Ammonium interaction with the epithelial sodium channel

Nazih L. Nakhoul, Kathleen S. Hering-Smith, Solange M. Abdulnour-Nakhoul, and L. Lee Hamm

Section of Nephrology, Department of Medicine, and Department of Physiology, Tulane University School of Medicine and Veterans Affairs Medical Center, New Orleans, Louisiana 70112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to investigate the direct effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on mouse epithelial Na+ channels (mENaC) expressed in Xenopus oocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na+ current, intracellular pH (pHi), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na+ reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pHi (0.43 ± 0.07) similar to the NH4Cl-induced pHi decrease (0.47 ± 0.12). Removal of Na+ in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pHi (in the absence of NH4Cl). Removal of external Na+ resulted in an outward current of 3.7 ± 0.8 µA (at -60 mV). The magnitude of this change in current was only 2.7 ± 0.7 µA when Na+ was removed in the presence of NH4Cl. In oocytes expressing mENaC, NH4Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na+ were not different from zero. These results indicate that NH4Cl inhibits Na+ transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH4Cl. Permeability and selectivity of ENaC to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> may play a role.

mouse epithelial sodium channel; intracellular pH; ammonium ion permeability


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ABSORPTION OF SODIUM across epithelia plays an essential role in maintaining Na+ and fluid homeostasis of the body. In these epithelia, Na+ transport is a two-step process involving diffusion of Na+ through channels across the apical membrane and active transport across the basolateral membrane via the Na+-K+-ATPase (19). In the kidney, although Na+ transport occurs throughout the length of the nephron, the fine regulation of Na+ excretion occurs in the collecting tubule. In this segment, as in several other tight epithelia, the epithelial Na+ channel (ENaC) (12, 27) is the rate-limiting pathway of luminal reabsorption of Na+. Mutations of ENaC can result in serious clinical problems, such as Liddle's syndrome and pseudohypoaldosteronism type 1 (14, 24, 40).

The ENaC is located at the apical membrane and is highly sensitive to amiloride. Biophysically, this channel is very selective for Na+ over K+ (with Na+ permeability-to-K+ permeability ratio > 10) but is selective for Li+ over Na+ by a factor of 1.3-1.5. The highly selective Na+ channels have low single-channel conductance and long open and closed times (21, 33).

Cloning of ENaC enabled the molecular characterization of the channel as consisting of three homologous subunits termed alpha -, beta -, and gamma -ENaC (13, 26, 27). More recently, cDNA encoding mouse alpha -, beta -, and gamma -ENaC (mENaC) were cloned (1). Coexpression of these subunits in Xenopus oocytes yielded an amiloride-sensitive Na+ channel with properties very similar to the rat ENaC. This channel was investigated in our study.

Regulation of Na+ channels by hormones has been recognized from physiological studies even before ENaC was cloned. Aldosterone is considered the primary hormone responsible for regulating channel activity through complex mechanisms that involve activation of preexisting channels and de novo synthesis of channels (4, 5, 29, 35). Vasopressin (and possibly insulin) is another hormone that is involved in ENaC regulation (8, 34, 39). Nonhormonal inhibition of ENaC occurs with elevated extracellular Na+ (leading to self-inhibition), increased intracellular Na+, and increased intracellular Ca2+, which has been postulated to be the mediator of self-inhibition (6, 15, 18, 25). On the other hand, hyperpolarization of the apical membrane seems to activate the channel. Intracellular acidification was reported to cause a decrease in apical Na+ conductance; however, the role of intracellular pH (pHi) in regulating Na+ channel activity may differ among tissues and is not yet fully understood (13, 26, 32). Regulation of ENaC has been addressed in several recent reviews (2, 8, 19, 35).

In the renal distal nephron, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport is critical for acid-base balance. Increases in NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and/or its effect on acid-base balance can have direct and indirect consequences on transport in general. For example, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> has been shown to inhibit transport across the tight epithelium of the toad bladder (20). In another study on the rabbit cortical collecting duct, NH4Cl was shown to decrease transepithelial voltage (Vte) and inhibit Na+ reabsorption (22). In this latter study, the effect of NH4Cl on Vte was not evident when Na+ reabsorption was inhibited by amiloride. This effect of NH4Cl could be due to one or several causes, including voltage-dependent changes in Na+ transport, pHi-mediated effect on Na+ conductance, and/or direct inhibition of the ENaC. The effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> may be physiologically relevant to the increased urinary Na+ observed with metabolic acidosis in vivo. This study was conducted to investigate the effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport through ENaC expressed in Xenopus oocytes.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and solutions. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. The standard bathing solution was ND96 medium containing (in mM) 100 NaCl, 2 KCl, and 1.8 CaCl2 and buffered with 5 mM HEPES to pH 7.5. The NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> solution contained 20 mM NH4Cl (replacing NaCl) at pH 7.5. In 80 mM Na+ solutions, 20 mM N-methyl-D-glucamine (NMDG+) replaced 20 mM Na+, and in 0 Na+ solutions, all Na+ was replaced with NMDG+. Osmolarity of all solutions was ~200 mosmol/l. OR3 medium (Leibovitz media, GIBCO BRL) contained glutamate and 500 U each of penicillin and streptomycin, with pH adjusted to 7.5 and osmolarity adjusted to ~200 mosmol/l.

Isolation of oocytes. We harvested oocytes in stage 5/6 from female Xenopus laevis. Briefly, this was done by anesthetizing the frog by mild hypothermia in water containing 0.2% tricaine (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO). A 1-cm incision was made in the abdominal wall, and one lobe of the ovary was externalized and the distal portion was cut. The wound was closed by a few stitches (5-0 catgut) in the muscular plane of the peritoneum followed by two to three stitches (6-0 silk) in the abdominal skin. The excised piece of ovary containing oocytes was rinsed several times with Ca2+-free ND96 solution until the solution was clear. The tissue was then agitated in ~15 ml of sterile-filtered Ca2+-free solution containing collagenase type 1A (Sigma) for 30-40 min. Free oocytes were rinsed several times with sterile OR3 medium, sorted, and then stored at 18°C.

