Transport of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> was added. However, after the cell was acidified (because of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>), 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<UP><SUB>4</SUB><SUP>+</SUP></UP>. In AQP1 oocytes, exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>, 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<UP><SUB>4</SUB><SUP>+</SUP></UP> transport. These results indicate that 1) the oocyte membrane, although highly permeable to NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, has a significant NH3 permeability and 2) NH3 permeability is enhanced by AQP1.

NH3 permeability; intracellular pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

TWO-THIRDS OF NET ACID secretion in the urine is via ammonium (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) where urinary levels of total ammonia can easily reach values in excess of 50 mmol/l. Along the nephron, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> transport across cell membranes assumes that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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 MOmega . 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP>-Induced pHi and Vm Changes in H2O-Injected Oocytes

To study NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> are permeable, as is the case in most cells, were well described by Boron and DeWeer (6). When NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is applied, there is usually an initial cellular alkalinization as a result of the influx of NH3 (usually faster than NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) that consumes intracellular H+ to form NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and thus raises cell pH. In many instances, when NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> reverses this process, causing a decrease in pHi (an acute acid load) as the accumulated intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>. 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<UP><SUB>4</SUB><SUP>+</SUP></UP> conductive pathway (7, 9, 12). Removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reversed these changes, with Vm recovering readily, whereas the recovery of pHi was slow (Fig. 1, segment bc).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on H2O-injected oocytes. In control oocytes (H2O injected), exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> influx. The absence of any initial pHi increase indicates low permeability to NH3. Removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reversed these changes (segment bc).

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<UP><SUB>4</SUB><SUP>+</SUP></UP>, 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<UP><SUB>4</SUB><SUP>+</SUP></UP> was -9.2 ± 1.1 × 10-4 pH/s (n = 18), whereas the rate of pHi recovery upon removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was 2.4 ± 0.3 × 10-4 pH/s (n = 10).

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> on H2O-Injected Oocytes in the Absence of External Na+

The working hypothesis for assessing NH3 and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport from pHi measurements predicts that a significant NH3 permeability would lead to an increase in pHi, whereas NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> influx, resulting in the observed pHi decrease. Because many pHi-regulating mechanisms that can affect the NH3- and/or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced pHi changes are Na+ linked, we exposed oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the absence of external Na+.

As shown in Fig. 2, exposing oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> still caused a pHi decrease (segment ef) and great depolarization of the oocyte. pHi and Vm recovered when NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was removed (segment fg). In seven experiments, the NH3- and/or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-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<UP><SUB>4</SUB><SUP>+</SUP></UP>-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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on H2O-injected oocytes in the absence of external Na+. NH3- and/or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> reversed these changes (segment fg).

In several membranes with low NH3 permeability and fast NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry, such as the thick ascending limb of Henle, one possible route of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport is thought to be through K+ channels (13, 21, 37). To check whether inhibiting K+ channels could block NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx, we performed the experiments depicted in Fig. 3. As shown in this experiment, exposure of H2O-injected oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced depolarization or illicit an NH3-induced intracellular alkalinization.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on H2O-injected oocytes in the presence of Ba2+. The first part of the experiment shows the usual and reversible effects of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. In the absence of bath Na+, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a decrease in pHi (segment ab) and a cellular depolarization, as shown earlier. pHi and Vm fully recovered upon removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> (segment gh) and Ba2+ (segment hi).

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> on Oocytes Expressing AQP1 in the Absence of External Na+

If NH3 permeability is enhanced by AQP1, then the NH3- and/or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>, 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<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-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<UP><SUB>4</SUB><SUP>+</SUP></UP> induced either no change or a pHi increase rather than the large decrease in pHi usually observed in control oocytes.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> in the absence of Na+ caused a small pHi increase (rather than a decrease) and significant depolarization (segment bc). The depolarization indicates that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport is probably not affected. The changes in Vm and pHi were reversed upon removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>.

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> at High Bath pH on H2O-Injected Oocytes

In AQP1 oocytes, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acidification to occur (see DISCUSSION). Nevertheless, this permeability is small compared with the apparently large NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeability, resulting in a net intracellular acidification and completely masking any alkalinization when oocytes are exposed to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> as observed above (see Figs. 1-3).

