Regulation of the epithelial Na+ channel by extracellular acidification

Mouhamed S. Awayda, Michael J. Boudreaux, Roxanne L. Reger, and L. Lee Hamm

Departments of Medicine and of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of extracellular acidification was tested on the native epithelial Na+ channel (ENaC) in A6 epithelia and on the cloned ENaC expressed in Xenopus oocytes. Channel activity was determined utilizing blocker-induced fluctuation analysis in A6 epithelia and dual electrode voltage clamp in oocytes. In A6 cells, a decrease of extracellular pH (pHo) from 7.4 to 6.4 caused a slow stimulation of the amiloride-sensitive short-circuit current (INa) by 68.4 ± 11% (n = 9) at 60 min. This increase of INa was attributed to an increase of open channel and total channel (NT) densities. Similar changes were observed with pHo 5.4. The effects of pHo were blocked by buffering intracellular Ca2+ with 5 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In oocytes, pHo 6.4 elicited a small transient increase of the slope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min) followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pHo stimulate the native ENaC by increasing NT but do not appreciably affect ENaC expressed in Xenopus oocytes. These effects are distinct from those observed with decreasing intracellular pH with permeant buffers that are known to inhibit ENaC.

epithelial sodium channel; A6 epithelia; Xenopus oocytes; noise analysis; channel density


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NA+ channel in the apical membrane of many native electrically tight Na+-absorbing epithelia is subjected to an environment with a highly dynamic extracellular pH (pHo). It is well established that large decreases (e.g., pH 2) of luminal pH (pHo) decrease Na+ absorption and that this inhibition is mediated via changes of intracellular pH (pHi), leading to inhibition of the apical Na+ channel (6-10, 16, 19, 20, 22, 25). Indeed, Palmer and Frindt (20) found that channel activity and presumably open probability (Po) is inhibited by pHi in excised membrane patches. Zeiske et al. (25) have also recently reported that open channel density (No) of the Na+ channel found in A6 epithelia is inhibited by decreasing pHi.

Leaf et al. (16) have an unexplainable finding that small decreases of pHo (down to 5.5) causes a stimulation, rather than inhibition, of Na+ transport in the toad bladder. This stimulation was observed in the absence of detectable effects on pHi and was clearly distinct from the inhibitory effects observed with intracellular acidification. The mechanism for this stimulation, and whether such observation is applicable to other Na+-absorbing epithelia, remains undetermined.

The regulation of the cloned epithelial Na+ channel (ENaC) by pH has been recently investigated by Chalfant et al. (4). These authors found that decreases of pHi cause a rapid (within minutes) inhibition of ENaC expressed in Xenopus oocytes through effects on channel activity. Similar changes of pHo were without appreciable short-term (<10 min) effects on the channel. Thus it appears that the cloned ENaC has the capability to rapidly (<5 min) respond to changes of pHi and that these effects may represent a direct interaction with H+, because an inhibition of Po was found for ENaC incorporated into planar lipid bilayers. Moreover, channel activity was relatively unaffected by short-term (<10 min) deceases of pHo.

We have recently observed that a small decrease of pHo from 7.4 to 6.4 causes stimulation of the short-circuit current (Isc) in the Xenopus kidney cell line A6. This stimulation was not a direct effect of the pH change in that the increase of Isc was not immediate. Moreover, this effect was similar in its time course to that observed by Leaf et al. (16) in toad bladder. Because the apical Na+ channel (ENaC) is rate limiting to transepithelial transport, this stimulation is likely mediated via effects on the native ENaC. To determine the single channel basis of this stimulation, we utilized blocker-induced transepithelial fluctuation analysis. Similar experiments were also carried out on the cloned ENaC expressed in Xenopus oocytes to determine if prolonged extracellular acidification also stimulates this channel in this system.

We report that stimulation of the Isc observed by small apical acidification in polarized A6 epithelia is due to increases of total channel density (NT). A small decrease of the single channel current (iNa) was also observed and is likely due to apical membrane depolarization. This decrease of iNa slightly underestimated the stimulation of the macroscopic Isc. The changes of NT and Isc were mediated via intracellular Ca2+-dependent mechanisms, since pretreatment of A6 epithelia with an intracellular Ca2+ buffer [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] prevented this stimulation.