Preparation of cRNA. Plasmid containing the appropriate template DNA of mouse ENaC (all a generous gift of Dr. Tom Kleyman) was purified by Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI). The plasmid was then digested with an appropriate restriction enzyme that has a cleavage site downstream of the insert to produce a linear template and then with proteinase K (1 mg/ml). DNA was then extracted twice with phenol-chloroform, extracted with chloroform, and precipitated with ethanol. cDNA was transcribed in vitro with T7 RNA polymerase. The in vitro synthesis of capped RNA (cRNA) transcripts was then accomplished using mCAP RNA Capping Kit (Stratagene, La Jolla, CA). The concentration of cRNA was determined by ultraviolet absorbance, and its quality was assessed by formaldehyde-MOPS-1% agarose gel electrophoresis (37).

Injection of oocytes. Oocytes in OR3 medium were visualized with a dissecting microscope and injected with 50 nl of full-length cRNA mix of mENaC containing cRNAs for the alpha -, beta -, and gamma -subunits. At 0.05 µg/µl concentration for each subunit, each oocyte was injected with 2.5 ng/subunit. Control oocytes were injected with 50 nl of sterile H2O. The sterile pipettes had tip diameters of 20-30 µm. They were backfilled with paraffin oil and were connected to a Nanoject displacement pipette (Drummond Scientific). Injected oocytes were used 2-5 days after injection with cRNA.

Electrophysiological measurements in frog oocytes. The pH microelectrodes were of the liquid ion-exchanger type, and the resin (H+ ionophore 1, cocktail B) was obtained from Fluka Chemical (Ronkonkoma, NY). Single-barreled microelectrodes were manufactured as described previously (31, 36). Briefly, alumina-silicate glass tubings (1.5 mm OD × 0.86 mm ID; Frederick Haer, Brunswick, MD) were pulled to a tip of <0.2 µm and dried in an oven at 200°C for 2 h. The electrodes were vapor silanized with bis(dimethylamino)-dimethyl silane in a closed vessel (300 ml). The exchanger was then introduced into the tip of the electrodes by means of a very fine glass capillary. pH electrodes were backfilled with a buffer solution (3). The electrodes were fitted with a holder with an Ag-AgCl pellet attached to a high-impedance probe of a WPI FD-223 electrometer. The pH electrodes were calibrated in standard solutions of pH 6 and 8. Only electrodes with a slope >58 mV/pH were used in our studies.

The oocyte, visualized with a dissecting microscope, was held on a nylon mesh in a special chamber where solutions flow continuously at a rate of ~4 ml/min. Solutions (6 possible) were switched by a combination of a six- and a four-way valve system, which was activated pneumatically. Very little dead space was present, and complete solution changes in the chamber occurred in 6-8 s.

Two-electrode voltage clamp. Whole cell currents were recorded using two-electrode voltage clamp (model OC-725, Warner Instruments, Hamden, CT). For these experiments, electrodes were pulled from borosilicate glass capillaries (OD 1.5 mm; Fredrick Haer) using a vertical puller (model 700C, David Kopf Instruments). Electrodes were filled with 3 M KCl solution and had resistances of 1-4 MOmega . Bath electrodes were also filled with 3 M KCl and were directly immersed in the chamber. In most cases, oocytes were clamped at -60 mV, and long-term readings of current were sampled at a rate of once per second. For current-voltage (I-V) relationships, oocytes were clamped at 0 mV and stepped from -80 to +60 mV in 20-mV steps (sampled at 10 times/s for 1 s at each step). Slope conductances were calculated from the slope of the I-V line between -60 and -40 mV. Inward flow of cations is defined by convention as inward (negative) current.

Statistics and data analysis. Values are means ± SE; n is the number of observations. Statistical significance was judged primarily from two-tailed Student's t-tests. Whenever feasible, measurements were determined under control and test conditions in the same cell, and each cell served as its own control (paired data). Results are considered statistically significant if P <=  0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular ionic compositions in control (water-injected) oocytes and oocytes expressing mENaC. The oocyte expression system has been extensively used to study the characteristics of expressed proteins, many of which are membrane transporters and ionic channels. The high level of expression usually achieved, the lack of substantial native transport in the oocyte, and the relative ease of measurements in this preparation make the oocyte a valuable system for study. A concern that has been inadequately addressed is that expressing an exogenous transporter may alter the cellular environment of the oocyte. An altered cellular environment may, by itself, be a factor affecting the function of the transporter. Because expressing an ion channel may cause such significant effects, we first characterized the cellular changes induced by expressing mENaC under steady-state conditions. As shown in Table 1, intracellular composition of the important monovalent ions was drastically and significantly altered. As expected, intracellular Na+ activity increased from 6.6 ± 0.6 to 30.0 ± 1.8 mM and steady-state membrane potential (Vm) was positive (+4.5 ± 1.6 mV). This was accompanied by substantial decrease in intracellular K+ activity (a<UP><SUB>i</SUB><SUP>K<SUP>+</SUP></SUP></UP>) from 74.3 ± 2.7 to 40.0 ± 0.5 mM, whereas intracellular Cl- activity (a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>) increased from 25.0 ± 0.3 to 75.3 ± 1.9 mM. pHi increased slightly from 7.34 ± 0.02 to 7.5 ± 0.02, but the difference was not statistically significant (P > 0.05).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   pHi, cellular ionic activities, and steady-state Vm in control oocytes and oocytes expressing mENaC

Because the ENaC is usually overexpressed, it is conceivable that acute changes in external Na+ may induce large changes in intracellular Na+ activity (a<UP><SUB>i</SUB><SUP>Na</SUP></UP>). To investigate this possibility, we measured the changes in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> by Na+-selective microelectrodes when external Na+ was removed (replaced by NMDG+). In control oocytes, removal of external Na+ (for ~10 min) hardly caused any detectable decrease in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> (Delta a<UP><SUB>i</SUB><SUP>Na</SUP></UP> = 0.9 ± 0.1 mM, n = 4). Unexpectedly, in mENaC oocytes, removal of external Na+ also caused a very small change in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> (Delta a<UP><SUB>i</SUB><SUP>Na</SUP></UP> = 1.8 ± 0.4 mM, n = 4). These experiments indicate that intracellular Na+ in the oocytes is not readily exchangeable, and no acute large changes in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> are expected to occur in response to Na+ removal, even when mENaC is expressed.

