Transport of NH3/NH
in oocytes
expressing aquaporin-1
Nazih L.
Nakhoul,
Kathleen S.
Hering-Smith,
Solange M.
Abdulnour-Nakhoul, and
L. Lee
Hamm
Section of Nephrology, Departments of Medicine and Physiology,
Tulane University School of Medicine, and Veterans Affairs Medical
Center, New Orleans, Louisiana 70112
 |
ABSTRACT |
The aim of this study was to determine whether
expressing aquaporin (AQP)-1 could affect transport of NH3.
Using ion-selective microelectrodes, the experiments were conducted on
frog oocytes (cells characterized by low NH3 permeability)
expressing AQP1. In H2O-injected oocytes, exposure to
NH3/NH
(20 mM, pH 7.5) caused a sustained
cell acidification and no initial increase in pHi (as
expected from NH3 influx), and the cell depolarized to near
0 mV. The absence of Na+, the presence of Ba2+,
or raising bath pH (pHB) did not inhibit the magnitude of
the pHi decrease or result in an initial increase in
pHi when NH3/NH
was added.
However, after the cell was acidified (because of
NH3/NH
), raising pHB to 8.0 caused a slow increase in pHi but had no effect on membrane
potential. The changes in pHi with raising pHB
did not occur in the absence of NH3/NH
. In AQP1 oocytes, exposure to NH3/NH
usually resulted in little or no change in pHi, and in the
absence of Na+ there was a small increase in
pHi (the cell still depolarized to near 0 mV). However,
after exposure to NH3/NH
, raising
pHB to 8.0 caused pHi to increase more than two
times faster than in control oocytes. This increase in pHi
is likely the result of increased NH3 entry and not the
result of NH
transport. These results indicate that
1) the oocyte membrane, although highly permeable to
NH
, has a significant NH3 permeability
and 2) NH3 permeability is enhanced by AQP1.
NH3 permeability; intracellular pH
 |
INTRODUCTION |
TWO-THIRDS OF NET
ACID secretion in the urine is via ammonium
(NH
) where urinary levels of total ammonia can easily
reach values in excess of 50 mmol/l. Along the nephron,
NH
is synthesized and secreted in the lumen of the
proximal tubule (22), reabsorbed in the thick ascending
limb of Henle's loop (15, 22), and is secreted again into
the lumen of the collecting duct (14, 19). The classic
model of NH3/NH
transport across cell
membranes assumes that NH
transport occurs via
channels (e.g., K+ channels) or transporters (e.g.,
Na-K-2Cl cotransport or Na/H exchange), but NH3 permeation
across cell membranes occurs by nonionic diffusion through the lipid
phase of the membrane. This concept has been supported by experiments
in many different cell types. However, several studies indicate that
certain nephron segments have restricted diffusion to NH3
(19, 22). This is particularly evident in the apical
membrane of the medullary thick ascending limb of Henle where
NH3/NH
transport plays an important role
in acid-base homeostasis. More recently, several other cell membranes
have been observed to have very low permeability to NH3
(21, 22, 29, 33, 36). Although the reasons for these
differences in NH3 permeability are not clear, variations
in lipid composition are certainly a possibility. Another strong
possibility is that certain membrane proteins, such as aquaporins
(AQPs), may facilitate NH3 transport and account for some
of the differences among membranes from different cell types.
Water, like ammonia, was traditionally thought to cross membranes of
most cells by solubility diffusion through the lipid bilayer. The
existence of membranes with restricted H2O permeability as
with many tight, barrier epithelia (21, 34, 41) and the discovery of H2O-selective channels (27, 28)
challenged the universality of this concept. AQPs belong to a family of
intrinsic membrane proteins functioning primarily as H2O
channels that facilitate significant transmembrane transport of
H2O in response to small osmotic gradients. The AQP(s)
family of mammalian H2O channels is growing and includes 10 homologs so far. All members have a major and common function as
selective pores through which H2O crosses plasma membranes.
They all contain structural motifs similar to AQP1, the most studied
AQP (3), with amino acid identities ranging from 20 to
52% (35). They are widely distributed in tissues but with
little overlap. Their functional and structural properties are being
actively investigated and have been reviewed lately (2, 5, 20,
35). For the most part, AQPs appear to function in a manner that
provides a transcellular route of H2O transport. However,
the selectivity of AQPs to transport of other solutes and/or gases has
not been well studied. AQPs are now classified into the following two
groups: those that primarily transport H2O (orthodox AQPs)
and others that can transport small molecules such as glycerol and urea
(1, 2). In a recent study, CO2 permeability in
oocytes expressing AQP1 was found to be ~40% higher than in control
oocytes, suggesting that CO2 can pass through AQP1
(24). These findings were later confirmed in studies
showing HgCl2 inhibition of CO2 transport
through AQP1 in oocytes (10) and in studies in
reconstituted proteoliposomes (26). These were the first
studies to demonstrate transport of a gas through a channel.
