Ammonium interaction with the epithelial sodium channel
Nazih L.
Nakhoul,
Kathleen
S.
Hering-Smith,
Solange M.
Abdulnour-Nakhoul, and
L. Lee
Hamm
Section of Nephrology, Department of Medicine, and Department of
Physiology, Tulane University School of Medicine and Veterans
Affairs Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
The
purpose of this study was to investigate the direct effect of
NH3/NH
on mouse epithelial
Na+ channels (mENaC) expressed in Xenopus
oocytes. Two-electrode voltage-clamp and ion-selective microelectrodes
were used to measure the Na+ current, intracellular pH
(pHi), and ion activities in oocytes expressing mENaC. In
oocytes expressing mENaC, removal of external Na+
reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in
the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV
in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a
decrease in pHi (0.43 ± 0.07) similar to the
NH4Cl-induced pHi decrease (0.47 ± 0.12).
Removal of Na+ in the presence of butyrate caused
hyperpolarization that was not significantly different from that in the
absence of butyrate at high pHi (in the absence of
NH4Cl). Removal of external Na+ resulted in an
outward current of 3.7 ± 0.8 µA (at
60 mV). The magnitude of
this change in current was only 2.7 ± 0.7 µA when Na+ was removed in the presence of NH4Cl. In
oocytes expressing mENaC, NH4Cl also caused a decrease in
whole cell conductance at negative potential and an outward current at
positive potential. In the presence of amiloride, steady-state current
and the change in current caused by removal of Na+ were not
different from zero. These results indicate that NH4Cl inhibits Na+ transport when mENaC is expressed in oocytes.
The inhibition of voltage changes is not due to intracellular
acidification caused by NH4Cl. Permeability and selectivity
of ENaC to NH
may play a role.
mouse epithelial sodium channel; intracellular pH; ammonium ion
permeability
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INTRODUCTION |
ABSORPTION OF
SODIUM across epithelia plays an essential role in maintaining
Na+ and fluid homeostasis of the body. In these epithelia,
Na+ transport is a two-step process involving diffusion of
Na+ through channels across the apical membrane and active
transport across the basolateral membrane via the
Na+-K+-ATPase (19). In the
kidney, although Na+ transport occurs throughout the length
of the nephron, the fine regulation of Na+ excretion occurs
in the collecting tubule. In this segment, as in several other tight
epithelia, the epithelial Na+ channel (ENaC) (12,
27) is the rate-limiting pathway of luminal reabsorption of
Na+. Mutations of ENaC can result in serious clinical
problems, such as Liddle's syndrome and pseudohypoaldosteronism type 1 (14, 24, 40).
The ENaC is located at the apical membrane and is highly sensitive to
amiloride. Biophysically, this channel is very selective for
Na+ over K+ (with Na+
permeability-to-K+ permeability ratio > 10) but is
selective for Li+ over Na+ by a factor of
1.3-1.5. The highly selective Na+ channels have low
single-channel conductance and long open and closed times (21,
33).
Cloning of ENaC enabled the molecular characterization of the channel
as consisting of three homologous subunits termed
-,
-, and
-ENaC (13, 26, 27). More recently, cDNA encoding mouse
-,
-, and
-ENaC (mENaC) were cloned (1).
Coexpression of these subunits in Xenopus oocytes yielded an
amiloride-sensitive Na+ channel with properties very
similar to the rat ENaC. This channel was investigated in our study.
Regulation of Na+ channels by hormones has been recognized
from physiological studies even before ENaC was cloned. Aldosterone is
considered the primary hormone responsible for regulating channel activity through complex mechanisms that involve activation of preexisting channels and de novo synthesis of channels (4, 5, 29,
35). Vasopressin (and possibly insulin) is another hormone that
is involved in ENaC regulation (8, 34, 39). Nonhormonal
inhibition of ENaC occurs with elevated extracellular Na+
(leading to self-inhibition), increased intracellular Na+,
and increased intracellular Ca2+, which has been postulated
to be the mediator of self-inhibition (6, 15, 18, 25). On
the other hand, hyperpolarization of the apical membrane seems to
activate the channel. Intracellular acidification was reported to cause
a decrease in apical Na+ conductance; however, the role of
intracellular pH (pHi) in regulating Na+
channel activity may differ among tissues and is not yet fully understood (13, 26, 32). Regulation of ENaC has been
addressed in several recent reviews (2, 8, 19, 35).
In the renal distal nephron, NH
transport is critical
for acid-base balance. Increases in
NH3/NH
and/or its effect on acid-base
balance can have direct and indirect consequences on transport in
general. For example, NH
has been shown to inhibit
transport across the tight epithelium of the toad bladder
(20). In another study on the rabbit cortical collecting
duct, NH4Cl was shown to decrease transepithelial voltage (Vte) and inhibit Na+ reabsorption
(22). In this latter study, the effect of
NH4Cl on Vte was not evident when
Na+ reabsorption was inhibited by amiloride. This effect of
NH4Cl could be due to one or several causes, including
voltage-dependent changes in Na+ transport,
pHi-mediated effect on Na+ conductance, and/or
direct inhibition of the ENaC. The effect of NH
may
be physiologically relevant to the increased urinary Na+
observed with metabolic acidosis in vivo. This study was conducted to
investigate the effect of NH
on Na+
transport through ENaC expressed in Xenopus oocytes.
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METHODS |
Reagents and solutions.