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> and removal of Na+ on water-injected oocytes. To study the effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport, we first characterized the changes caused by addition of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or removal of external Na+ in water-injected oocytes. As shown in Fig. 1, exposing control oocytes to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a decrease in pHi (segment ab) and large depolarization of the cell. These changes were fully reversed when NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was removed from the bath (segment bc). Removal of bath Na+ caused a small, sustained hyperpolarization of the cell, but pHi did not change (segment cd). Exposing the oocyte to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the continued absence of Na+ resulted in changes similar to those observed in the presence of Na+; pHi decreased substantially (segment de), and the cell depolarized to near 0 mV. Removal of bath NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> fully reversed these changes (segment ef). These experiments demonstrated the following important features: 1) There is a small but significant voltage change induced by Na+ transport. 2) NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused substantial voltage and pHi changes. These changes occur in the water-injected oocytes and are independent of mENaC expression. Both the pHi and the voltage changes caused by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> can potentially affect Na+ transport through mENaC.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on membrane potential (Vm) and intracellular pH (pHi) in the presence and absence of Na+. Exposing control (H2O-injected) oocytes to 20 mM NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the presence of Na+ decreased pHi by 0.15 and substantially depolarized the cell from -72 to -4 mV (segment ab). This effect was fully reversed when NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was removed from the bath (segment bc). Removal of external Na+ did not affect pHi (segment cd) but caused moderate hyperpolarization (Delta Vm = -20 mV). Exposing oocytes to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the absence of Na+ still decreased pHi by 0.18 and hyperpolarized the cell to near 0 mV (segment de). Removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reversed these changes (segment ef).

Na+ removal in the presence and absence of NH3/NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP> in oocytes expressing mENaC. By comparison to water-injected oocytes, Na+ removal and exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused substantially different effects when mENaC was expressed. The basic observation indicating the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport through mENaC was indicated in the experiments depicted in Fig. 2. As shown in Fig. 2A, in oocytes expressing mENaC, removal of bath Na+, caused a large hyperpolarization from +16 to -112 mV (segment ab), which readily recovered when Na+ was returned (segment bc). Exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (at point c) caused a small hyperpolarization of ~5 mV (segment cd). In the continued presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, subsequent removal of bath Na+ hyperpolarized the oocyte to -89 mV (segment de), which readily recovered on readdition of Na+ to the bath (segment ef). The effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was also reversed when NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was removed from the bath (segment fg). Removal and readdition of external Na+ in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> for a second time caused the same hyperpolarization (segment gh) and recovery (segment hi) observed in the first pulse (compare with segments ab and bc, respectively). In 10 paired experiments, the average hyperpolarization caused by Na+ removal was -129 ± 5.3 mV in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and -100 ± 7.8 mV in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (P < 0.001). These experiments indicate that NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> may inhibit Na+ transport in oocytes expressing mENaC.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   A: effect of Na+ removal in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in mouse oocytes expressing the epithelial Na+ channel (ENaC). Trace represents 10 experiments showing that removal of Na+ hyperpolarized the oocyte by 129 ± 5.3 mV (segment ab), which was readily reversed when Na+ was returned to normal (segment bc). Exposing oocytes expressing mENaC to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (segment cd) caused a slight hyperpolarization (Delta Vm = -4.3 ± 1.4 mV). Removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> hyperpolarized the oocyte by 100 ± 7.8 mV (segment de), which was significantly less than the hyperpolarization in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (P < 0.05). Hyperpolarization was reversed on readdition of Na+ to the bath (segment ef). Removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> from the bath caused a small depolarization (segment fg). Removal of Na+ again in the absence of NH3/NH4+ caused a larger hyperpolarization (segment gh) as seen in the first pulse (compare with segment ab). Readdition of Na+ fully reversed this effect on Vm (segment hi). B: Na+ removal in the presence and absence of NH3/NH4+ in oocytes expressing mENaC. When bath Na+ at 100 mM was removed (from 100 to 0 mM), Vm hyperpolarized significantly (segment ab) as seen in A and was reversed on restoration of bath Na+ to 100 mM (segment bc). Replacing 20 mM Na+ with N-methyl-D-glucamine (NMDG, 80 mM Na+) caused a small hyperpolarization (segment cd). Subsequent exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (at 80 mM Na+) caused a small depolarization (segment de). Removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a smaller hyperpolarization (segment ef) than that observed with Na+ removal in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (compare with segment ab). Readdition of 80 mM Na+ to the bath reversed this effect on Vm (segment fg). Removal of bath NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a small hyperpolarization (segment gh). When bath Na+ at 80 mM was removed again (going from 80 mM to 0 mM), Vm hyperpolarized (segment hi) to the same extent as in the first pulse (compare with segment ab). C: effect of amiloride on Vm in oocytes expressing mENaC. Exposing oocytes to amiloride (100 µM) caused hyperpolarization of 77 ± 4.1 mV (segment ab). Addition of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the presence of amiloride caused a transient hyperpolarization (segment bc) followed by sustained depolarization of 23 ± 8.7 mV (segment cd). Removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and amiloride caused a small but statistically insignificant (P > 0.05) depolarization of Vm (segment de). All changes were completely reversible: readdition of Na+ in the presence of amiloride hyperpolarized Vm slightly (segment ef) and removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> hyperpolarized Vm even further (segment fg).