The aim of this study was to determine whether expressing AQP1 could
facilitate transport of NH3. The experiments were conducted on oocytes because of the ease of expressing AQP and the very low
permeability of oocytes to NH3. Because NH3 (a
gas) has a molar volume (24.9 cm3/mol) that is similar to
that of H2O (18 cm3/mol), permeation of
NH3 through AQP1 was hypothesized.
 |
METHODS |
General Methods
Oocytes were used to address the specific aims of this study
because they provide distinct advantages that are not available in
other preparations. First, the oocyte is a powerful system for
expressing cloned genes. In this case, expression of H2O
channels by injecting cRNA for AQP1 has proven to be very effective and highly efficient. Second, oocytes can be easily injected with RNA
or other substances on multiple occasions without suffering extensive
or irreversible damage. Third, oocytes have low basal permeability to
NH3. This property is essential for this study where we are
looking for a differential effect on the relative permeability of this
molecule. Finally, intracellular measurements using ion-selective
microelectrodes or electrical measurements using voltage clamp are very
stable, reproducible, and relatively easy in oocytes.
Solutions.
The standard bathing solution used was ND-96 medium containing (in mM)
96 NaCl, 2 KCl, and 1.8 CaCl2 buffered with 5 HEPES to pH
7.5. The NH3/NH
solution contained 20 mM
NH4Cl (replacing NaCl) at the desired pH. Osmolarity of all
solutions was ~200 mosmol/l. OR3 medium (GIBCO-BRL Leibovitz media)
contained glutamate and 500 units each of penicillin/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 H2O containing 0.2% tricaine
(3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO). A 1-cm
incision was made in the abdominal wall, one lobe of the ovary was
externalized, and the distal portion was cut. The wound was closed by a
few stitches in the muscular plane of the peritoneum using 5-0
catgut followed by two to three stitches in the abdominal skin using
6-0 silk. The excised piece of ovary containing oocytes was rinsed
several times with Ca-free ND-96 solution until the solution was clear.
The tissue was then agitated in ~15 ml sterile filtered Ca-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 was purified by the
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, followed by proteinase K (1 mg/ml) digestion. DNA was then
phenol-chloroform extracted two times followed by chloroform extraction
and ethanol precipitation. cDNA was transcribed in vitro with T7 RNA
polymerase. The in vitro synthesis of capped RNA (cRNA) transcripts was
then accomplished using the mCAPTM 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 (31).
Injection of oocytes.
Oocytes in OR3 medium were visualized with a dissecting microscope and
were injected with 50 nl of cRNA for AQP1 (0.02 µg/µl, for a total
of 1 ng of RNA). 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 connected to a
Drummond nanoject displacement pipette (Drummond Scientific). Injected
oocytes were used 3-5 days after injection with RNA.
Electrophysiological measurements in frog oocytes.
The pH microelectrodes were of the liquid ion exchanger type, and the
resin (hydrogen ionophore I, cocktail B) was obtained from Fluka
Chemical (Ronkonkoma, NY). Single-barreled microelectrodes were
manufactured as described earlier (30). Briefly,
alumina-silicate glass tubings (1.5 mm OD × 0.86 mm ID; Frederick
Haer, Brunswick, MD) were pulled to a tip <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 (4). 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. The average slope
of 50 electrodes used in our studies was 59.0 ± 1.0.
Two-electrode voltage clamp.
Whole cell currents were recorded using two-electrode voltage clamp
(OC-725; Warner Instruments, Hamden, CT). For those 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. For current measurements, oocytes were clamped at
60 mV, and long-term readings of current were sampled at a rate of one per second. Inward flow of
cations is defined by convention as inward current (negative current).
For measurement of whole cell conductance, oocytes were periodically
pulsed (6 times/min) with a constant current (100 nA), and voltage
deflections were recorded. Whole cell conductance was calculated from
the current-to-voltage ratio.
The oocyte, visualized with a dissecting microscope, was held on nylon
mesh in a special chamber through which solutions flow continuously at
a rate of 3-5 ml/min. Incoming solution passed through a
H2O-jacketed stainless steel tube (22°C). Solutions (6 possible) were switched by a combination of a six-way and a four-way
valve system that was activated pneumatically. Very little dead space
was present, and complete solution changes in the chamber occurred in
6-8 s.
Curve fitting, statistics, and data analysis.
