinkage (2).
No shrinkage-activated anion channels have yet been identified in
Xenopus oocytes. In addition,
Cl
-free ND-20 medium would
be expected to diminish the endogenous Cl
channel activity. The
endogenous volume-sensitive cation channels and the mechanogated cation
channels that have been previously described in
Xenopus oocytes were difficult to
demonstrate under our experimental conditions in the present study
(39). However, the small whole cell current magnitude (~100 nA), the
poor cation selectivity (equal for
Na+,
K+, and
Ca2+), and the rapid adaptation
of their mechanosensitivity are very different from the properties
associated with expression of rENaC. Although these native channels are
blocked by amiloride and Gd3+ (21,
39), Ki (0.5 mM)
was 120- to 625-fold higher than those of rENaC (500 vs. 0.8 µM in
hypotonic ND-48 medium; 500 vs. 4.19 µM in hypotonic ND-20 medium).
The presence of stretch-activated endogenous cation channels has not
been verified by two-electrode voltage-clamping record, and, under our
experimental conditions, the channels were not activated in
H2O-injected or uninjected oocytes
(Fig. 2).
The present study demonstrates that 

-rENaC expressed in
Xenopus oocytes is sensitive to the
mechanogated channel inhibitors La3+ and
Gd3+.
La3+ and
Gd3+, as specific antagonists of
stretch-activated (mechanosensitive) channels, have been used to
inhibit many epithelial or nonepithelial cation channels (11, 25, 28,
31). Our observations show that
Gd3+ and
La3+ block rENaC-associated
currents elicited by changes in osmotic pressure. The mechanisms of
Gd3+ and
La3+ inhibition of rENaC are not
known. It is conceivable that cryptic binding sites for
Gd3+ and
La3+ could be revealed by membrane
conformational change as a result of changes in bath solution
tonicities.
We confirmed that amiloride sensitivity
(Ki) of the
cloned rENaC expressed in Xenopus
oocytes was altered after exposure to anisosmotic media. Similar
findings have been reported for bENaC (3) and rENaC (14). These earlier
studies showed that the activation of the epithelial
amiloride-sensitive Na+
conductances were linearly dependent on the hydrostatic pressure across
an artificial membrane. In the present study, amiloride Ki values were
related to the extracellular osmolarity; as depicted in Fig. 5,
Ki values
estimated with 450, 200, 100, and 70 mosM were 55, 114, 800, and 4,199 nM, respectively, indicating that as osmolarity decreased,
the Ki of
amiloride for the channel increased in parallel.
The ionic permeability of a channel is a critical parameter for channel
characterization. Ionic selectivity studies revealed that expressed
rENaC was highly selective for Na+
and was dependent on the extracellular
Na+ concentration. After exposure
to 70 or 450 mosM ND-20 medium, the monovalent cation permeability of
the channel (Na+,
K+,
Li+, and
Cs+) was blunted. Observations
from both cell-free (3, 14) and whole cell systems (1) on ENaC behavior
under similar conditions are consistent with the present study.
Similarly, stable expression of the
-subunit of ENaC in an
osteoblast cell line was associated with the appearance of a
stretch-activated nonselective cation channel (18).
The cellular mechanisms underlying volume regulation in oocytes
expressing 

-rENaC are unknown. However, in the cell, ENaC is
structurally connected to actin, spectrin, and their associated proteins (29, 32). Evidence that actin can regulate ENaC (5, 27)
supports the idea that hypertonicity may activate rENaC through a
mechanism involving G-actin. The underlying signal may involve an
increase in intracellular Ca2+
concentration
([Ca2+]i).
An increase in
[Ca2+]i
has been shown in several cell lines to be a consequence of hypotonic
shock (22, 33). As rENaC is converted to a less selective or
nonselective cation channel by changing osmotic pressures, an increase
in the permeability of Ca2+
through this pathway could give rise to an increase in
[Ca2+]i.
In the present study, we demonstrate that expressed amiloride-sensitive
rENaC is involved in RVI in shrunken oocytes (Fig. 8). When the degree
of amiloride sensitivity of whole cell currents (>90%) is compared
with amiloride-inhibited changes in cell volume (~60%), it is clear
that some apparently "amiloride-resistant" pathways were
sensitive to osmotic signals outside the cell. A native
Na+/H+
antiporter that was sensitive to amiloride and to hypertonicity (34)
could contribute to RVI in shrunken oocytes. The relative contributions
of epithelial Na+ channel,
Na+/H+
exchange, and
Na+-K+-Cl
symport to RVI in rat hepatocytes were reported to be in the proportions 4:1:1 (37). This observation that the
Na+ channel makes a major
contribution to RVI in rat hepatocytes is consistent with the findings
of the present study that expression of ENaC enhances RVI in oocytes.
In conclusion, we report that the cloned ENaC isolated from the rat
colon is responsive to changes in external osmotic pressure when
expressed in Xenopus oocytes. The
sensitivity of expressed rENaC in oocytes to osmotic pressure relates
to RVI under hypertonic conditions, but the role of native


-rENaC in the physiological regulation of epithelial cell
volume remains to be determined.
 |
ACKNOWLEDGEMENTS |
We acknowledge Eddie J. Walthall (Dept. of Physiology and
Biophysics) for superb technical assistance, Dr. Michael DuVall (Dept.
of Anesthesiology) for helpful discussions of the manuscript, Shawn
Williams (Dept. of Cell Biology) for excellent assistance in imaging
Xenopus oocytes, and Dr. Lan Chen
(Dept. of Anesthesiology) for help in the dissection of
Xenopus ovaries.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-37206 and National Heart, Lung, and Blood
Institute Grant HL-50487.
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. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Benos, Department of Physiology and
Biophysics, The University of Alabama at Birmingham, BHSB 706, 1918 University Blvd., Birmingham, AL 35294-0005.
Received 6 April 1998; accepted in final form 8 July 1998.
 |
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