In contrast, decreasing pHo in ENaC-expressing oocytes caused a small transient stimulation of the amiloride-sensitive conductance followed by recovery to below control levels. Thus additional Ca2+-dependent mechanisms may be present in tight epithelia and may be responsible for the sustained stimulation of NT with an acidic luminal environment. We speculate that the increase of NT observed in A6 cells may serve as a protective mechanism whereby an epithelium subjected to large acid loads, which would normally inhibit Na+ transport through changes of pHi, is more capable of resuming its Na+ reabsorption after recovery from this acidic environment.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A6 epithelia. Cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured to confluency in 75-cm2 flasks and were subcultured on permeable polyester membrane inserts (Transwell Clear; Costar). Cells were trypsinized, and the equivalent of 1/360th of cells from a single flask were seeded on a single insert (area 4.7 cm2). Cell polarity was assessed by their ability to develop a transepithelial potential difference (Voc) and appreciable transepithelial resistance (RT). With the use of the solutions described below, monolayers exhibited Voc in the range of -40 to -60 mV and RT in the range of 7-12 kOmega · cm2 within 10-14 days after plating. These values were stable for ~2 mo.

Cells were grown at 26°C in a humidified incubator containing ambient air with 1.2% CO2. The culture media was similar to that previously described (23) and of the following composition: 26.2% L-15 Leibovitz, 26.2% Ham's F-12, 7.6% FBS, 1.5% L-glutamine (200 mM solution), 0.3% penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin), and 0.3% of a 7% sodium-bicarbonate solution. ddH2O was added (~38%) to a final solution osmolarity of ~200 mosmol/l. Media in both flasks and membrane inserts was changed two times weekly.

Oocyte isolation and injection. Toads were obtained from Xenopus Express (Beverly Hills, FL) and were kept in dechlorinated tap water at 18°C. Conditions for oocyte removal, processing, injection, and cRNA synthesis were as previously described (2). Injected oocytes were incubated at 18°C for 1-3 days until recording. All recordings were performed at 19-21°C.

Solutions and chemicals. All solutions and chemicals were as previously described by Awayda and Subramanyam (3). ND-96 (96 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 2 mM KCl, and 5 mM HEPES) at pH 7.4 was used for initial recording from both oocytes and A6 epithelia. HCl was used to alter pH in ND-96 to pH 6.4 or 5.4. Amiloride was a gift from Merck-Sharp & Dohme (Rahway, NJ). BAPTA-AM was obtained from Molecular Probes (Eugene, OR). All other chemicals were of the highest grade and were obtained from Sigma Chemical (St. Louis, MO).

Dual electrode clamp. Defoliculated Xenopus oocytes were injected with cRNAs for rat alpha -, beta -, and gamma -ENaC (rENaC). Injected oocytes were cultured as previously described (3). Whole cell currents were recorded in oocytes held at 0 mV and pulsed from -100 to +40 mV. Slope conductance (gm) was summarized between -80 and -100 mV (2). By convention, inward flow of cations is designated as inward current (negative current), and all voltages are reported with respect to ground or bath. Except where noted, all data are reported as means ± SE.

Fluctuation analysis. Membranes were placed in a modified Ussing chamber, and the transepithelial voltage was clamped to 0 mV using a low-noise, direct current-powered, four-electrode voltage clamp. Short 2-mV pulses were used to measure the transepithelial resistance.

Noise or fluctuation analysis was carried out as previously described by Helman et al. (13). After Isc was allowed to stabilize, noise analysis was conducted using the uncharged amiloride analog 6-chloro-3,5-diamino-2-pyrazinecarboxamide (CDPC). CDPC was pulsed into the apical side of the Ussing chamber at concentrations of 20 and 80 µM or at 15 and 40 µM. Current noise was filtered at Nyquist frequency (~1,900 Hz). The filtered signal was amplified and stored digitally. Signals were Fourier transformed to yield the power-density spectra.

pH changes were made to the apical side of the tissue while the pH of the basolateral side was held constant. All solutions used a HEPES-based buffer and were therefore not expected to affect pHi. When used, BAPTA-AM was added to both sides of the tissue at a final concentration of 5 µM in 0.05% DMSO (tissue culture grade).

pHi. These measurements used the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). BCECF was purchased from Molecular Probes in its membrane-permeable BCECF-AM form. BCECF-AM was dissolved in DMSO on the day of experiments and was used at a final concentration of 10 µM in ND-96. Cells were allowed at least 60 min to uptake the dye.