In these experiments, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> solutions contain 20 mM NH4Cl obtained by replacing 20 mM NaCl from the control ND96 solution. Because Na+-induced voltage changes in oocytes expressing mENaC are substantial (Fig. 2A), it is conceivable that this partial removal of Na+ could be responsible in part for the reduced hyperpolarization induced by removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. The next set of experiments was designed to investigate this possibility. As shown in Fig. 2B, removal of external Na+ (from 100 to 0 mM in the bath) caused the usual large and reversible hyperpolarization observed earlier (segments ab and bc). At point c, 20 mM Na+ was removed from the bath (replaced by NMDG+), resulting in a small hyperpolarization of ~4 mV (segment cd). On switching to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> solution at point d, no Na+ was replaced, and the external solution still contained 80 mM Na+. This maneuver caused a small depolarization (segment de). Removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused hyperpolarization (segment ef) that was substantially smaller than in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Readdition of Na+ to the bath caused Vm to recover (segment fg). At point g, the bath solution was switched to a solution free of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> but containing 80 mM Na+, which caused a small hyperpolarization (segment gh). Removal of bath Na+ again (from 80 to 0 mM Na+) caused hyperpolarization of Vm similar to the first pulse (from 100 to 0 mM Na+), which was significantly larger than the hyperpolarization of Vm in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (segment hi). In three paired experiments, Delta Vm (from 80 to 0 mM Na+) was -120 ± 4.0 mV in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and only -61 ± 10.9 mV in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. These experiments further indicate that the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is affecting the 0 Na+ response in oocytes expressing mENaC.

Effect of amiloride on Vm. Inhibition of the ENaC by amiloride is one of the main properties that characterize this channel. The next set of experiments was conducted to investigate the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Vm when mENaC was inhibited by amiloride. As shown in Fig. 2C, in oocytes expressing mENaC, addition of amiloride (100 µM) to the bath caused a significant hyperpolarization (segment ab), as expected if ENaC is inhibited. In the presence of amiloride, exposing the oocyte to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused depolarization of the oocyte, which was preceded by a small transient hyperpolarization (segment bcd). At point d, removal of bath Na+, in the presence of amiloride and NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, caused a sustained further depolarization of Vm (segment de). This is in contrast to the usual hyperpolarization observed when Na+ was removed in the absence of amiloride (compare with Fig. 2, A and B). These changes were reversed on readdition of Na+ to the bath (segment ef) and then on removal of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> from the external solution (segment fg). These experiments indicated that, in oocytes expressing mENaC, amiloride inhibits the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on the Na+-induced changes in Vm.

Na+ removal at low pHi or in the presence of NH3/NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP> in oocytes expressing mENaC. In contrast to most other cells, where exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> causes an initial increase in pHi [NH<UP><SUB>4</SUB><SUP>+</SUP></UP> prepulse (9)], in the oocytes, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> causes a significant sustained decrease in pHi (11, 38). One possibility is, therefore, that the low pHi is responsible for the inhibition of the Na+-induced change in Vm in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. To investigate this possibility, we decreased pHi of oocytes expressing mENaC, independently of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, and measured the Vm changes induced by removal of bath Na+. As shown in Fig. 3, removal of external Na+ caused no change in pHi (segment ab) except the usual large hyperpolarization, which readily recovered on readdition of Na+ (segment bc). At point c, butyrate (20 mM) was added to the bath, causing a significant decrease in pHi (segment cd) and very little change in Vm. At low pHi, Na+ was again removed from the bath, which did not cause a significant further change in pHi, but Vm became more negative (segment de). In five paired experiments, the hyperpolarization caused by removal of Na+ was 146 ± 2.7 mV in the absence of butyrate, a value not significantly different from 149 ± 5.4 mV in the presence of butyrate (P > 0.05). All these changes were completely reversible: Vm depolarized again on readdition of Na+ to the bath (segment ef), and pHi fully recovered on removal of butyrate (segment fg). At point g, the oocytes were exposed to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, which caused a small change in Vm, but pHi decreased substantially (segment gh). When bath Na+ was removed again, pHi decreased a little further and the cell hyperpolarized, as shown previously (segment hi). The pHi decrease caused by NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (0.47 ± 0.12) was not statistically different from the pHi decrease of 0.43 ± 0.07 caused by butyrate (P > 0.05). However, the cell hyperpolarization caused by removal of Na+ in the presence of butyrate (149 ± 5.4 mV) was significantly larger than that in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (109 ± 5.5 mV, P < 0.05). These experiments are consistent with a significant effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+-induced Vm changes, which are independent of pHi.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Na+ removal at low pHi in oocytes expressing mENaC. Because NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> causes substantial intracellular acidification, this experiment examined the effect of low pHi on changes caused by Na+ removal. Na+ removal at steady-state pHi caused the usual large hyperpolarization of Vm but had no effect on pHi (segment ab). Readdition of bath Na+ completely reversed this effect (segment bc). pHi was then acidified by exposing the oocyte to 20 mM butyrate (segment cd), and Vm was not affected . At low pHi (point d), removal of Na+ (in the continued presence of butyrate) hyperpolarized the oocyte to a value similar to that at high pHi in the absence of butyrate (segment de). On readdition of Na+ in the presence of butyrate, Vm recovered, but pHi was not affected (segment ef). On removal of butyrate, pHi recovered, but Vm was not affected (segment fg). Exposing the oocyte to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a substantial decrease in pHi comparable to that caused by butyrate and slightly depolarized Vm (segment gh). Subsequent removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused hyperpolarization (segment hi) that was significantly less than that caused by removal of Na+ in the presence of butyrate (compare with segment de).