To maximize the NH3-induced signal (in the face of a substantial NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acidification), we conducted the experiments shown in Fig. 5. In H2O-injected oocytes, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP>), pHi increased again (segment de) with no significant change in Vm. The pHi increase was reversed when external NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was returned to pH 7.5 (segment ef).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at higher bath pH (8.0 and 8.5) on H2O-injected oocytes. Exposing control oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> transport. Switching back to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at pH 7.5 reversed the changes in pHi (segment ef).

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> at High Bath pH on Oocytes Expressing AQP1

Preequilibrating oocytes with NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at higher bath pH (8.0) on oocytes expressing AQP1. Exposing AQP1 oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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).

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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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<UP><SUB>4</SUB><SUP>+</SUP></UP> (20 mM NH4Cl). In such an approach, as NH3 enters the cell, it combines with intracellular H+ to form NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, which will lead to an increase in pHi. In contrast, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry into the cell would lead to a decrease in pHi when NH<UP><SUB>4</SUB><SUP>+</SUP></UP> dissociates intracellularly, releasing NH3 and H+. Therefore, an increase in NH3 permeability would cause an alkaline shift in pHi. Whereas a decrease in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>, and 3) expression of AQP1 increases permeability of NH3 without substantially affecting NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> is particularly high. The evidence for this is based on three observations. First, exposure of oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a substantial decrease in pHi consistent with a big influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and its subsequent release of intracellular H+, leading to intracellular acidification. Second, exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a huge depolarization of the oocyte to near 0 mV, indicating an electrogenic pathway also consistent with NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> exposure of oocytes induced an inward current in voltage-clamp experiments (23). These observations confirm previous studies which suggested that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx in oocytes occurs through a nonselective cationic channel (7, 8, 12).

It is also likely that at least a fraction of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> is probably the major contributor to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx. Sasaki et al. (32) could only slightly inhibit NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acidification in the presence of furosemide, and Burckhardt and Fromter (8) showed no significant effect of bumetanide on NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced Vm changes or cellular acidification. Our own experiments, with bumetanide, agree with the above studies.

This high permeability of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and its effects on Vm and pHi complicates examination of NH3 permeability in the oocyte. The path of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced depolarization. Other inhibitors, including Cs+, tetramethylammonium, and quinidine were tried by other investigators (8, 12) and also did not inhibit the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced depolarization of the cell. In the absence of a specific inhibitor of this NH<UP><SUB>4</SUB><SUP>+</SUP></UP> pathway, an NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>. 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<UP><SUB>4</SUB><SUP>+</SUP></UP> cannot be achieved unless NH3, generated intracellularly from the dissociation of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, can exit the cell. In control oocytes, for example, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>, with no significant permeability to NH3, intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentration has to reach an impossibly high level of ~571 mM (see APPENDIX). In other words, for the influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> right-arrow 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> influx because Vm was not affected at all. To further verify this latter point, we ruled out a major change in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeability in oocytes expressing AQP1 by two ways. First, in voltage-clamped experiments, we measured the current induced by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the presence and absence of expressed AQP1. Exposure to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced current is opposite to what would be expected if NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP> (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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acidification (consistently observed in control oocytes) was usually absent when AQP1 was expressed (see Fig. 4). Because NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport was presumably unchanged (voltage change not altered with AQP1), an alkalinization secondary to NH3 influx likely masked the acidification from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry. Second, when oocytes were preequilibrated with NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, raising pHB (and therefore increasing the ratio of NH3 to NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) 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<UP><SUB>4</SUB><SUP>+</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The concentration of intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced pHi change of 0.29 was caused solely by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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
[H<SUP>+</SUP>]<IT>=</IT>&bgr;<IT>×</IT>&Dgr;pH<SUB>i</SUB><IT>=</IT>3.60 mM
Because H+ is formed from the dissociation of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, an equivalent amount of NH3 (3.60 mM) must also be generated intracellularly. Assuming no loss of NH3 with a pKa of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> of 9.25 then at pHi 7.05 (pHi after exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentration ([NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]) is equal to
[NH<SUP>+</SUP><SUB>4</SUB>]<IT>=</IT>[NH<SUB>3</SUB>]<IT>×</IT>10<SUP>p<IT>K</IT>−pH</SUP>
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
NH<SUP>+</SUP><SUB>4</SUB><IT> ↔ </IT>NH<SUB>3</SUB><IT>+</IT>H<SUP>+</SUP>
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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