Fluorescence studies were carried out on an inverted Nikon Diaphot-TMD microscope as previously described (14). Fluorescence ratio was measured using the Nikon/PTI Photoscan II system, with excitation at 490 and 440 nm and emission at 530 nm. Background fluorescence was measured in BCECF-free cells and was <10% of the fluorescence observed in BCECF-loaded cells. All experimental data were corrected for background fluorescence.

pHi experiments were also carried out on polarized A6 monolayers grown on the same filters used for the noise analysis experiments to allow us to correlate these two measurements. Cells were studied in special chambers designed to allow pHi measurements in cultured polarized cells and offered separate access to the apical and basolateral compartments in addition to a means of rapid solution exchange in each of these compartments (17). Because the cells were grown on transparent membrane supports, it was expected that the filter material would not obstruct the pHi measurements. However, to circumvent any potential problems, the filter and accompanying cells were inverted in the chamber so that the apical membrane and the cells directly faced the excitation source.

Statistical analysis was carried out using paired Student's t-test where appropriate. Significance was determined at the 95% confidence level (P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of decreasing pHo on the macroscopic properties of the native ENaC. Confluent A6 cells were short circuited according to conventional methods in symmetrical ND-96 at pH 7.4. In these cells, Isc is essentially all attributed to Na+ transport through the apical native ENaC and is blocked by 10 µM amiloride. To noninvasively calculate the single channel properties, we used blocker-induced fluctuation analysis. This protocol is similar to that used by Helman et al. (13) and allows the assessment of the time course of changes of the single channel parameters. To utilize these methods, cells were continuously perfused in Ringer solution containing a low blocker concentration (20 or 15 µM CDPC) and were periodically (every 10 min) pulsed for ~3 min with solution containing a higher blocker concentration (80 or 40 µM CDPC). This protocol is shown in Fig. 1 along with a continuous recording of the Isc and the effect of a decrease of apical pHo. It is evident from the examples in Fig. 1 that decreasing pHo to 6.4 or 5.4 caused a gradual stimulation of the Isc and presumably the apical Na+ channels.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Noise analysis protocol and representative effect of extracellular acidification on Na+ transport in A6 monolayers. Continuous recording of the short-circuit current (Isc) at a holding potential of 0 mV. A 2-s pulse was applied at fixed intervals to determine monolayer resistance. This pulse was removed during acquisition of the power density spectra. A: effect of pH 6.4 on the Isc. B: effect of pH 5.4 on the Isc. In these particular examples, a 6-chloro-3,5-diamino-2-pyrazinecarboxamide (CDPC) dose response (20-100 µM) was carried out at the beginning and end of the experiment. To carry out noise analysis during the control and experimental periods, tissues were incubated in 20 µM CDPC and pulsed for ~3 min with 80 µM CDPC (see text for details). Both pH 6.4 and 5.4 caused a gradual stimulation of the Isc. [CDPC], CDPC concentration.

The time course of the effects of decreasing pHo is summarized in Fig. 2. These data summarize the changes of the amiloride-sensitive currents (INa). Extracellular acidification with HEPES-based buffer caused a gradual stimulation of the INa that reached a plateau within 30-40 min. To determine whether similar effects are observed for the cloned channel, we carried out experiments in the Xenopus oocytes expression system.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of the stimulation of amiloride-sensitive Na+ transport with extracellular acidification. A: pH 6.4 caused a sustained but gradual stimulation of the amiloride-sensitive short-circuit current (INa) that reached a relative plateau within ~40 min. B: similar effects were observed with pH 5.4, except that the stimulation of INa was slightly faster. All data are summarized in the presence of the lower [CDPC]; n = 9 and n = 6 experiments for pH 6.4 and 5.4, respectively.