Initial rates of change in intracellular pH (pHi;
dpHi/dt) were determined from the slope of the
line obtained by fitting pHi vs. time to a linear
regression line. In all the experiments, values were reported as
means ± SE. 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);
"n" is the number of observations and is shown in
parentheses. Results are considered statistically significant at
P
0.05.
 |
RESULTS |
NH3/NH
-Induced
pHi and Vm Changes in
H2O-Injected Oocytes
To study NH3/NH
transport, we relied
on measurements of pHi and cell voltage
(Vm) changes induced by exposing the oocyte to a
solution containing 20 mM NH4Cl at the same osmolality
(~200 mosmol/lH2O) and pH (7.5) as the control (ND-96)
solution. The pHi changes that occur when both
NH3 and NH
are permeable, as is the case
in most cells, were well described by Boron and DeWeer
(6). When NH3/NH
is applied,
there is usually an initial cellular alkalinization as a result of the
influx of NH3 (usually faster than NH
)
that consumes intracellular H+ to form NH
and thus raises cell pH. In many instances, when NH
is also permeable, the initial rise in pHi is followed by a
slow decrease in pHi known as "plateau phase
acidification." The removal of external
NH3/NH
reverses this process, causing a
decrease in pHi (an acute acid load) as the accumulated intracellular NH
splits into NH3 (which leaves the cell), leaving behind H+, which acidifies the
cell. In our experiments (Fig. 1),
exposure of control oocytes (H2O injected) to
NH3/NH
caused a substantial
pHi decrease (segment ab) with very little or no
initial increase in pHi. Our data also indicate a huge
depolarization of the cell, leading to almost a complete collapse of
Vm. These observations have been reported
previously (8, 12, 32) and indicate that the permeability
of the native or control oocytes to NH3 is very low
compared with the entry of NH
. The low
NH3 permeability in the oocyte resembles that of other NH3-impermeable cells, such as those of the medullary thick
ascending limb of Henle's loop. The huge depolarization, however, is
unique in the oocyte and is consistent with an NH
conductive pathway (7, 9, 12). Removal of
NH3/NH
reversed these changes, with
Vm recovering readily, whereas the recovery of
pHi was slow (Fig. 1, segment bc).

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Fig. 1.
Effect of NH3/NH on
H2O-injected oocytes. In control oocytes (H2O
injected), exposure to NH3/NH (20 mM)
decreased intracellular pH (pHi) by 0.29 ± 0.05 (segment ab) and depolarized the cell by 48 ± 2.3 mV.
The pHi decrease and depolarization of the cell are
consistent with a significant NH influx. The absence
of any initial pHi increase indicates low permeability to
NH3. Removal of NH3/NH
reversed these changes (segment bc).
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Results from experiments similar to those of Fig. 1 indicated that
steady-state Vm and pHi of
H2O-injected oocytes bathed in HEPES-Ringer averaged
55 ± 1.1 mV (n = 30) and 7.31 ± 0.03 (n = 28), respectively. In the presence of
NH3/NH
, the oocyte depolarized to
7 ± 1.8 mV (n = 22), and pHi
decreased to 7.03 ± 0.05 (n = 20). The rate of
pHi decrease caused by NH3/NH
was
9.2 ± 1.1 × 10
4 pH/s (n = 18), whereas the rate of pHi recovery upon removal of
NH3/NH
was 2.4 ± 0.3 × 10
4 pH/s (n = 10).
Effect of NH3/NH
on
H2O-Injected Oocytes in the Absence of External
Na+
The working hypothesis for assessing NH3 and
NH
transport from pHi measurements
predicts that a significant NH3 permeability would lead to
an increase in pHi, whereas NH
permeability would cause a decrease in pHi. Figure 1
clearly shows that in control oocytes NH3 permeability (and
therefore any pHi increase associated with it) is masked by
a bigger NH
influx, resulting in the observed
pHi decrease. Because many pHi-regulating mechanisms that can affect the NH3- and/or
NH
-induced pHi changes are
Na+ linked, we exposed oocytes to
NH3/NH
in the absence of external
Na+.
As shown in Fig. 2, exposing oocytes to
NH3/NH
caused a pHi decrease
(segment abc) and depolarization, as observed previously in
Fig. 1. In this experiment (as with several other experiments), there
was a small pHi increase (segment ab),
consistent with an apparent small NH3 permeability. These changes were fully reversible upon removal of
NH3/NH
(segment cd). At
point d, Na+ was removed from the external
solution (replaced with
N-methyl-D-glucamine), which caused a
sustained hyperpolarization, but pHi did not change (segment de). In the continued absence of Na+,
addition of NH3/NH
still caused a pHi decrease (segment ef) and great
depolarization of the oocyte. pHi and
Vm recovered when
NH3/NH
was removed (segment
fg). In seven experiments, the NH3- and/or
NH
-induced pHi decrease in the absence of
Na+ was 0.18 ± 0.06, and the rate of acidification
was
6.4 ± 1.7 × 10
4 pH/s. Although both
values were less than the NH3- and/or
NH
-induced pHi decrease in the presence
of Na+ (0.28 ± 0.05 and
9.2 ± 1.1 × 10
4 pH/s, n = 20, respectively), the
differences were not statistically significant (P > 0.05).

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Fig. 2.