1.   Abrami, L, Tacnet F, and Ripoche P. Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels. Pflügers Arch 430: 447-458, 1995[ISI][Medline].

2.   Agre, P, and Homer W. Smith award lecture. Aquaporin water channels in kidney. J Am Soc Nephrol 11: 764-777, 2000[Free Full Text].

3.   Agre, P, Mathai JC, Smith BL, and Preston GM. Functional analyses of aquaporin water channel proteins. Methods Enzymol 294: 550-572, 1999[Medline].

4.   Ammann, D, Lanter F, Steiner RA, Schulthess P, Shijo Y, and Simon W. Neutral carrier based hydrogen ion selective microelectrode for extra- and intracellular studies. Anal Chem 53: 2267-2269, 1981[ISI][Medline].

5.   Borgnia, M, Nielsen S, Engel A, and Agre P. Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 68: 425-458, 1999[ISI][Medline].

6.   Boron, WF, and De Weer P. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 67: 91-112, 1976[Abstract].

7.   Burckhardt, BC, and Burckhardt G. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> conductance in Xenopus laevis oocytes. I. Basic observations. Pflügers Arch 434: 306-312, 1997[ISI][Medline].

8.   Burckhardt, BC, and Fromter E. Pathways of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation across Xenopus laevis oocyte cell membrane. Pflügers Arch 420: 83-86, 1992[ISI][Medline].

9.   Burckhardt, BC, and Thelen P. Effect of primary, secondary and tertiary amines on membrane potential and intracellular pH in Xenopus laevis oocytes. Pflügers Arch 429: 306-312, 1995[ISI][Medline].

10.   Cooper, GJ, and Boron WF. Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin-1 or its C189S mutant. Am J Physiol Cell Physiol 275: C1481-C1486, 1998[Abstract/Free Full Text].

11.   Cooper, GJ, Virkki LV, and Boron WF. Effect of expressing nodulin 26 on the membrane permeability of Xenopus oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and acetic acid. FASEB J 13, Suppl: L26, 1999.

12.   Cougnon, M, Bouyer P, Hulin P, Anagnostopoulos T, and Planelles G. Further investigation of ionic diffusive properties and of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> pathways in Xenopus laevis oocyte cell membrane. Pflügers Arch 431: 658-667, 1996[ISI][Medline].

13.   Good, DW. Active absorption of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> by rat medullary thick ascending limb: inhibition by potassium. Am J Physiol Renal Fluid Electrolyte Physiol 255: F78-F87, 1988[Abstract/Free Full Text].

14.   Good, DW, and Knepper MA. Ammonia transport in the mammalian kidney. Am J Physiol Renal Fluid Electrolyte Physiol 248: F459-F471, 1985[Abstract/Free Full Text].

15.   Good, DW, Knepper MA, and Burg MB. Ammonia and bicarbonate transport by thick ascending limb of rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 247: F35-F44, 1984[ISI][Medline].

16.   Hamm, LL. Ammonia transportin the rabbit medullary collecting tubule (Abstract). Kidney Int 29: 367A, 1986.

17.   Hamm, LL, and Simon EE. Ammonia transport in the proximal tubule in vivo. Am J Kidney Dis 14: 253-257, 1989[ISI][Medline].

18.   Hamm, LL, and Simon EE. Ammonia transport in the proximal tubule. Miner Electrolyte Metab 16: 283-290, 1990[ISI][Medline].

19.   Hamm, LL, Trigg D, Martin D, Gillespie C, and Buerkert J. Transport of ammonia in the rabbit cortical collecting tubule. J Clin Invest 75: 478-485, 1985[ISI][Medline].