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless
otherwise noted. The standard bathing solution was ND96 medium
containing (in mM) 100 NaCl, 2 KCl, and 1.8 CaCl2 and
buffered with 5 mM HEPES to pH 7.5. The
NH3/NH
solution contained 20 mM
NH4Cl (replacing NaCl) at pH 7.5. In 80 mM Na+
solutions, 20 mM N-methyl-D-glucamine
(NMDG+) replaced 20 mM Na+, and in 0 Na+ solutions, all Na+ was replaced with
NMDG+. Osmolarity of all solutions was ~200 mosmol/l. OR3
medium (Leibovitz media, GIBCO BRL) contained glutamate and 500 U each
of penicillin and streptomycin, with pH adjusted to 7.5 and osmolarity
adjusted to ~200 mosmol/l.
Isolation of oocytes.
We harvested oocytes in stage 5/6 from female Xenopus
laevis. Briefly, this was done by anesthetizing the frog by mild
hypothermia in water containing 0.2% tricaine (3-aminobenzoic acid
ethyl ester; Sigma, St. Louis, MO). A 1-cm incision was made in the
abdominal wall, and one lobe of the ovary was externalized and the
distal portion was cut. The wound was closed by a few stitches (5-0 catgut) in the muscular plane of the peritoneum followed by two to
three stitches (6-0 silk) in the abdominal skin. The excised piece of ovary containing oocytes was rinsed several times with
Ca2+-free ND96 solution until the solution was clear. The
tissue was then agitated in ~15 ml of sterile-filtered
Ca2+-free solution containing collagenase type 1A (Sigma)
for 30-40 min. Free oocytes were rinsed several times with sterile
OR3 medium, sorted, and then stored at 18°C.
Preparation of cRNA.
Plasmid containing the appropriate template DNA of mouse ENaC (all a
generous gift of Dr. Tom Kleyman) was purified by Wizard Plus Minipreps
DNA Purification System (Promega, Madison, WI). The plasmid was then
digested with an appropriate restriction enzyme that has a cleavage
site downstream of the insert to produce a linear template and then
with proteinase K (1 mg/ml). DNA was then extracted twice with
phenol-chloroform, extracted with chloroform, and precipitated with
ethanol. cDNA was transcribed in vitro with T7 RNA polymerase. The in
vitro synthesis of capped RNA (cRNA) transcripts was then accomplished
using mCAP RNA Capping Kit (Stratagene, La Jolla, CA). The
concentration of cRNA was determined by ultraviolet absorbance, and its
quality was assessed by formaldehyde-MOPS-1% agarose gel
electrophoresis (37).
Injection of oocytes.
Oocytes in OR3 medium were visualized with a dissecting microscope and
injected with 50 nl of full-length cRNA mix of mENaC containing cRNAs
for the
-,
-, and
-subunits. At 0.05 µg/µl concentration
for each subunit, each oocyte was injected with 2.5 ng/subunit. Control
oocytes were injected with 50 nl of sterile H2O. The
sterile pipettes had tip diameters of 20-30 µm. They were
backfilled with paraffin oil and were connected to a Nanoject displacement pipette (Drummond Scientific). Injected oocytes were used
2-5 days after injection with cRNA.
Electrophysiological measurements in frog oocytes.
The pH microelectrodes were of the liquid ion-exchanger type, and the
resin (H+ ionophore 1, cocktail B) was obtained from Fluka
Chemical (Ronkonkoma, NY). Single-barreled microelectrodes were
manufactured as described previously (31, 36). Briefly,
alumina-silicate glass tubings (1.5 mm OD × 0.86 mm ID; Frederick
Haer, Brunswick, MD) were pulled to a tip of <0.2 µm and dried in an
oven at 200°C for 2 h. The electrodes were vapor silanized with
bis(dimethylamino)-dimethyl silane in a closed vessel (300 ml). The
exchanger was then introduced into the tip of the electrodes by means
of a very fine glass capillary. pH electrodes were backfilled with a
buffer solution (3). The electrodes were fitted with a
holder with an Ag-AgCl pellet attached to a high-impedance probe of a
WPI FD-223 electrometer. The pH electrodes were calibrated in standard
solutions of pH 6 and 8. Only electrodes with a slope >58 mV/pH were
used in our studies.
The oocyte, visualized with a dissecting microscope, was held on a
nylon mesh in a special chamber where solutions flow continuously at a
rate of ~4 ml/min. Solutions (6 possible) were switched by a
combination of a six- and a four-way valve system, which was activated
pneumatically. Very little dead space was present, and complete
solution changes in the chamber occurred in 6-8 s.
Two-electrode voltage clamp.
Whole cell currents were recorded using two-electrode voltage clamp
(model OC-725, Warner Instruments, Hamden, CT). For these experiments,
electrodes were pulled from borosilicate glass capillaries (OD 1.5 mm;
Fredrick Haer) using a vertical puller (model 700C, David Kopf
Instruments). Electrodes were filled with 3 M KCl solution and had
resistances of 1-4 M
. Bath electrodes were also filled with 3 M
KCl and were directly immersed in the chamber. In most cases, oocytes
were clamped at
60 mV, and long-term readings of current were sampled
at a rate of once per second. For current-voltage (I-V)
relationships, oocytes were clamped at 0 mV and stepped from
80 to
+60 mV in 20-mV steps (sampled at 10 times/s for 1 s at each
step). Slope conductances were calculated from the slope of the
I-V line between
60 and
40 mV. Inward flow of cations is
defined by convention as inward (negative) current.