Effect of NH3/NH on
H2O-injected oocytes in the absence of external
Na+. NH3- and/or NH -induced
pHi decrease and depolarization are not inhibited in the
absence of external Na+. In paired experiments, exposing
control (H2O-injected) oocytes to 20 mM
NH3/NH in the presence of Na+
decreased pHi by 0.15 ± 0.02 and substantially
depolarized the cell by 45 ± 5.9 mV (segment abc). In
this experiment, there was a small initial increase in pHi
(segment ab) consistent with a small NH3
permeability. These effects were reversed upon removal of
NH3/NH from the bathing solution
(segment cd). Removal of external Na+ did not
affect pHi (segment de) but moderately
hyperpolarized the cell (change in Vm = 15 ± 2.6 mV). Exposing oocytes to
NH3/NH in the absence of Na+
still decreased pHi by 0.18 ± 0.06 (segment
ef) and depolarized the cell by 74 ± 3.3 mV. Removal of
NH3/NH reversed these changes
(segment fg).
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In several membranes with low NH3 permeability and fast
NH
entry, such as the thick ascending limb of Henle,
one possible route of NH
transport is thought to be
through K+ channels (13, 21, 37). To check
whether inhibiting K+ channels could block
NH
influx, we performed the experiments depicted in
Fig. 3. As shown in this experiment,
exposure of H2O-injected oocytes to
NH3/NH
in the absence of Na+
caused a pHi acidification (segment ab), and
Vm became more positive, as shown in Fig. 2.
Removal of NH3/NH
reversed these changes
with a prompt Vm recovery and a slow
pHi increase toward the initial steady-state value
(segment bc). Readdition of external Na+ (at
point c) did not affect pHi, which continued to
recover (segment cd), but the cell depolarized slightly.
When Na+ was removed again, there was no change in
pHi but a small hyperpolarization of ~10 mV
(segment de). Addition of Ba2+ (1 mM)
depolarized the cell by ~33 mV but did not affect pHi (segment ef). Addition of
NH3/NH
in the presence of
Ba2+ and the absence of Na+ still caused a
substantial depolarization and acidification of the cell (segment
fg). All changes were fully reversible (segment ghi).
In three similar experiments, Ba2+ slowed down the rate of
pHi decrease but did not prevent the NH
-induced depolarization or illicit an
NH3-induced intracellular alkalinization.

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Fig. 3.
Effects of NH3/NH on
H2O-injected oocytes in the presence of Ba2+.
The first part of the experiment shows the usual and reversible effects
of NH3/NH . In the absence of bath
Na+, NH3/NH caused a decrease
in pHi (segment ab) and a cellular
depolarization, as shown earlier. pHi and
Vm fully recovered upon removal of
NH3/NH (segment bc) and
addition of Na+ (segment cd). In the second
part, Ba2+, in the absence of Na+, depolarized
the oocyte by 30 mV but did not affect pHi (segment
ef). In the continued presence of Ba2+,
NH3/NH still caused a decrease in
pHi (segment fg), and Vm
became more positive. The changes in pHi and
Vm were reversed upon removal of
NH3/NH (segment gh) and
Ba2+ (segment hi).
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Effect of NH3/NH
on
Oocytes Expressing AQP1 in the Absence of External
Na+
If NH3 permeability is enhanced by AQP1, then the
NH3- and/or NH
-induced intracellular
acidification is expected to be affected but the depolarization of the
cell is not necessarily affected. To investigate this possibility, we
exposed oocytes expressing AQP1 to NH3/NH
in the absence of external Na+ as was done in the
experiments of Fig. 2 on control oocytes. As shown in Fig.
4, removal of external Na+
caused hyperpolarization from
59 ± 4.0 to
70 ± 5.3 mV
(n = 3), and there was no significant effect on
pHi (segment ab). However, subsequent exposure
to NH3/NH
, in the absence of external
Na+, caused a small pHi increase (segment
bc) rather than the significant acidification observed in control
oocytes (compare with Fig. 2, segment ef), even though the
oocyte depolarized from
70 ± 5.3 to 1 ± 2.0 mV
(n = 5). These changes were reversed upon removal of
NH3/NH
(segment cd). One
likely possibility indicated by these experiments is that, in oocytes expressing AQP1, NH3 permeability was enhanced, which
resulted in a complete inhibition of cell acidification.
NH
influx was apparently not affected, as
demonstrated by the significant depolarization of the cell and as shown
previously in control oocytes. In some experiments, the
NH3- and/or NH
-induced acidification was
still evident in AQP1-expressing oocytes. In those experiments, it was
also evident that the pHi recovery, as well as the recovery
of Vm, was much slower than usual, and pHi did not recover completely. In the majority of
experiments on AQP1 oocytes, however,
NH3/NH
induced either no change or a
pHi increase rather than the large decrease in
pHi usually observed in control oocytes.

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Fig. 4.