Current changes in response to Na+ removal in the presence and absence of NH3/NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP> in oocytes expressing mENaC. To further study the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport in oocytes expressing mENaC, we measured the changes in whole cell current in response to removal of Na+. As can be seen in Fig. 4, removal of bath Na+ caused an outward deflection in whole cell current (Im; oocyte clamped at -60 mV, segment ab) that was readily reversed when Na+ was returned to the bath (segment bc). Exposing the oocyte to 20 mM total NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused an inward change in Im of 383 ± 5.1 nA (segment cd). This current is supposedly caused by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry through a nonselective cationic channel as observed in water-injected oocytes (10, 11, 36). In the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, removal of bath Na+ again resulted in outward deflection of Im (segment de), which was reversed when Na+ was restored to the bath (segment ef). In nine paired experiments, the outward deflection in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was 3.7 ± 0.8 µA but was only 2.7 ± 0.7 µA in its presence (P < 0.01). These experiments are consistent with the original observation that NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibits Na+ transport in the oocyte when mENaC is expressed.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Whole cell currents (Im) in oocytes expressing mENaC in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Representative experiment indicates that oocytes expressing mENaC had a large inward current (-2.9 ± 0.6 µA) at -60 mV. Removal of external Na+ caused an outward deflection of the current to ~1 µA (segment ab) that was readily reversed on readdition of Na+ (segment bc). Exposure to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a small increase in the inward current of 0.38 ± 0.08 µA (segment cd). Removal of Na+ in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a smaller outward deflection in the current (segment de) compared with that in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (change in current = 2.7 ± 0.7 µA, P < 0.01). Inward current was restored on readdition of bath Na+ (segment ef).

Amiloride inhibition of Na+ and NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP> whole cell currents in oocytes expressing mENaC. The data presented so far suggest that, in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, Na+ transport through the epithelial Na+ channel expressed in oocytes is partially inhibited. Because mENaC is blocked by amiloride, we investigated whether inhibiting mENaC would abolish the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+-induced changes in Im. As shown in Fig. 5, exposing oocytes expressing mENaC (clamped at -60 mV) to amiloride completely blocked the inward current (segment abc). In the presence of amiloride, no significant change in Im occurred when Na+ was removed (segment cd) or readded (segment de) to the bath. The small changes in Im in response to Na+ removal or addition in the bath were much different from those in the absence of amiloride and resemble the Na+-induced effects on Im that occur in water-injected oocytes. In the continued presence of amiloride, addition of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (segment ef) or its removal (segment fg) also did not cause significant changes in Im. It is important to note that the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> effect in the presence of amiloride seems to be smaller than the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Im when mENaC is not blocked (compare with Fig. 4, segment cd). Removal of amiloride restored the inward current to steady-state value (segment gh). These experiments indicate that inhibiting mENaC also abolished the NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-mediated effect on Na+ transport.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5.   Amiloride inhibition of Na+ and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> currents in mENaC oocytes. Amiloride (100 µM) completely inhibited the inward current in oocytes expressing mENaC (segment abc). In the presence of amiloride, Na+ removal (segment cd) or addition of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (segment ef) did not cause any significant changes in the current. The oocyte was clamped at -60 mV.

Effect of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP> on Na+ current at positive potential. NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> application to oocytes results in two main effects: 1) a significant intracellular acidification and 2) a huge depolarization of Vm. Both effects are attributed to the influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and a relatively low permeability of NH3. In oocytes expressing mENaC, NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> could influence Na+ transport by several mechanisms: 1) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> permeation into the cell results in a pHi decrease, which in turn inhibits the Na+ channel. 2) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> could be permeating through ENaC and, therefore, affecting Na+ transport through the channel. 3) Extracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> modulates mENaC. In the next set of experiments, we investigated whether limiting NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry could influence the Na+-induced response in oocytes expressing mENaC. To do so, we clamped the oocyte at positive potential (+10 mV) and measured the whole cell current in response to Na+. As shown in Fig. 6, clamping the oocyte at +10 mV, the whole cell current was positive. Removal of bath Na+ (from 80 to 0 mM) caused an outward deflection in Im (segment ab) that readily recovered when bath Na+ was restored to control (segment bc). At point c, exposing the oocyte to NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a sharp transient increase in Im (segment cd) that settled to a value more positive than that before addition of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, indicating an outward current (segment de). This also indicates that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry through a conductive pathway probably did not occur. When bath Na+ was removed in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, there was the usual positive shift in Im (segment ef), and readdition of Na+ to the bath reversed this effect with an inward shift of Im to its original value (segment fg). Removal of bath NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (80 Na+) caused Im to become more negative (segment gh), reaching a value not significantly different from the original value (at point a) before addition of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. In four paired experiments, the average change in Im caused by removal of bath Na+ in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was 3.4 ± 0.72 µA but was only 1.7 ± 0.23 µA in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (P < 0.05). These experiments indicate that even when NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx is limited, the presence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> still reduces the Na+ response in oocytes expressing mENaC.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (at positive potential) on Na+ current in oocytes expressing mENaC. At positive potential (clamped at +10 mV), the whole cell current in the presence of 80 Na+ in the bath was 1.64 ± 0.8 µA. Removal of bath Na+ in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a larger outward deflection in Im (segment ab) than in its presence (segment ef). Addition of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at positive potential did not cause an inward current but, rather, a transient outward deflection that settled at a more positive value of Im (segment cde).