Effects of decreasing pHo on the macroscopic properties of the cloned channel. Figure 3 is a representative example of the whole cell currents in an ENaC-expressing oocyte and their block by 10 µM amiloride. Amiloride causes a decrease of the whole cell currents to levels not different from those observed in control water-injected oocytes. The whole cell currents between -100 and -80 mV were used to calculate the inward gm. As evident from Fig. 3, the majority of the gm is amiloride sensitive and is attributed to ENaC.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Representative whole cell currents in an epithelial Na+ channel (ENaC)-expressing oocyte and its block by amiloride. A: currents in an ENaC-expressing oocyte bathed in control Ringer solution. B: currents in the same oocyte after the addition of 10 µM amiloride. C: INa calculated as A - B. The slope conductance (gm) was calculated from the currents at -100 and -80 mV.

The effects of a small decrease of pHo with HEPES-buffered solution on the gm are summarized in Fig. 4. Within the resolution of the first measurement (30 s), pHo of 6.4 caused an increase of gm. This increase reached a peak value at 2 min and then steadily declined to values below control. Thus the response to decreasing pHo was different between oocytes and A6 cells. The origin of these differences is unclear but may be due to one or a combination of differences in the expression system (oocytes vs. epithelial cells) or differences in the channel itself (cloned vs. native).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Time course of the effects of extracellular acidification on the cloned ENaC expressed in oocytes. Data are summarized as gm and are normalized in each oocyte to the value of gm immediately before the pHo change. Decreasing pHo caused a transient stimulation of gm. This stimulation reached a peak value of ~12% at 2 min and was followed by inhibition to below control values (n = 6). E/C, mean ratio of the paired control and experimental groups.

It is possible that the initial stimulation of the cloned ENaC is similar to that observed from the native channel in A6 cells. It is clear, however, that the cloned ENaC expressed in oocytes did not exhibit a sustained stimulation. To further investigate the mechanisms of the sustained stimulation observed in A6 cells, we used blocker-induced noise analysis.

Effects of decreasing pHo on the single channel properties of the native channel. In the absence of blockers, the spontaneous rates of ENaC transition are too slow for the resolution of noise analysis. This is indeed consistent with single channel patch-clamp data that indicate open and close times on the order of seconds [see Garty and Palmer (9)]. These slow kinetics are manifested by the absence of a spontaneous Lorentzian function in the power density spectrum (Fig. 5A). To resolve channel properties, a blocker is used to interact with the channel and speed up its rates of opening and closing. CDPC is used as it is uncharged and results in small inhibition of macroscopic currents. The on and off rates for CDPC are also sufficiently fast enough and allow for better resolution of the blocker-induced Lorentzian (Fig. 5B). The corner frequency of the Lorentzian function exhibits a linear relationship with CDPC concentration, as expected from a first-order reaction (Fig. 5C). The corner frequencies, macroscopic currents, and power plateaus are used to calculate the channel properties as described by Helman et al. (13). The effects of pHo 6.4 and 5.4 on the channel properties are summarized below.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Noise or fluctuation analysis of the native ENaC in A6 epithelia. A: background power density spectrum in the absence of a blocker. Consistent with the known kinetics of ENaC, there are no observed spontaneous Lorentzian functions. B: addition of a blocker, such as CDPC, induces the appearance of a Lorentzian function. This function can be described by power plateau and a corner frequency. C: as expected from a first-order reaction, the corner frequency is linearly related to blocker concentration, and this relationship can be used to calculate the blocker on and off rates. See text for more details.

Figure 6 shows the effects of decreasing pHo on the blocker equilibrium constant (KB). This constant is calculated from the ratio of the blocker off and on rates and is therefore independent of Po, which can affect the apparent equilibrium constant calculated from the half-maximal block of the macroscopic currents. CDPC is electroneutral at pH 7.4 and down to approximately pH 4, and therefore its net charge is unaffected by a change of pH from 7.4 to 5.4. In this respect, it becomes of additional advantage to use CDPC in the current experiments since any pH-related changes of KB are not due to simple changes of the net charge on this blocker. As evident from Fig. 6, KB is unaffected by changes of pHo, and thus pHo does not affect the interaction between CDPC with the externally accessible portion of ENaC. Because amiloride, the parent molecule for CDPC, behaves as a plug (18) that protrudes ~25% of the way into the mouth of the channel (24), the lack of effects on KB may also indicate that the outer portion of the channel that interacts with amiloride and CDPC is not modified by these changes of pHo. This, however, does not rule out pHo-induced modification of an accessory protein or of different extracellular regions of the channel that do not interact with CDPC.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Lack of effects of extracellular acidification on the blocker equilibrium constant (KB). KB was insensitive to pHo 6.4 (A) and 5.4 (B); n = 9 and 6 for pHo 6.4 and 5.4, respectively.