Effect of NH3/NH on oocytes
expressing aquaporin (AQP) 1 in the absence of external
Na+. In oocytes expressing AQP1, removal of Na+
from the bath hyperpolarized the oocyte but did not cause any
significant change in pHi (segment ab). Exposure
to NH3/NH in the absence of
Na+ caused a small pHi increase (rather than a
decrease) and significant depolarization (segment bc). The
depolarization indicates that NH transport is
probably not affected. The changes in Vm and
pHi were reversed upon removal of
NH3/NH .
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Effect of NH3/NH
at
High Bath pH on H2O-Injected Oocytes
In AQP1 oocytes, NH3/NH
induced a
slight increase in pHi in contrast to the usual large
acidification observed in the absence of AQP1. Any pHi
increase is likely the result of an increase in NH3
permeability. Even in control oocytes with a minimal transient
NH3-induced intracellular alkalinization, a significant
NH3 permeability has to exist for the observed magnitude of
NH
-induced acidification to occur (see
DISCUSSION). Nevertheless, this permeability is small
compared with the apparently large NH
permeability, resulting in a net intracellular acidification and completely masking
any alkalinization when oocytes are exposed to
NH3/NH
as observed above (see Figs.
1-3).
To maximize the NH3-induced signal (in the face of a
substantial NH
-induced acidification), we conducted
the experiments shown in Fig. 5. In
H2O-injected oocytes, NH3/NH
(20 mM) at external pH [bath pH (pHB)] of 7.5 decreased
pHi (segment abc) and depolarized the cell, as
previously described. In this experiment, as occasionally seen, a
transient small alkalinization (segment ab), presumably resulting from NH3 entry, was observed. At point
c, switching the external solution to
NH3/NH
(20 mM) at pHB of 8.0 (which raises outside NH3 ~3-fold) caused an increase in
pHi (segment cd) with no significant change in
Vm, suggesting that the pHi change
is primarily the result of NH3. When pHB was
subsequently raised to 8.5 (thus raising external NH3
further at the expense of NH
), pHi
increased again (segment de) with no significant
change in Vm. The pHi increase
was reversed when external NH3/NH
was
returned to pH 7.5 (segment ef).

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Fig. 5.
Effect of NH3/NH at higher bath pH
(8.0 and 8.5) on H2O-injected oocytes. Exposing control
oocytes to NH3/NH (at bath pH 7.5) caused
a small transient alkalinization (segment ab) followed by a
substantial decrease in pHi (segment bc), and
the cell depolarized significantly as shown previously. After a new
steady state was reached, raising bath pH from 7.5 to 8.0 (and
therefore increasing external NH3 concentration) caused a
slow increase in pHi (segment cd) at a rate of
3.1 × 10 4 pH/min. Elevating bath pH further from
8.0 to 8.5 caused another increase in pHi (segment
de) at a rate of 7.6 × 10 4 pH/min.
Vm did not change significantly throughout,
indicating that the pHi increase is the result of increased
NH3 entry rather than a change in NH
transport. Switching back to NH3/NH at pH
7.5 reversed the changes in pHi (segment ef).
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Effect of NH3/NH
at
High Bath pH on Oocytes Expressing AQP1
Preequilibrating oocytes with NH3/NH
(at pHB of 7.5) and then raising pHB, thus
elevating external NH3, resulted in unmasking the
NH3 permeability. If NH3 permeability is
facilitated by AQP1, then the same protocol should cause a faster rate
of alkalinization in response to elevated external NH3. As
shown in Fig. 6, exposing AQP1 oocytes to
NH3/NH
at pHB of 7.5 caused
an increase, not a decrease, in pHi (segment ab), and the cell depolarized substantially. The pHi
increase is consistent with increased NH3 permeability, as
described earlier. In 10 experiments, pHi increased from
7.26 ± 0.03 to 7.32 ± 0.04, and the rate of pHi
increase was 4.4 ± 1.8 × 10
4 pH/s. The oocyte
depolarized from
52 ± 2.3 to
3 ± 1.6 mV. Switching to
NH3/NH
at pHB of 8.0 significantly increased pHi from 7.32 ± 0.04 to
7.66 ± 0.08 (segment bc), and the rate of
pHi increase was 9.1 ± 1.5 × 10
4
pH/s. Lowering external NH3, by switching back the bath
solution to NH3/NH
at pHB of
7.5, reversed the change in pHi (segment cd).
Exposing the oocyte to control solution caused a full recovery of
pHi and Vm (segment de).

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Fig. 6.
Effect of NH3/NH at higher
bath pH (8.0) on oocytes expressing AQP1. Exposing AQP1 oocytes to
NH3/NH (at bath pH 7.5) caused a small
and slow increase in pHi (segment ab).