Statistics and data analysis.
Values are means ± SE; n is the number of
observations. Statistical significance was judged primarily from
two-tailed Student's t-tests. Whenever feasible,
measurements were determined under control and test conditions in the
same cell, and each cell served as its own control (paired data).
Results are considered statistically significant if P
0.05.
 |
RESULTS |
Intracellular ionic compositions in control (water-injected)
oocytes and oocytes expressing mENaC.
The oocyte expression system has been extensively used to study the
characteristics of expressed proteins, many of which are membrane
transporters and ionic channels. The high level of expression usually
achieved, the lack of substantial native transport in the oocyte, and
the relative ease of measurements in this preparation make the oocyte a
valuable system for study. A concern that has been inadequately
addressed is that expressing an exogenous transporter may alter the
cellular environment of the oocyte. An altered cellular environment
may, by itself, be a factor affecting the function of the transporter.
Because expressing an ion channel may cause such significant effects,
we first characterized the cellular changes induced by expressing mENaC
under steady-state conditions. As shown in Table
1, intracellular composition of the
important monovalent ions was drastically and significantly altered. As expected, intracellular Na+ activity increased from
6.6 ± 0.6 to 30.0 ± 1.8 mM and steady-state membrane
potential (Vm) was positive (+4.5 ± 1.6 mV). This was accompanied by substantial decrease in intracellular
K+ activity (a
)
from 74.3 ± 2.7 to 40.0 ± 0.5 mM, whereas intracellular
Cl
activity (a
) increased
from 25.0 ± 0.3 to 75.3 ± 1.9 mM. pHi increased
slightly from 7.34 ± 0.02 to 7.5 ± 0.02, but the difference
was not statistically significant (P > 0.05).
Because the ENaC is usually overexpressed, it is conceivable that acute
changes in external Na+ may induce large changes in
intracellular Na+ activity
(a
). To investigate this possibility, we
measured the changes in a
by
Na+-selective microelectrodes when external Na+
was removed (replaced by NMDG+). In control oocytes,
removal of external Na+ (for ~10 min) hardly caused any
detectable decrease in a
(
a
= 0.9 ± 0.1 mM,
n = 4). Unexpectedly, in mENaC oocytes, removal of
external Na+ also caused a very small change in
a
(
a
= 1.8 ± 0.4 mM, n = 4). These experiments indicate
that intracellular Na+ in the oocytes is not readily
exchangeable, and no acute large changes in
a
are expected to occur in response to
Na+ removal, even when mENaC is expressed.
Effect of NH3/NH
and removal of Na+ on water-injected
oocytes.
To study the effect of NH
on Na+
transport, we first characterized the changes caused by addition of
NH
or removal of external Na+ in
water-injected oocytes. As shown in Fig.
1, exposing control oocytes to
NH
caused a decrease in pHi
(segment ab) and large depolarization of the cell. These
changes were fully reversed when NH3/NH
was removed from the bath (segment bc). Removal of bath
Na+ caused a small, sustained hyperpolarization of the
cell, but pHi did not change (segment cd).
Exposing the oocyte to NH3/NH
in the
continued absence of Na+ resulted in changes similar to
those observed in the presence of Na+; pHi
decreased substantially (segment de), and the cell
depolarized to near 0 mV. Removal of bath
NH3/NH
fully reversed these changes
(segment ef). These experiments demonstrated the
following important features: 1) There is a small but
significant voltage change induced by Na+ transport.
2) NH3/NH
caused substantial voltage and pHi changes. These changes occur in the
water-injected oocytes and are independent of mENaC expression. Both
the pHi and the voltage changes caused by
NH
can potentially affect Na+ transport
through mENaC.

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Fig. 1.
Effect of NH3/NH on
membrane potential (Vm) and intracellular pH
(pHi) in the presence and absence of Na+.
Exposing control (H2O-injected) oocytes to 20 mM
NH3/NH in the presence of Na+
decreased pHi by 0.15 and substantially depolarized the
cell from 72 to 4 mV (segment ab). This effect was fully
reversed when NH3/NH was removed from the
bath (segment bc). Removal of external Na+ did
not affect pHi (segment cd) but caused moderate
hyperpolarization ( Vm = 20 mV).
Exposing oocytes to NH3/NH in the absence
of Na+ still decreased pHi by 0.18 and
hyperpolarized the cell to near 0 mV (segment de). Removal
of NH3/NH reversed these changes
(segment ef).
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Na+ removal in the presence and
absence of NH3/NH
in
oocytes expressing mENaC.
By comparison to water-injected oocytes, Na+ removal and
exposure to NH3/NH
caused substantially different effects when mENaC was expressed. The basic observation indicating the effect of NH3/NH
on Na+ transport through mENaC was indicated in the
experiments depicted in Fig.
2. As shown in Fig.
2A, in oocytes expressing mENaC, removal of bath
Na+, caused a large hyperpolarization from +16 to
112 mV
(segment ab), which readily recovered when Na+
was returned (segment bc). Exposure to
NH3/NH
(at point c) caused a
small hyperpolarization of ~5 mV (segment cd). In the
continued presence of NH3/NH
, subsequent
removal of bath Na+ hyperpolarized the oocyte to
89 mV
(segment de), which readily recovered on readdition of
Na+ to the bath (segment ef). The effect
of NH3/NH
was also reversed when
NH3/NH
was removed from the bath
(segment fg). Removal and readdition of external Na+ in the absence of NH3/NH
for a second time caused the same hyperpolarization (segment
gh) and recovery (segment hi) observed in the first
pulse (compare with segments ab and bc,
respectively). In 10 paired experiments, the average hyperpolarization
caused by Na+ removal was
129 ± 5.3 mV in the
absence of NH3/NH
and
100 ± 7.8 mV in the presence of NH3/NH
(P < 0.001). These experiments indicate that
NH3/NH
may inhibit Na+
transport in oocytes expressing mENaC.