To determine the mechanisms of stimulation of the INa, we calculated the single channel properties and channel density. As shown in Fig. 7, the stimulation observed with pHo 6.4 is predominantly due to a stimulation of No. These changes were accompanied by a compensatory decrease of iNa. These effects on iNa are likely due to a decrease of the electrochemical gradient across the channel rather than a change of the single channel conductance (see DISCUSSION).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Time course of the effects of pHo 6.4 on the single channel properties. A: pHo 6.4 caused a small but significant decrease of the single channel current (iNa). These changes appeared to be more rapid than those of the INa. B: open channel probability (No) was stimulated with a similar time course to that observed for the changes of INa. C: no effects were observed on open probability (Po; n = 9).

The mechanisms underlying the stimulation of INa by pHo 5.4 were similar to those observed above with pHo 6.4. As shown in Fig. 8, pHo 5.4 caused a stimulation of No and a compensatory decrease of iNa. Consistent with the slightly faster changes of INa with pHo 5.4, these changes of No appear to reach a relative plateau within 30 min. The relatively slow time course of these changes may indicate the presence of channel trafficking events resulting from the involvement of second messenger cascades. The two most prominent second messenger cascades that affect Na+ transport involve cAMP and Ca2+. We focused our attention on the Ca2+ pathway because of our ongoing interest in ENaC regulation by Ca2+ and/or protein kinase C. 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8.   Time course of the effects of pHo 5.4 on the single channel properties. A: pHo 5.4 also caused a small but significant decrease of iNa. The time course of the changes of iNa was clearly more rapid than that of the changes of INa. B: No was stimulated with a similar time course to that observed for the changes of INa. C: no effects were observed on Po (n = 6). Both No and iNa exhibited similar but more rapid changes compared with pHo 6.4 (see Fig. 7).

Role of intracellular Ca2+ concentration in the observed stimulation. It is well known that large increases of the intracellular Ca2+ concentration ([Ca2+]i) inhibit ENaC (5, 7, 19, 20). However, Ca2+ is also involved in many second messenger-mediated signaling cascades, including those resulting in vesicular trafficking. To determine the role of [Ca2+]i, A6 monolayers were incubated with 5 µM BAPTA-AM, an intracellular Ca2+ chelator. This chelator is added in a membrane-permeable form that allows it to enter the cell. Intracellular BAPTA is then deesterified, which renders it membrane impermeant, and is trapped within the cell where it binds free Ca2+ and buffers the changes of [Ca2+]i. Figure 9 is a representative effect of BAPTA followed by pHo 6.4 on the Isc. Within minutes, the addition of BAPTA caused a marked decrease of transport. These monolayers were challenged with pHo 6.4 1 h after the addition of BAPTA. In this example, there were no changes of the Isc with pHo 6.4. 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 9.   Representative effect of extracellular acidification on Na+ transport in A6 monolayers pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Addition of 5 µM BAPTA caused a gradual inhibition of the Isc. Subsequent extracellular acidification ~1 h after the initial BAPTA treatment was without effect. Thus the stimulation observed with pHo 6.4 is dependent on [Ca2+]i. See Fig. 1 legend for additional details.