Increasing bath pH from 7.5 to 8.0, thus increasing external
NH3 concentration, resulted in a significant increase in
pHi (segment bc) at a rate of 12 × 10 4 pH/min. Switching bath solution to
NH3/NH at pH 7.5 reversed the change in
pHi, causing a decrease in pHi (segment
cd) with no effect on Vm. Both
pHi and Vm fully recovered when bath
solution was switched to control (segment de).
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The change in pHi (0.32 ± 0.06) at pHB of
8.0 was significantly more than that in control oocytes (0.05 ± 0.02), and the rate of pHi increase was more than two times
faster (9.1 ± 1.5 vs. 4.3 ± 1.2 × 10
4
pH/s). In fact, in AQP1 oocytes, the rate of the
NH3-induced increase at pHB of 7.5 was faster
than that at pHB of 8.0 in control oocytes. During these
maneuvers, Vm was stable and did not change significantly toward a more negative value; hence, the flux of NH
was likely unchanged. Therefore, the difference in
the change in pHi (and the rate) in response to increased
NH3 concentration in the bath (as when pHB is
raised) can be used to estimate the change in NH3
permeability secondary to AQP1 expression. These experiments
demonstrate that expressing AQP1 significantly enhanced NH3
permeability in oocytes.
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DISCUSSION |
In investigating NH3 transport and the role of AQP1,
we measured changes in pHi and Vm
induced by exposing oocytes to solutions equilibrated with
NH3/NH
(20 mM NH4Cl). In such
an approach, as NH3 enters the cell, it combines with intracellular H+ to form NH
, which will
lead to an increase in pHi. In contrast,
NH
entry into the cell would lead to a decrease in
pHi when NH
dissociates intracellularly,
releasing NH3 and H+. Therefore, an increase in
NH3 permeability would cause an alkaline shift in
pHi. Whereas a decrease in NH
permeability could cause an increase in pHi, it would also
lead to a change in Vm (with the cell expected
to hyperpolarize). Relying on such measurements, the results of the
present study indicate that 1) oocytes have low but finite
permeability to NH3, 2) oocytes have high
apparent permeability to NH
, and 3)
expression of AQP1 increases permeability of NH3 without substantially affecting NH
transport in the oocyte.
We used Xenopus oocytes in this study because of the
distinct advantages they provide. First, the oocyte is a very
convenient system for expressing exogenous proteins and transporters.
Second, it is easy to obtain long-time and stable measurements by
microelectrodes. This is highly advantageous because pHi
measurements by microelectrodes are very reliable and more accurate
than other methods. Third, pHi changes that occur in the
oocytes are relatively slow; therefore, accurate measurements can be
obtained. This contrasts with measurements in other preparations in
which NH3-induced pHi changes are extremely fast, as is the case in most mammalian cells (25), or in
which NH3 permeability varies dramatically (and the
background permeability is very high) with different lipid composition
as is the case with studies in lipid bilayers (40).
In the kidney, NH3 transport is very important, yet not
well studied. In the proximal tubule, significant NH3
transport occurs in addition to NH
transport
(18). NH3 transport is the predominant mode of
entry of total ammonia in the collecting duct (21). The
predominant mechanism of NH3 transport in most tubular
segments is presumed to be lipid phase diffusion through the membrane.
However, NH3 permeability varies considerably along the
nephron. For example, whereas significant NH3 diffusion
occurs in the proximal tubule (18), permeability to
NH3 is substantially less and diffusion of NH3
is more restricted in the cortical and medullary collecting ducts
(16, 19, 22). Other studies (21) reported
very little permeability of the apical membrane to NH3 in
the thick ascending limb of Henle's loop. Various studies proposed
several mechanisms to account for this variability in NH3
transport ranging from lipid solubility differences to specific
channels or carriers to nonspecific pathways that may mediate
NH3 transport. Although speculative, the correlation between low H2O permeability and low NH3
permeability in the thick ascending limb and the collecting duct has
been noted (21) and is suggestive that NH3 may
be transported through specific carriers, perhaps even H2O channels.
In this study, the data indicate that, in control
(H2O-injected) oocytes and in AQP1 oocytes, permeability of
NH
is particularly high. The evidence for this is
based on three observations. First, exposure of oocytes to
NH3/NH
caused a substantial decrease in
pHi consistent with a big influx of NH
and its subsequent release of intracellular H+, leading to
intracellular acidification. Second, exposure to NH3/NH
caused a huge depolarization of
the oocyte to near 0 mV, indicating an electrogenic pathway also
consistent with NH
influx. The amount of cell
depolarization caused by 20 mM NH4Cl (48 ± 1.9 mV) is
substantially bigger than the depolarization caused by 20 mM
K+ (18 ± 1.1 mV, n = 3) and is not
inhibited by Ba2+ (see Fig. 3). Third,
NH3/NH
exposure of oocytes induced an
inward current in voltage-clamp experiments (23). These
observations confirm previous studies which suggested that
NH
influx in oocytes occurs through a nonselective
cationic channel (7, 8, 12).