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Fig. 2.
A: effect of Na+ removal in the
presence and absence of NH3/NH in mouse
oocytes expressing the epithelial Na+ channel (ENaC). Trace
represents 10 experiments showing that removal of Na+
hyperpolarized the oocyte by 129 ± 5.3 mV (segment
ab), which was readily reversed when Na+ was returned
to normal (segment bc). Exposing oocytes expressing mENaC to
NH3/NH (segment cd) caused a
slight hyperpolarization ( Vm = 4.3 ± 1.4 mV). Removal of Na+ in the presence of
NH3/NH hyperpolarized the oocyte by
100 ± 7.8 mV (segment de), which was significantly
less than the hyperpolarization in the absence of
NH3/NH (P < 0.05).
Hyperpolarization was reversed on readdition of Na+ to the
bath (segment ef). Removal of
NH3/NH from the bath caused a small
depolarization (segment fg). Removal of Na+
again in the absence of NH3/NH4+
caused a larger hyperpolarization (segment gh) as seen in
the first pulse (compare with segment ab). Readdition of
Na+ fully reversed this effect on Vm
(segment hi). B: Na+ removal in the
presence and absence of NH3/NH4+ in
oocytes expressing mENaC. When bath Na+ at 100 mM was
removed (from 100 to 0 mM), Vm hyperpolarized
significantly (segment ab) as seen in A and was
reversed on restoration of bath Na+ to 100 mM
(segment bc). Replacing 20 mM Na+ with
N-methyl-D-glucamine (NMDG, 80 mM
Na+) caused a small hyperpolarization (segment
cd). Subsequent exposure to NH3/NH
(at 80 mM Na+) caused a small depolarization (segment
de). Removal of Na+ in the presence of
NH3/NH caused a smaller hyperpolarization
(segment ef) than that observed with Na+
removal in the absence of NH3/NH (compare
with segment ab). Readdition of 80 mM Na+ to the
bath reversed this effect on Vm (segment
fg). Removal of bath NH3/NH caused a
small hyperpolarization (segment gh). When bath
Na+ at 80 mM was removed again (going from 80 mM to 0 mM),
Vm hyperpolarized (segment hi) to the
same extent as in the first pulse (compare with segment ab).
C: effect of amiloride on Vm in
oocytes expressing mENaC. Exposing oocytes to amiloride (100 µM)
caused hyperpolarization of 77 ± 4.1 mV (segment ab).
Addition of NH3/NH in the presence of
amiloride caused a transient hyperpolarization (segment bc)
followed by sustained depolarization of 23 ± 8.7 mV
(segment cd). Removal of Na+ in the presence of
NH3/NH and amiloride caused a small but
statistically insignificant (P > 0.05) depolarization
of Vm (segment de). All changes were
completely reversible: readdition of Na+ in the presence of
amiloride hyperpolarized Vm slightly
(segment ef) and removal of
NH3/NH hyperpolarized
Vm even further (segment fg).
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In these experiments, NH3/NH
solutions
contain 20 mM NH4Cl obtained by replacing 20 mM NaCl from the control ND96 solution. Because Na+-induced voltage
changes in oocytes expressing mENaC are substantial (Fig.
2A), it is conceivable that this partial removal of
Na+ could be responsible in part for the reduced
hyperpolarization induced by removal of Na+ in the presence
of NH3/NH
. The next set of experiments
was designed to investigate this possibility. As shown in Fig.
2B, removal of external Na+ (from 100 to 0 mM in
the bath) caused the usual large and reversible hyperpolarization
observed earlier (segments ab and bc). At
point c, 20 mM Na+ was removed from the bath
(replaced by NMDG+), resulting in a small hyperpolarization
of ~4 mV (segment cd). On switching to
NH3/NH
solution at point d, no
Na+ was replaced, and the external solution still contained
80 mM Na+. This maneuver caused a small depolarization
(segment de). Removal of Na+ in the presence of
NH3/NH
caused hyperpolarization (segment ef) that was substantially smaller than in
the absence of NH3/NH
. Readdition of
Na+ to the bath caused Vm to recover
(segment fg). At point g, the bath solution was
switched to a solution free of NH3/NH
but
containing 80 mM Na+, which caused a small
hyperpolarization (segment gh). Removal of bath
Na+ again (from 80 to 0 mM Na+) caused
hyperpolarization of Vm similar to the first
pulse (from 100 to 0 mM Na+), which was significantly
larger than the hyperpolarization of Vm in the
presence of NH3/NH
(segment hi). In three paired experiments,
Vm
(from 80 to 0 mM Na+) was
120 ± 4.0 mV in the
absence of NH3/NH
and only
61 ± 10.9 mV in the presence of NH3/NH
. These
experiments further indicate that the presence of
NH3/NH
is affecting the 0 Na+
response in oocytes expressing mENaC.
Effect of amiloride on Vm.