Whole cell conductance in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP><UP>+</UP></SUP></UP>. The last set of experiments was conducted to examine the effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on whole cell conductance of oocytes expressing mENaC. To do so, we plotted the whole cell currents in relation to test potentials used to clamp the oocyte between -80 and +40 mV in steps of 20 mV. Figure 7 shows the I-V relationship in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. The results of this and similar experiments indicate that in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> the slope of the curve (i.e., conductance) is smaller at negative potentials. However, the current at positive potentials was significantly higher in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, indicating the possible activation of an outward current. The nature of the increased outward current at positive potentials in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is not known. In eight experiments, the whole cell conductance calculated between -60 and -40 mV was 71.3 ± 17 µS in the absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and 55.4 ± 15.3 µS in its presence (P < 0.05). These results are consistent with an inhibitory effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ currents in oocytes expressing mENaC, at least at negative potentials.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 7.   Current-voltage (I-V) relationship for mENaC in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Representative whole cell recordings show I-V relationships of oocytes expressing mENaC in the presence and absence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Control oocytes are oocytes expressing mENaC bathed in standard solution containing 80 mM Na+. The same oocyte was then exposed to a solution containing 20 mM total NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and 80 mM Na+. The experiment indicates a small negative shift in reversal potential and a significant outward current in the presence of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at positive potentials.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The ENaC plays a pivotal role in regulation of Na+ balance, which in turn is critical for regulation of extracellular fluid volume and blood pressure. Regulation of ENaC is accomplished by a complex interaction of several mechanisms, including hormonal factors such as aldosterone and vasopressin and nonhormonal factors such as pHi, Na+, and Ca2+ (8, 19, 35).

Ionic activities. In this study we report the first measurements of the changes in intracellular ionic activities that occur as a result of expressing ENaC in oocytes. As shown in Table 1, these changes are very significant. Three important observations are evident from these results. First, although an increase in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> is expected when ENaC is expressed, the decrease in a<UP><SUB>i</SUB><SUP>K</SUP></UP> and the increase in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> were not known or measured previously. Because the oocyte has a significant permeability to K+ and Cl-, ignoring these changes in studies involving measurements in the intact oocyte could result in significant errors. For instance, one such possibility is an erroneous estimation of the Na+ reversal potential, which will vary depending on the Cl- content of the bathing medium. The second important observation is the value of a<UP><SUB>i</SUB><SUP>Na</SUP></UP> of 30 ± 1.8 mM shown in Table 1. This value is almost five times more than that of control oocytes; however, it is much smaller than the value (50-60 mM) usually calculated from reversal potential of Na+ in oocytes expressing ENaC. In such studies, it has been argued that the high a<UP><SUB>i</SUB><SUP>Na</SUP></UP> (calculated from the reversal potential) represents the microscopic value of Na+ activity at the tip of the channel (6). Although some compartmentalization may occur, it is very likely that the contribution of other ions (and their respective membrane permeabilities) to the reversal potential is underestimated in oocytes expressing ENaC. A third point to note is that, in our experiments, complete removal of external Na+ resulted in only small changes in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> (Delta a<UP><SUB>i</SUB><SUP>Na</SUP></UP> = 1.8 mM). Although the volume of the oocyte is large, this was unexpected and indicates that even though ENaC is usually overexpressed, total intracellular Na+ is not readily exchangeable through the channel. In support of this observation, we calculated the change in intracellular Na+ from the change in current caused by removal of external Na+ (Fig. 4). For a change in current of 3.7 µA over a period of ~100 s, the number of coulombs (Q) presumably carried by movement of Na+ can be calculated as
Q=I×t

=3.7×10<SUP>−6</SUP>×100

=370×10<SUP>−6</SUP> coulombs
where current is measured in amperes and time in seconds. If Na+ transfer is responsible for all this charge, then the number of moles of Na+ that would result in this change can be calculated from dividing by Faraday's constant, which results in
370×10<SUP>−6</SUP>/96,500 mol Na<SUP>+</SUP>
If the oocyte volume is ~0.9 µl (with the assumption of a spherical volume with a diameter of 1.2 mm), then the calculated change in intracellular Na+ concentration is
(370×10<SUP>−6</SUP>/96,500)×(1/0.9)

×(10<SUP>6</SUP>) &mgr;mol<IT>/&mgr;</IT>l (M)<IT>=</IT>4.2 mM
This value is much smaller than the presumed large changes in a<UP><SUB>i</SUB><SUP>Na</SUP></UP> and within the small range of intracellular Na+ change measured with microelectrodes.

Inhibition of Na+ transport with NH3/NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP>. The present study suggests that NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibits Na+ transport via ENaC. Urinary excretion of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is very important for acid-base homeostasis. Two-thirds of net acid excretion in the urine is via NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, and in systemic acidosis intratubular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentrations and renal cortical ammonia are increased (10). Under these conditions, urinary levels of total ammonia can easily exceed 50 mmol/l. In the cortical collecting duct, peritubular NH4Cl was shown to inhibit transepithelial Na+ transport (22, 23). In other tight epithelia, NH4Cl was also reported to influence Na+ transport (20). This effect of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ transport and its importance in regulation of acid-base homeostasis raise the possibility that NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> may directly or indirectly affect transport through ENaC.

The first evidence for the inhibitory affect on ENaC by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was obtained from measurements of voltage changes induced by Na+ removal in the presence and absence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. The studies showed that 1) in the presence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, the hyperpolarization caused by removal of bath Na+ was significantly inhibited, 2) the voltage inhibition by NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is not due to intracellular acidification caused by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, and 3) the effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on Na+ removal was completely blocked by amiloride, demonstrating that the inhibitory effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is probably through an effect on ENaC. The effects of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> are not due to significant changes in a<UP><SUB>i</SUB><SUP>Na</SUP></UP>. Evidence for this comes from the experiment of Fig. 2B, where NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibited Na+-induced Vm changes irrespective of whether initial external Na+ was 100 or 80 mM. This is further supported by our previous observation that complete removal of external Na+ caused only a small change in a<UP><SUB>i</SUB><SUP>Na</SUP></UP>, measured with microelectrodes.