The effects of pHo 6.4 on the macroscopic and single channel parameters in BAPTA-pretreated monolayers are summarized in Fig. 10. BAPTA treatment (5 µM) caused a decline of INa. This trend was not altered after treatment with pHo 6.4, and INa showed no appreciable evidence of stimulation (Fig. 10A). Similarly, no significant effects of pHo were observed on iNa, No, and Po in BAPTA-pretreated cells within the first 30 min. A significant increase of Po was observed in measurements >30 min after the pHo change. The reason for this increase is unknown. It may be unrelated to the change of pHo but related to prolonged intracellular Ca2+ depletion. Nevertheless, the effects of pHo on No appear to involve [Ca2+]i.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 10.   BAPTA pretreatment eliminated the effects of extracellular acidification on the macroscopic and single channel properties. A: addition of BAPTA caused a gradual and continuous decrease of INa. This trend was not appreciably affected by pHo 6.4. B: no appreciable effects were observed on iNa. C: No exhibited a small transient tendency for an increase after pHo 6.4. However, this effect was not statistically significant. D: no effects were observed on Po (n = 6).

A major advantage of noise analysis is that this technique allows the calculation of single channel parameters and NT. This circumvents problems with variable channel gating, as observed with ENaC, and allows a more accurate estimate of Po and NT. The effects on NT of decreasing pHo and its block by buffering [Ca2+]i are summarized in Fig. 11. This figure demonstrates that pHo causes a greater than twofold increase of NT via mechanisms that involve increases of [Ca2+]i. As observed from Figs. 1-10, the changes of NT with pHo 5.4 were more rapid than those observed with pHo 6.4. 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 11.   Time course of the stimulation of total channel density (NT) by extracellular acidification. Extracellular acidification caused a >2-fold increase of NT. The magnitude of the changes was similar between pHo 6.4 (circles, n = 9) and 5.4 (squares, n = 6); however, the effects of pHo 5.4 were significantly faster. Pretreatment of cells with BAPTA caused a decrease of NT (triangles, n = 6) that was unaffected by subsequent acidification, as NT continued to decrease to ~30% on control within 60 min.

Table 1 summarizes our findings with extracellular acidification. The only significant changes observed at both decreased pHo were those involving iNa and NT. No significant changes were observed in the BAPTA-pretreated cells. In both cases, and in the absence of BAPTA, decreasing pHo caused an ~60% increase of INa mediated via an approximately twofold increase of NT and a small ~20% decrease of iNa.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effects of decreasing pHo in HEPES-buffered solution on ENaC in A6 epithelia

Measurements of pHi. It is highly possible that prolonged changes of pHo, even in HEPES-buffered solutions, could lead to appreciable changes of pHi. To determine whether such a hypothesis is tenable in the present experiments, we utilized fluorescence measurements of pHi in polarized A6 monolayers. As shown in the representative example in Fig. 12, decreasing apical pHo did not have any detectable changes of the BCECF fluorescence ratio, indicating lack of effects on pHi. On the other hand, an appreciable and reversible effect could be observed with the permeable ion NH4. These observations do not rule out small local changes of pHi but indicate the lack of large changes of pHi.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 12.   Representative example of the effects of apical acidification on pHi. In all experiments, a control period of ~20 min was established. The apical solution was then switched to one with a pH of 6.4. Ammonium chloride (20 mM) was then added as a positive control to verify that the measured fluorescence ratio reflected pHi. This maneuver resulted in a reversible change of pHi. Data are representative of 6 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We used A6 epithelia and the Xenopus oocyte expression system to study the effects of extracellular acidification on ENaC. Consistent with a previous report by Leaf et al. (16) in the toad bladder, extracellular acidification caused a stimulation of Na+ transport in A6 epithelia. This response was sustained over 60 min. In contrast, currents in oocytes expressing the cloned ENaC were only slightly and transiently stimulated by extracellular acidification. The stimulation in A6 cells was reflected as a large increase of NT and a small compensatory decrease of iNa. These effects were dependent on [Ca2+]i, as pretreatment with BAPTA prevented the changes of INa, iNa, and NT.

Role of pHi. It is established in a variety of preparations that the native channel is inhibited by decreasing pHi (7-10, 16, 19, 20, 22). Moreover, recent evidence and our own unpublished observations indicate that the channel in cultured A6 epithelia is also inhibited by decreasing pHi (25). However, aside from a report in toad bladder (16), pHo is not thought to affect ENaC. Thus the present findings indicate that this phenomenon is not restricted to toad bladder and may be present in other Na+-absorbing epithelia. Our findings also provide the single channel basis for this increase along with potential mechanisms. The changes of pHo to 5.4 are well within the range of those encountered in the urinary bladder and in the distal nephron; thus, these findings represent an important physiological effect of external H+.