Inhibition of the ENaC by amiloride is one of the main properties that
characterize this channel. The next set of experiments was conducted to
investigate the effect of NH3/NH
on
Vm when mENaC was inhibited by amiloride. As
shown in Fig. 2C, in oocytes expressing mENaC, addition of
amiloride (100 µM) to the bath caused a significant hyperpolarization
(segment ab), as expected if ENaC is inhibited. In the
presence of amiloride, exposing the oocyte to
NH3/NH
caused depolarization of the
oocyte, which was preceded by a small transient hyperpolarization
(segment bcd). At point d, removal of bath
Na+, in the presence of amiloride and
NH3/NH
, caused a sustained further
depolarization of Vm (segment de). This is in contrast to the usual hyperpolarization observed when Na+ was removed in the absence of amiloride (compare with
Fig. 2, A and B). These changes were reversed on
readdition of Na+ to the bath (segment
ef) and then on removal of
NH3/NH
from the external solution
(segment fg). These experiments indicated that, in oocytes
expressing mENaC, amiloride inhibits the effect of
NH3/NH
on the Na+-induced
changes in Vm.
Na+ removal at low pHi or
in the presence of
NH3/NH
in oocytes
expressing mENaC.
In contrast to most other cells, where exposure to
NH3/NH
causes an initial increase in
pHi [NH
prepulse (9)], in
the oocytes, NH3/NH
causes a significant
sustained decrease in pHi (11, 38). One possibility is, therefore, that the low pHi is responsible
for the inhibition of the Na+-induced change in
Vm in the presence of
NH3/NH
. To investigate this possibility,
we decreased pHi of oocytes expressing mENaC, independently
of NH3/NH
, and measured the
Vm changes induced by removal of bath
Na+. As shown in Fig. 3,
removal of external Na+ caused no change in pHi
(segment ab) except the usual large hyperpolarization, which
readily recovered on readdition of Na+ (segment
bc). At point c, butyrate (20 mM) was added to the
bath, causing a significant decrease in pHi (segment
cd) and very little change in Vm. At low
pHi, Na+ was again removed from the bath, which
did not cause a significant further change in pHi, but
Vm became more negative (segment de). In five paired experiments, the hyperpolarization caused by removal of
Na+ was 146 ± 2.7 mV in the absence of butyrate, a
value not significantly different from 149 ± 5.4 mV in the
presence of butyrate (P > 0.05). All these changes
were completely reversible: Vm depolarized again on readdition of Na+ to the bath (segment
ef), and pHi fully recovered on removal of
butyrate (segment fg). At point g, the oocytes
were exposed to NH3/NH
, which caused a
small change in Vm, but pHi
decreased substantially (segment gh). When bath Na+ was removed again, pHi decreased a little
further and the cell hyperpolarized, as shown previously (segment
hi). The pHi decrease caused by
NH3/NH
(0.47 ± 0.12) was not
statistically different from the pHi decrease of 0.43 ± 0.07 caused by butyrate (P > 0.05). However, the
cell hyperpolarization caused by removal of Na+ in the
presence of butyrate (149 ± 5.4 mV) was significantly larger than
that in the presence of NH3/NH
(109 ± 5.5 mV, P < 0.05). These experiments are consistent
with a significant effect of NH3/NH
on
Na+-induced Vm changes, which are
independent of pHi.

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Fig. 3.
Na+ removal at low pHi in oocytes
expressing mENaC. Because NH3/NH causes
substantial intracellular acidification, this experiment examined the
effect of low pHi on changes caused by Na+
removal. Na+ removal at steady-state pHi caused
the usual large hyperpolarization of Vm but had
no effect on pHi (segment ab). Readdition of
bath Na+ completely reversed this effect (segment
bc). pHi was then acidified by exposing the oocyte to
20 mM butyrate (segment cd), and Vm
was not affected . At low pHi (point d), removal
of Na+ (in the continued presence of butyrate)
hyperpolarized the oocyte to a value similar to that at high
pHi in the absence of butyrate (segment de). On
readdition of Na+ in the presence of butyrate,
Vm recovered, but pHi was not
affected (segment ef). On removal of butyrate,
pHi recovered, but Vm was not
affected (segment fg). Exposing the oocyte to
NH3/NH caused a substantial decrease in
pHi comparable to that caused by butyrate and slightly
depolarized Vm (segment gh).
Subsequent removal of Na+ in the presence of
NH3/NH caused hyperpolarization
(segment hi) that was significantly less than that caused by
removal of Na+ in the presence of butyrate (compare with
segment de).
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Current changes in response to Na+
removal in the presence and absence of
NH3/NH
in oocytes
expressing mENaC.
To further study the effect of NH3/NH
on
Na+ transport in oocytes expressing mENaC, we measured the
changes in whole cell current in response to removal of
Na+. As can be seen in Fig.
4, removal of bath Na+ caused
an outward deflection in whole cell current (Im;
oocyte clamped at
60 mV, segment ab) that was readily
reversed when Na+ was returned to the bath (segment
bc). Exposing the oocyte to 20 mM total NH
caused an inward change in Im of 383 ± 5.1 nA (segment cd). This current is supposedly caused by
NH
entry through a nonselective cationic channel as
observed in water-injected oocytes (10, 11, 36). In the
presence of NH3/NH
, removal of bath
Na+ again resulted in outward deflection of
Im (segment de), which was reversed
when Na+ was restored to the bath (segment
ef). In nine paired experiments, the outward deflection in
the absence of NH3/NH
was 3.7 ± 0.8 µA but was only 2.7 ± 0.7 µA in its presence
(P < 0.01). These experiments are consistent with the
original observation that NH3/NH
inhibits
Na+ transport in the oocyte when mENaC is expressed.