Measurement of current in response to Na+ removal further demonstrated the inhibition of ENaC by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Again, in the presence of 100 or 80 mM external Na+, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibited the outward current caused by removal of bath Na+. In the presence of amiloride, neither Na+ removal nor NH<UP><SUB>4</SUB><SUP>+</SUP></UP> resulted in significant changes in current. The voltage and current data indicate that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibits ENaC.

Many studies (7, 19, 33, 42) have addressed the role of pH in regulating ENaC activity. As reviewed by Lyall et al. (28), decreases in extracellular pH and/or pHi have usually been reported to decrease Na+ transport in tight epithelia. The direct studies of Chalfant et al. (13) on oocytes expressing ENaC recently reported that a decrease in pHi, but not extracellular pH, reduced single-channel open probability and open time without altering single-channel conductance. This study further indicated that the alpha -subunit of ENaC is directly regulated by pHi. In contrast, however, Awayda et al. (7) reported that luminal acidosis over a period of minutes stimulates Na+ current in A6 cells. On the other hand, more prolonged changes in pH, such as in vivo systemic acidosis, may have complicated effects. For example, Kim et al. (26) reported that long-term acid loading in vivo by NH4Cl produced a large decrease in the abundance of beta - and gamma -subunits of ENaC but had no effect on the alpha -subunit.

In our study, the presence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> clearly affects ENaC in a manner distinct from a pHi inhibition of the channel. Although NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibited the whole cell conductance (consistent with a pHi effect), a decrease in pHi is unlikely to account for all the inhibitory effect of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on the basis of the studies with butyrate in Fig. 3 and the studies with voltage clamp at positive potential in Fig. 6. These latter experiments demonstrated that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> still inhibited the Na+ current, even though there was no apparent conductive NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx as evidenced from the absence of inward current on exposure to NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. This raises the likelihood that external NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, and not necessarily intracellular acidification, is responsible for inhibiting the Na+ current.

At positive potentials, the presence of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> caused a significant activation of an outward current not seen in control oocytes. This raises the possibility of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> interacting with ENaC in a manner more complicated than simple inhibition. The nature of this voltage-dependent outward current is not clear and needs to be pursued further.

Although the interaction of several cations, such as Li+ and K+, with ENaC has been investigated, that of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> has not been studied to our knowledge. A complicating factor in addressing this issue is the significant change in pHi caused by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>.

Possible physiological implications. The effects of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> are particularly relevant in states of systemic acidosis during which cortical and medullary interstitial ammonia concentrations rise. Acidosis is well known to be associated with natriuresis and diuresis. Although this has been attributed in part to decreases in proximal tubular reabsorption, effects of acidosis on distal nephron transport are also reported. Metabolic acidosis has been shown to inhibit K+ secretion and Na+ reabsorption in the distal tubule and collecting duct (30, 41). As discussed above, low pHi has been shown to inhibit Na+ transport (presumably via ENaC inhibition) in a variety of tight epithelia (reviewed in Ref. 28). Long-term acidosis has recently been reported to decrease the distal tubule Na-Cl cotransporter and beta - and gamma -subunits of ENaC (26). Low pH has also been shown to inhibit Na+-K+-ATPase (16). In addition to the effects of pH and systemic acidosis, the present studies and some prior work suggest that the increased levels of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> that occur with acidosis may have additive effects to inhibit Na+ transport. We previously demonstrated that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibited transepithelial Na+ and K+ transport in cortical collecting ducts in vitro. The present studies extend these findings to demonstrate that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibits Na+ transport via ENaC. Therefore, acidosis inhibits renal Na+ transport via a variety of mechanisms, including the effects of NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, acting in concert. Recently, Frank et al. (17) suggested that renal ammonia content can act as a type of extracellular signaling molecule in integrating the response to systemic acidosis; the present studies would be consistent with such a role.

In conclusion, the results of this study demonstrate that NH<UP><SUB>4</SUB><SUP>+</SUP></UP> inhibits Na+ transport when ENaC is expressed in the oocyte. Although some pHi effect cannot be ruled out, it is likely that a decrease in pHi is not the major cause of this inhibition. These results are important for understanding the role of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in modulating Na+ transport in vivo in states of systemic acidosis.


    ACKNOWLEDGEMENTS

We thank Dr. Emile L. Boulpaep for important suggestions and valuable discussions, Dr. Keith Elmslie for critical comments on the data, and Stephanie Palmer for secretarial and technical help.


    FOOTNOTES

This work was supported by American Heart Association Grant 0050547N, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54952, the Department of Veterans Affairs, and by DCI, Inc.

Address for reprint requests and other correspondence: N. L. Nakhoul, Dept. of Medicine, Sect. of Nephrology, SL-45, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: nakhoul{at}tulane.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 February 2001; accepted in final form 7 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahn, YJ, Brooker DR, Kosari F, Harte BJ, Li J, Mackler SA, and Kleyman TR. Cloning and functional expression of the mouse epithelial sodium channel. Am J Physiol Renal Physiol 277: F121-F129, 1999[Abstract/Free Full Text].

2.   Alvarez de la Rosa, D, Canessa CM, Fyfe GK, and Zhang P. Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62: 573-594, 2000[ISI][Medline].

3.   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].

4.   Asher, C, Eren R, Kahn L, Yeger O, and Garty H. Expression of the amiloride-blockable Na+ channel by RNA from control versus aldosterone-stimulated tissue. J Biol Chem 267: 16061-16065, 1992[Abstract/Free Full Text].

5.   Asher, C, and Garty H. Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms. Proc Natl Acad Sci USA 85: 7413-7417, 1988[Abstract].