It is also likely that at least a fraction of NH
influx could occur through an electroneutral pathway such as Na-K-2Cl
cotransport. Several studies have addressed this issue and concluded
that the conductive pathway of NH
is probably the
major contributor to NH
influx. Sasaki et al.
(32) could only slightly inhibit
NH
-induced acidification in the presence of
furosemide, and Burckhardt and Fromter (8) showed no
significant effect of bumetanide on NH
-induced Vm changes or cellular acidification. Our own
experiments, with bumetanide, agree with the above studies.
This high permeability of NH
and its effects on
Vm and pHi complicates examination
of NH3 permeability in the oocyte. The path of
NH
transport has been poorly defined and difficult to
inhibit but is presumably a cationic conductive pathway. In preliminary
experiments, cinnamate, diphenylamine-2-carboxylic acid, and bumetanide
did not inhibit the NH
-induced depolarization. Other inhibitors, including Cs+, tetramethylammonium, and
quinidine were tried by other investigators (8, 12) and
also did not inhibit the NH
-induced depolarization of
the cell. In the absence of a specific inhibitor of this
NH
pathway, an NH
-induced pHi decrease could conceivably mask an
NH3-induced pHi increase. Our data, however,
indicate that oocytes have a low but finite permeability to
NH3.
The low permeability of oocytes to NH3 compared with that
of NH
is evident because there is usually no apparent
NH3-induced alkalinization. In some experiments, however, there was a small transient alkalinization that preceded the sustained pHi decrease in response to
NH3/NH
. However, our data indicate that,
unlike other virtually NH3-impermeable membranes such as
the distal colon or gastric glands or even the medullary thick
ascending loop, the oocyte membrane is permeable to NH3.
The evidences for this are the following observations. First, the
significant decrease in pHi in response to
NH3/NH
cannot be achieved unless
NH3, generated intracellularly from the dissociation of
NH
, can exit the cell. In control oocytes, for
example, NH3/NH
exposure causes a
pHi decrease of 0.29 ± 0.04 (n = 20).
With an estimated buffering power of 12.4 mM/pH, calculated from
CO2 pulses (24), and assuming that this
pHi decrease is caused solely by the influx of
NH
, with no significant permeability to
NH3, intracellular NH
concentration has
to reach an impossibly high level of ~571 mM (see
APPENDIX). In other words, for the influx of
NH
to cause this amount of decrease in
pHi, the newly generated NH3 has to leave the
oocyte, hence, a permeable membrane, thus driving the dissociation
reaction of NH
NH3 + H+ to the right. The second evidence is more direct and is
derived from the protocol employed in the experiments of Figs. 5 and 6. In these experiments, increasing the ratio of NH3 to
NH
by raising pHB resulted in a direct
increase in pHi. Thus an increase in the NH3
gradient across the membrane is reflected in an increase in the
NH3-induced pHi change as expected if
NH3 is permeable through the membrane. This increase in
pHi is not likely caused by a decrease in
NH
influx because Vm was not
affected at all. To further verify this latter point, we ruled out a
major change in NH
permeability in oocytes expressing
AQP1 by two ways. First, in voltage-clamped experiments, we measured
the current induced by NH
in the presence and absence
of expressed AQP1. Exposure to NH
caused an inward
current of 169 ± 20.4 nA (n = 3) in AQP1 oocytes and 145.3 ± 9.2 nA (n = 3) in
H2O-injected oocytes. These values were not significantly
different (P > 0.05), and this small change in
NH
-induced current is opposite to what would be
expected if NH
conductance decreased. Second, we
assessed total membrane conductance in the presence and absence of AQP1
from voltage deflections in response to a constant current pulse. These
results show that total membrane conductance in oocytes expressing AQP1
(1.43 ± 0.09 µS, n = 3) was not statistically
different (P > 0.05) from that of
H2O-injected oocytes (1.09 ± 0.15 µS,
n = 3).
Our experiments on oocytes expressing AQP1 indicate that expressing the
H2O channel enhanced NH3 transport with little
or no effect on NH
transport. The possibility that
AQP1 may be permeable to NH3 has its parallel in previous studies, which demonstrated that another gas, CO2, can also
be transported through AQP1 (10, 24, 26). NH3
has a small molar volume (24.9 cm3/mol) that is close to
that of H2O (18 cm3/mol); therefore, it is
conceivable that H2O channels may also be permeable to
NH3 as well.
The permeability of AQP(s) in general to solutes other than
H2O remains controversial. Various AQP(s), although highly
permeable to H2O, have been reported to transport other
solutes as well. Abrami and coworkers (1) reported that
AQP1 has a low permeability to glycerol, ethelene glycol, and
1,3-propanediol. On the basis of their ability to transport glycerol
and various other solutes, a set of AQP(s) is referred to as
"aquaglyceroporins" and include several mammalian AQP(s), such as
human AQP3, rat AQP7, and rat AQP9 (for a review see Refs.