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Fig. 4.
Whole cell currents (Im) in oocytes
expressing mENaC in the presence and absence of
NH3/NH . Representative experiment
indicates that oocytes expressing mENaC had a large inward current
( 2.9 ± 0.6 µA) at 60 mV. Removal of external
Na+ caused an outward deflection of the current to ~1
µA (segment ab) that was readily reversed on readdition of
Na+ (segment bc). Exposure to
NH3/NH caused a small increase in the
inward current of 0.38 ± 0.08 µA (segment cd).
Removal of Na+ in the presence of
NH3/NH caused a smaller outward
deflection in the current (segment de) compared with that in
the absence of NH3/NH (change in
current = 2.7 ± 0.7 µA, P < 0.01). Inward
current was restored on readdition of bath Na+
(segment ef).
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|
Amiloride inhibition of Na+ and
NH
whole cell currents in oocytes
expressing mENaC.
The data presented so far suggest that, in the presence of
NH3/NH
, Na+ transport through
the epithelial Na+ channel expressed in oocytes is
partially inhibited. Because mENaC is blocked by amiloride, we
investigated whether inhibiting mENaC would abolish the effect of
NH3/NH
on Na+-induced changes
in Im. As shown in Fig.
5, exposing oocytes expressing mENaC
(clamped at
60 mV) to amiloride completely blocked the inward current
(segment abc). In the presence of amiloride, no significant
change in Im occurred when Na+ was
removed (segment cd) or readded (segment de) to
the bath. The small changes in Im in response to
Na+ removal or addition in the bath were much different
from those in the absence of amiloride and resemble the
Na+-induced effects on Im that occur
in water-injected oocytes. In the continued presence of amiloride,
addition of NH3/NH
(segment
ef) or its removal (segment fg) also did not
cause significant changes in Im. It is important
to note that the NH
effect in the presence of
amiloride seems to be smaller than the effect of
NH3/NH
on Im when
mENaC is not blocked (compare with Fig. 4, segment cd).
Removal of amiloride restored the inward current to steady-state value
(segment gh). These experiments indicate that inhibiting
mENaC also abolished the NH3/NH
-mediated
effect on Na+ transport.

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Fig. 5.
Amiloride inhibition of Na+ and
NH currents in mENaC oocytes. Amiloride (100 µM)
completely inhibited the inward current in oocytes expressing mENaC
(segment abc). In the presence of amiloride, Na+
removal (segment cd) or addition of
NH3/NH (segment ef) did
not cause any significant changes in the current. The oocyte was
clamped at 60 mV.
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Effect of NH3/NH
on
Na+ current at positive potential.
NH3/NH
application to oocytes results in
two main effects: 1) a significant intracellular
acidification and 2) a huge depolarization of
Vm. Both effects are attributed to the influx of
NH
and a relatively low permeability of
NH3. In oocytes expressing mENaC,
NH3/NH
could influence Na+
transport by several mechanisms: 1) NH
permeation into the cell results in a pHi decrease, which
in turn inhibits the Na+ channel. 2)
NH
could be permeating through ENaC and, therefore,
affecting Na+ transport through the channel. 3)
Extracellular NH
modulates mENaC. In the next set of
experiments, we investigated whether limiting NH
entry could influence the Na+-induced response in oocytes
expressing mENaC. To do so, we clamped the oocyte at positive potential
(+10 mV) and measured the whole cell current in response to
Na+. As shown in Fig. 6,
clamping the oocyte at +10 mV, the whole cell current was positive.
Removal of bath Na+ (from 80 to 0 mM) caused an outward
deflection in Im (segment ab) that
readily recovered when bath Na+ was restored to control
(segment bc). At point c, exposing the oocyte to
NH3/NH
caused a sharp transient increase
in Im (segment cd) that settled to a
value more positive than that before addition of NH
,
indicating an outward current (segment de). This also
indicates that NH
entry through a conductive pathway
probably did not occur. When bath Na+ was removed in the
presence of NH3/NH
, there was the usual
positive shift in Im (segment
ef), and readdition of Na+ to the bath reversed
this effect with an inward shift of Im to its
original value (segment fg). Removal of bath
NH3/NH
(80 Na+) caused
Im to become more negative (segment
gh), reaching a value not significantly different from the
original value (at point a) before addition of
NH3/NH
. In four paired experiments, the
average change in Im caused by removal of bath
Na+ in the absence of NH3/NH
was 3.4 ± 0.72 µA but was only 1.7 ± 0.23 µA in the
presence of NH3/NH
(P < 0.05). These experiments indicate that even when NH
influx is limited, the presence of NH
still reduces
the Na+ response in oocytes expressing mENaC.

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Fig. 6.
Effect of NH3/NH (at
positive potential) on Na+ current in oocytes expressing
mENaC. At positive potential (clamped at +10 mV), the whole cell
current in the presence of 80 Na+ in the bath was 1.64 ± 0.8 µA. Removal of bath Na+ in the absence of
NH3/NH caused a larger outward deflection
in Im (segment ab) than in its
presence (segment ef). Addition of
NH3/NH at positive potential did not
cause an inward current but, rather, a transient outward deflection
that settled at a more positive value of Im
(segment cde).