6.   Awayda, MS. Regulation of the epithelial Na+ channel by intracellular Na+. Am J Physiol Cell Physiol 277: C216-C224, 1999[Abstract/Free Full Text].

7.   Awayda, MS, Boudreaux MJ, Reger RL, and Hamm LL. Regulation of the epithelial Na+ channel by extracellular acidification. Am J Physiol Cell Physiol 279: C1896-C1905, 2000[Abstract/Free Full Text].

8.   Benos, DJ, Awayda MS, Ismailov II, and Johnson JP. Structure and function of amiloride-sensitive Na+ channels. J Membr Biol 143: 1-18, 1995[ISI][Medline].

9.   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].

10.   Buerkert, J, Martin D, and Trigg D. Ammonium handling by superficial and juxtamedullary nephrons in the rat: evidence for an ammonia shunt between the loop of Henle and the collecting duct. J Clin Invest 70: 1-12, 1982[ISI][Medline].

11.   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].

12.   Canessa, CM, Horisberger JD, and Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[ISI][Medline].

13.   Chalfant, ML, Denton JS, Berdiev BK, Ismailov II, Benos DJ, and Stanton BA. Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel. Am J Physiol Cell Physiol 276: C477-C486, 1999[Abstract/Free Full Text].

14.   Chang, SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996[ISI][Medline].

15.   Chase, HS, Jr, and Al-Awqati Q. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder: studies using a fast-reaction apparatus. J Gen Physiol 81: 643-665, 1983[Abstract].

16.   Eaton, DC, Hamilton KL, and Johnson KE. Intracellular acidosis blocks the basolateral Na-K pump in rabbit urinary bladder. Am J Physiol Renal Fluid Electrolyte Physiol 247: F946-F954, 1984[ISI][Medline].

17.   Frank, AE, Wingo CS, and Weiner ID. Effects of ammonia on bicarbonate transport in the cortical collecting duct. Am J Physiol Renal Physiol 278: F219-F226, 2000[Abstract/Free Full Text].

18.   Fuchs, W, Larsen EH, and Lindemann B. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J Physiol (Lond) 267: 137-166, 1977[ISI][Medline].

19.   Garty, H, and Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77: 359-396, 1997[Abstract/Free Full Text].

20.   Guggenheim, SJ, Bourgoignie J, and Klahr S. Inhibition by ammonium of sodium transport across isolated toad bladder. Am J Physiol 220: 1651-1659, 1971[ISI][Medline].

21.   Hamilton, KL, and Eaton DC. Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels. Membr Biochem 6: 149-171, 1986[ISI][Medline].

22.   Hamm, LL, Gillespie C, and Klahr S. Ammonium chloride inhibits Na+ and K+ transport in the cortical collecting tubule. Contrib Nephrol 47: 125-129, 1985[Medline].

23.   Hamm, LL, Gillespie C, and Klahr S. NH4Cl inhibition of transport in the rabbit cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 248: F631-F637, 1985[Abstract/Free Full Text].

24.   Hansson, JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, and Lifton RP. Hypertension caused by a truncated epithelial sodium channel gamma -subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11: 76-82, 1995[ISI][Medline].

25.   Ismailov, II, Berdiev BK, and Benos DJ. Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers. J Gen Physiol 106: 445-466, 1995[Abstract].

26.   Kim, GH, Martin SW, Fernandez-Llama P, Masilamani S, Packer RK, and Knepper MA. Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake. Am J Physiol Renal Physiol 279: F459-F467, 2000[Abstract/Free Full Text].

27.   Lingueglia, E, Renard S, Voilley N, Waldmann R, Chassande O, Lazdunski M, and Barbry P. Molecular cloning and functional expression of different molecular forms of rat amiloride-binding proteins. Eur J Biochem 216: 679-687, 1993[Abstract].

28.   Lyall, V, Feldman GM, and Biber TU. Regulation of apical Na+ conductive transport in epithelia by pH. Biochim Biophys Acta 1241: 31-44, 1995[ISI][Medline].

29.   Masilamani, S, Kim GH, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC alpha -, beta -, and gamma -subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999[Abstract/Free Full Text].

30.   Molony, DA, Kokko JP, Seldin DW, and Jacobson HR. Acid peritubular pH suppresses sodium reabsorption in cortical collecting tubules (Abstract). Kidney Int 25: 279A, 1984.

31.   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].

32.   Palmer, LG, and Frindt G. Effects of cell Ca and pH on Na channels from rat cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 253: F333-F339, 1987[Abstract/Free Full Text].

33.   Palmer, LG, and Frindt G. Epithelial sodium channels: characterization by using the patch-clamp technique. Fed Proc 45: 2708-2712, 1986[ISI][Medline].

34.   Rossier, BC. Mechanisms of aldosterone action on sodium and potassium transport. In: The Kidney: Physiology and Pathophysiology, edited by Giebisch DW.. New York: Raven, 1992, p. 1373-1409.

35.   Rossier, BC, Canessa CM, Schild L, and Horisberger JD. Epithelial sodium channels. Curr Opin Nephrol Hypertens 3: 487-496, 1994[Medline].

36.   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].

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

38.   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].

39.   Schafer, JA, and Hawk CT. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int 41: 255-268, 1992[ISI][Medline].

40.   Shimkets, RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR, Jr, Ulick S, Milora RV, and Findling JW. Liddle's syndrome: heritable human hypertension caused by mutations in the beta -subunit of the epithelial sodium channel. Cell 79: 407-414, 1994[ISI][Medline].

41.   Stanton, BA, and Giebisch G. Effects of pH on potassium transport by renal distal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 242: F544-F551, 1982[Abstract/Free Full Text].

42.   Zeiske, W, Smets I, Ameloot M, Steels P, and van Driessche W. Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport. Am J Physiol Cell Physiol 277: C469-C479, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(3):F493-F502
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society