2, 20, 35). Although
CO2 permeability through AQP1 (24) has been
confirmed by other studies (10, 26), studies on red blood
cells from AQP1-deficient mice failed to elucidate an effect on
CO2 transport (38). This last study also
failed to show a change in NH3 transport across red blood cells from AQP1-deficient mice. Another study (40) on
proteoliposomes with reconstituted AQP1 did not show a significant
effect on NH3 permeability either. Both studies, however,
were conducted on membranes with very high baseline permeability to
NH3. Nodulin 26, a plant AQP, was recently reported to
transport NH3 and NH
(11). A
more recent study on AQP6 indicates that it may function as an anion
conductive pathway under certain conditions (39). Non-H2O transport function of some AQP(s) is increasingly
recognized as a property that may have important implications (2,
20, 35).
In our study, two lines of evidences indicate increased NH3
permeability in oocytes expressing AQP1. First, NH3- and/or
NH
-induced acidification (consistently observed in
control oocytes) was usually absent when AQP1 was expressed (see Fig.
4). Because NH
transport was presumably unchanged
(voltage change not altered with AQP1), an alkalinization secondary to
NH3 influx likely masked the acidification from
NH
entry. Second, when oocytes were preequilibrated
with NH3/NH
, raising pHB (and
therefore increasing the ratio of NH3 to
NH
) induced a pHi increase at a rate that
was more than twofold faster in AQP1 oocytes compared with control
oocytes. In fact, the rate of pHi increase at pH 8.0 in
AQP1 oocytes was faster than the rate of pHi increase
induced by raising pHB to 8.5 in control oocytes. Both sets
of experiments are consistent with an increased NH3
transport when AQP1 is expressed.
Several observations indicate that the increased NH3
transport with the expression of AQP1 may potentially be
physiologically significant. For example, in the mammalian proximal
tubule, where AQP1 H2O channels are highly expressed,
significant transport of NH3 also occurs (17,
18). H2O channels could, in principle, be a major
component of the pathway of NH3 transport in the proximal tubule. On the other hand, in the thick ascending limb, NH3
transport across the apical membrane is limited because of a low
relative permeability to NH3. This limited NH3
transport does correlate with an apparent lack of H2O
channels in the apical membrane of this segment, but other factors,
such as the lipid composition of the membrane, may also play a role.
Further along the nephron, in the collecting duct, entry of total
ammonia is thought to be mediated by both NH3 and
NH
across the basolateral membrane and
NH3 diffusion across the apical membrane. The basolateral
membrane has AQP3 and AQP4, and the apical membrane has AQP2 water
channels inserted in the presence of antidiuretic hormone vasopressin.
The permeation of NH3 through AQP2, -3, and -4 has not yet
been documented but certainly deserves further investigation,
particularly since this would represent a route for regulation of
NH3 transport via H2O channels. In summary, there is correspondence between the presence of H2O
channels in various membranes along the nephron and NH3
transport. The present findings indicate that H2O channels
may be important in mediating NH3 transport, but this
awaits further investigation. To date, AQP knockout animals have not
demonstrated acid-base abnormalities, but whether defects in ammonia
transport could be uncovered with more direct testing awaits further studies.
 |
APPENDIX |
The concentration of intracellular NH
is
calculated from changes in pHi and the buffering power. The
intrinsic buffering power was calculated from the pHi
changes caused by acutely acid loading the cell by CO2
pulses as described by Boron and DeWeer (6) and averaged
12.4 ± 1.6 mM/pH (n = 19). Assuming that the
NH3/NH
-induced pHi change of
0.29 was caused solely by NH
influx, and no apparent
permeability of NH3, then with a buffering power of 12.4 mM/pH the resulting H+ concentration ([H+])
generated intracellularly can be calculated as
Because H+ is formed from the dissociation of
NH
, an equivalent amount of NH3 (3.60 mM)
must also be generated intracellularly. Assuming no loss of
NH3 with a pKa of
NH
of 9.25 then at pHi 7.05 (pHi after exposure to
NH3/NH
) intracellular NH
concentration ([NH
]) is equal
to
yielding a value of 571 mM, which is unrealistically high.
Therefore, there is almost certainly loss of NH3, implying
a finite permeability. In fact, loss of NH3 acidifies the
cell as the reaction
is "pulled" to the right, generating H+.
 |
ACKNOWLEDGEMENTS |
We thank Theresa DiCarlo for secretarial assistance.
 |
FOOTNOTES |
This work was supported by Grant AHA 0050547N from the American Heart
Association (National), by the Department of Veterans Affairs, and by
DCI, Inc.
Address for reprint requests and other correspondence: N. L. Nakhoul, Dept. of Medicine, Section of Nephrology, SL-45, Tulane Univ. 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 14 August 2000; accepted in final form 27 March 2001.
 |
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