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|
Whole cell conductance in the presence and absence of
NH3/NH
.
The last set of experiments was conducted to examine the effect of
NH3/NH
on whole cell conductance of
oocytes expressing mENaC. To do so, we plotted the whole cell currents
in relation to test potentials used to clamp the oocyte between
80
and +40 mV in steps of 20 mV. Figure 7
shows the I-V relationship in the presence and absence of
NH3/NH
. The results of this and similar
experiments indicate that in the presence of
NH3/NH
the slope of the curve (i.e.,
conductance) is smaller at negative potentials. However, the current at
positive potentials was significantly higher in the presence of
NH3/NH
, indicating the possible
activation of an outward current. The nature of the increased outward
current at positive potentials in the presence of
NH3/NH
is not known. In eight
experiments, the whole cell conductance calculated between
60 and
40 mV was 71.3 ± 17 µS in the absence of
NH3/NH
and 55.4 ± 15.3 µS in its
presence (P < 0.05). These results are consistent with
an inhibitory effect of NH3/NH
on
Na+ currents in oocytes expressing mENaC, at least at
negative potentials.

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Fig. 7.
Current-voltage (I-V) relationship for mENaC
in the presence and absence of NH3/NH .
Representative whole cell recordings show I-V relationships
of oocytes expressing mENaC in the presence and absence of
NH3/NH . Control oocytes are oocytes
expressing mENaC bathed in standard solution containing 80 mM
Na+. The same oocyte was then exposed to a solution
containing 20 mM total NH and 80 mM Na+.
The experiment indicates a small negative shift in reversal potential
and a significant outward current in the presence of
NH3/NH at positive potentials.
|
|
 |
DISCUSSION |
The ENaC plays a pivotal role in regulation of Na+
balance, which in turn is critical for regulation of extracellular
fluid volume and blood pressure. Regulation of ENaC is accomplished by
a complex interaction of several mechanisms, including hormonal factors
such as aldosterone and vasopressin and nonhormonal factors such as
pHi, Na+, and Ca2+ (8, 19,
35).
Ionic activities.
In this study we report the first measurements of the changes in
intracellular ionic activities that occur as a result of expressing
ENaC in oocytes. As shown in Table 1, these changes are very
significant. Three important observations are evident from these
results. First, although an increase in a
is expected when ENaC is expressed, the decrease in
a
and the increase in
a
were not known or measured previously.
Because the oocyte has a significant permeability to K+ and
Cl
, ignoring these changes in studies involving
measurements in the intact oocyte could result in significant errors.
For instance, one such possibility is an erroneous estimation of the
Na+ reversal potential, which will vary depending on the
Cl
content of the bathing medium. The second important
observation is the value of a
of 30 ± 1.8 mM shown in Table 1. This value is almost five times more than
that of control oocytes; however, it is much smaller than the value
(50-60 mM) usually calculated from reversal potential of
Na+ in oocytes expressing ENaC. In such studies, it has
been argued that the high a
(calculated
from the reversal potential) represents the microscopic value of
Na+ activity at the tip of the channel (6).
Although some compartmentalization may occur, it is very likely that
the contribution of other ions (and their respective membrane
permeabilities) to the reversal potential is underestimated in oocytes
expressing ENaC. A third point to note is that, in our experiments,
complete removal of external Na+ resulted in only small
changes in a
(
a
= 1.8 mM). Although the volume of
the oocyte is large, this was unexpected and indicates that even though
ENaC is usually overexpressed, total intracellular Na+ is
not readily exchangeable through the channel. In support of this
observation, we calculated the change in intracellular Na+
from the change in current caused by removal of external
Na+ (Fig. 4). For a change in current of 3.7 µA over a
period of ~100 s, the number of coulombs (Q) presumably
carried by movement of Na+ can be calculated as
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where current is measured in amperes and time in seconds. If
Na+ transfer is responsible for all this charge, then the
number of moles of Na+ that would result in this change can
be calculated from dividing by Faraday's constant, which results in
If the oocyte volume is ~0.9 µl (with the assumption of a
spherical volume with a diameter of 1.2 mm), then the calculated change
in intracellular Na+ concentration is
This value is much smaller than the presumed large changes in
a
and within the small range of
intracellular Na+ change measured with microelectrodes.
Inhibition of Na+ transport with
NH3/NH
.
The present study suggests that NH3/NH
inhibits Na+ transport via ENaC. Urinary excretion of
NH3/NH
is very important for acid-base
homeostasis. Two-thirds of net acid excretion in the urine is via
NH
, and in systemic acidosis intratubular
NH
concentrations and renal cortical ammonia are
increased (10). Under these conditions, urinary levels of
total ammonia can easily exceed 50 mmol/l. In the cortical collecting
duct, peritubular NH4Cl was shown to inhibit
transepithelial Na+ transport (22, 23). In
other tight epithelia, NH4Cl was also reported to influence
Na+ transport (20). This effect of
NH3/NH
on Na+ transport and
its importance in regulation of acid-base homeostasis raise the
possibility that NH3/NH
may directly or
indirectly affect transport through ENaC.