20.   Heymann, JB, and Engel A. Structural clues in the sequences of the aquaporins. J Mol Biol 295: 1039-1053, 2000[ISI][Medline].

21.   Kikeri, D, Sun A, Zeidel ML, and Hebert SC. Cell membranes impermeable to NH3. Nature 339: 478-480, 1989[ISI][Medline].

22.   Knepper, MA, Packer R, and Good DW. Ammonium transport in the kidney. Physiol Rev 69: 179-249, 1989[Free Full Text].

23.   Nakhoul, NL, Abdulnour-Nakhoul S, Hering-Smith K, and Hamm LL. Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport in Xenopus oocytes expressing mENaC (Abstract). J Am Soc Nephrol 10: 41A, 1999.

24.   Nakhoul, NL, Davis BA, Romero MF, and Boron WF. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am J Physiol Cell Physiol 274: C543-C548, 1998[Abstract/Free Full Text].

25.   Nakhoul, NL, Lopes AG, Chaillet JR, and Boron WF. Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. J Gen Physiol 92: 369-393, 1988[Abstract].

26.   Prasad, GV, Coury LA, Finn F, and Zeidel ML. Reconstituted aquaporin 1 water channels transport CO2 across membranes. J Biol Chem 273: 33123-33126, 1998[Abstract/Free Full Text].

27.   Preston, GM, and Agre P. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. Proc Natl Acad Sci USA 88: 11110-11114, 1991[Abstract].

28.   Preston, GM, Carroll TP, Guggino WB, and Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385-387, 1992[ISI][Medline].

29.   Priver, NA, Rabon EC, and Zeidel ML. Apical membrane of the gastric parietal cell: water, proton, and nonelectrolyte permeabilities. Biochemistry 32: 2459-2468, 1993[ISI][Medline].

30.   Sackin, H, and Boulpaep EL. Isolated perfused salamander proximal tubule: methods, electrophysiology, and transport. Am J Physiol Renal Fluid Electrolyte Physiol 241: F39-F52, 1981[ISI][Medline].

31.   Sambrook, J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

32.   Sasaki, S, Ishibashi K, Nagai T, and Marumo F. Regulation mechanisms of intracellular pH of Xenopus laevis oocyte. Biochim Biophys Acta 1137: 45-51, 1992[ISI][Medline].

33.   Singh, SK, Binder HJ, Geibel JP, and Boron WF. An apical permeability barrier to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in isolated, perfused colonic crypts. Proc Natl Acad Sci USA 92: 11573-11577, 1995[Abstract].

34.   Strange, K, and Spring KR. Cell membrane water permeability of rabbit cortical collecting duct. J Membr Biol 96: 27-43, 1987[ISI][Medline].

35.   Verkman, AS, and Mitra AK. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 278: F13-F28, 2000[Abstract/Free Full Text].

36.   Waisbren, SJ, Geibel JP, Modlin IM, and Boron WF. Unusual permeability properties of gastric gland cells. Nature 368: 332-335, 1994[ISI][Medline].

37.   Wall, SM, and Koger LM. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport mediated by Na+-K+-ATPase in rat inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 267: F660-F670, 1994[Abstract/Free Full Text].

38.   Yang, B, Fukuda N, van Hoek A, Matthay MA, Ma T, and Verkman AS. Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes. J Biol Chem 275: 2686-2692, 2000[Abstract/Free Full Text].

39.   Yasui, M, Kwon TH, Knepper MA, Nielsen S, and Agre P. Aquaporin-6: an intracellular vesicle water channel protein in renal epithelia. Proc Natl Acad Sci USA 96: 5808-5813, 1999[Abstract/Free Full Text].

40.   Zeidel, ML, Nielsen S, Smith BL, Ambudkar SV, Maunsbach AB, and Agre P. Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes. Biochemistry 33: 1606-1615, 1994[ISI][Medline].

41.   Zeidel, M, Strange K, Emma F, and Harris HWJ Mechanisms and regulation of water transport in the kidney. Semin Nephrol 13: 155-167, 1993[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F255-F263
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society