The first evidence for the inhibitory affect on ENaC by
NH
was obtained from measurements of voltage changes
induced by Na+ removal in the presence and absence of
NH
. The studies showed that 1) in the
presence of NH
, the hyperpolarization caused by
removal of bath Na+ was significantly inhibited,
2) the voltage inhibition by NH
is not due
to intracellular acidification caused by NH
, and
3) the effect of NH
on Na+
removal was completely blocked by amiloride, demonstrating that the
inhibitory effect of NH
is probably through an effect
on ENaC. The effects of NH
are not due to significant
changes in a
. Evidence for this comes
from the experiment of Fig. 2B, where NH
inhibited Na+-induced Vm changes
irrespective of whether initial external Na+ was 100 or 80 mM. This is further supported by our previous observation that complete
removal of external Na+ caused only a small change in
a
, measured with microelectrodes.
Measurement of current in response to Na+ removal further
demonstrated the inhibition of ENaC by NH
. Again, in
the presence of 100 or 80 mM external Na+,
NH
inhibited the outward current caused by removal of
bath Na+. In the presence of amiloride, neither
Na+ removal nor NH
resulted in
significant changes in current. The voltage and current data indicate
that NH
inhibits ENaC.
Many studies (7, 19, 33, 42) have addressed the role of pH
in regulating ENaC activity. As reviewed by Lyall et al. (28), decreases in extracellular pH and/or pHi
have usually been reported to decrease Na+ transport in
tight epithelia. The direct studies of Chalfant et al.
(13) on oocytes expressing ENaC recently reported that a
decrease in pHi, but not extracellular pH, reduced
single-channel open probability and open time without altering
single-channel conductance. This study further indicated that the
-subunit of ENaC is directly regulated by pHi. In
contrast, however, Awayda et al. (7) reported that luminal
acidosis over a period of minutes stimulates Na+ current in
A6 cells. On the other hand, more prolonged changes in pH, such as in
vivo systemic acidosis, may have complicated effects. For example, Kim
et al. (26) reported that long-term acid loading in vivo
by NH4Cl produced a large decrease in the abundance of
-
and
-subunits of ENaC but had no effect on the
-subunit.
In our study, the presence of NH
clearly affects ENaC
in a manner distinct from a pHi inhibition of the channel.
Although NH
inhibited the whole cell conductance
(consistent with a pHi effect), a decrease in
pHi is unlikely to account for all the inhibitory effect of NH
on the basis of the studies with butyrate in Fig.
3 and the studies with voltage clamp at positive potential in Fig. 6.
These latter experiments demonstrated that NH
still
inhibited the Na+ current, even though there was no
apparent conductive NH
influx as evidenced from the
absence of inward current on exposure to NH
. This
raises the likelihood that external NH
, and not
necessarily intracellular acidification, is responsible for inhibiting
the Na+ current.
At positive potentials, the presence of NH
caused a
significant activation of an outward current not seen in control
oocytes. This raises the possibility of NH
interacting with ENaC in a manner more complicated than simple inhibition. The nature of this voltage-dependent outward current is not
clear and needs to be pursued further.
Although the interaction of several cations, such as Li+
and K+, with ENaC has been investigated, that of
NH
has not been studied to our knowledge. A
complicating factor in addressing this issue is the significant change
in pHi caused by NH
.
Possible physiological implications.
The effects of NH
are particularly relevant in states
of systemic acidosis during which cortical and medullary interstitial
ammonia concentrations rise. Acidosis is well known to be associated
with natriuresis and diuresis. Although this has been attributed in
part to decreases in proximal tubular reabsorption, effects of acidosis
on distal nephron transport are also reported. Metabolic acidosis has
been shown to inhibit K+ secretion and Na+
reabsorption in the distal tubule and collecting duct (30, 41). As discussed above, low pHi has been shown to
inhibit Na+ transport (presumably via ENaC inhibition) in a
variety of tight epithelia (reviewed in Ref. 28).
Long-term acidosis has recently been reported to decrease the distal
tubule Na-Cl cotransporter and
- and
-subunits of ENaC
(26). Low pH has also been shown to inhibit
Na+-K+-ATPase (16). In addition to
the effects of pH and systemic acidosis, the present studies and some
prior work suggest that the increased levels of
NH3/NH
that occur with acidosis may have
additive effects to inhibit Na+ transport. We previously
demonstrated that NH
inhibited transepithelial
Na+ and K+ transport in cortical collecting
ducts in vitro. The present studies extend these findings to
demonstrate that NH
inhibits Na+
transport via ENaC. Therefore, acidosis inhibits renal Na+
transport via a variety of mechanisms, including the effects of
NH3/NH
, acting in concert. Recently, Frank et al. (17) suggested that renal ammonia content can
act as a type of extracellular signaling molecule in integrating the response to systemic acidosis; the present studies would be consistent with such a role.
In conclusion, the results of this study demonstrate that
NH
inhibits Na+ transport when ENaC is
expressed in the oocyte. Although some pHi effect cannot be
ruled out, it is likely that a decrease in pHi is not the
major cause of this inhibition. These results are important for
understanding the role of NH
in modulating
Na+ transport in vivo in states of systemic acidosis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Emile L. Boulpaep for important suggestions and
valuable discussions, Dr. Keith Elmslie for critical comments on the
data, and Stephanie Palmer for secretarial and technical help.
 |
FOOTNOTES |
This work was supported by American Heart Association Grant 0050547N,
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-54952, the Department of Veterans Affairs, and by DCI, Inc.
Address for reprint requests and other correspondence: N. L. Nakhoul, Dept. of Medicine, Sect. of Nephrology, SL-45, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: nakhoul{at}tulane.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.
Received 26 February 2001; accepted in final form 7 May 2001.
 |
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