We cannot rule out with absolute certainty that the observed stimulation of No was due to small localized changes of pHi, despite the fact that we could not detect any changes of the BCECF fluorescence ratio and therefore bulk pHi. However, three additional lines of indirect evidence argue against this possibility. First, both pHo 6.4 and 5.4 were without effects on Po, which is shown to be rapidly inhibited by small intracellular acidification (4, 19, 20). Second, a decrease of pHi is expected to decrease No in A6 cells (25), which is opposite to our observed effects with decreasing pHo. Third, similar experiments in toad bladder found that a pHo down to 5.4 was also without detectable effects on pHi (16).

Effects in oocytes vs. A6 cells. It is well established that many of the ENaC properties found in native and cultured epithelia are well reproduced for the cloned channel expressed in Xenopus oocytes. However, overexpression of the three cloned ENaC subunits may result in the formation of a channel that reproduces the basic native Na+ channel properties but lacks the regulation conferred by association with other endogenous proteins. One such example was proposed by Awayda et al. (2) to explain the lack of regulation of the cloned ENaC by protein kinase A in Xenopus oocytes. It is possible that this may also be applicable to the observed differences in the response to pHo. However, at the present time, we cannot distinguish whether the variance in the response to pHo is due to differences between the native and cloned ENaC or oocytes and A6 cells. In either case, a better understanding of the origins of these differences will ultimately depend on elucidation of the mechanisms for sensing pHo and/or the role of Ca2+.

Effects on NT (role of Ca2+). The observed effects of pHo on NT were gradual, and appreciable changes were observed up to 30 min at pHo 5.4 and 40 min at pHo 6.4. This time course rules out an effect of external H+ on the channel, leading to direct activation of electrically silent but membrane-resident channels. However, it is possible that external H+ activates the channel via indirect effects on channel-related regulatory mechanisms. Decreasing pHi may protonate an accessory or a regulatory protein, e.g., a kinase or a phosphatase, that may be involved in channel trafficking. Alternatively, ENaC itself may be modified to alter its membrane residency to decrease its rates of endocytosis. At present, we are not able to select a likely mechanism among these; however, the finding that these changes were dependent on [Ca2+]i may indicate the potential involvement of a cell signaling cascade in the observed increase of NT.

Stimulation of Na+ transport in A6 epithelia by various mechanisms has been linked to Ca2+ mobilization. Hayslett and colleagues (11, 12) found that stimulation by adenosine and by vasopressin is linked to Ca2+ mobilization, since this effect could be blocked by BAPTA pretreatment. These authors measured the equivalent Isc; therefore, their methods did not allow for an assessment of the single channel properties and channel density. Nevertheless, our data are consistent with their observations and establish a role for channel trafficking or channel activation by [Ca2+]i. This may occur via a simple dependence of the cellular trafficking machinery on [Ca2+]i similar to that observed in many exocytic fusion events. Alternatively, it may involve a more complicated second messenger cascade. In any case, the potential roles of Ca2+, as delineated by buffering with BAPTA, should be distinguished from those observed with large and sometimes pharmacological increases of Ca2+ with ionophores that are known to inhibit ENaC.

Assuming that pHi is not altered, how do we envision an increase of external H+ concentration causing an increase of [Ca2+]i? There are no known H+-sensing proteins in A6 cells. However, these cells are thought to contain an external Ca2+ sensor (15). This sensor is mildly Ca2+ selective in that it can also respond to Mg2+ and other multivalent cations (1, 21). Moreover, is it also known that this sensor is coupled to intracellular Ca2+-signaling cascades and to G protein-coupled cascades (1). It is unclear if such a sensor is also affected by extracellular H+ concentration; however, such a process could account for the effects of pHo on NT and the involvement of [Ca2+]i.

A notable alternative hypothesis worth mentioning is that pHo may not alter [Ca2+]i but may affect the interaction of intracellular Ca2+ with ENaC. To our knowledge, such a mechanism has not been described previously. However, a relevant mechanism was described by Garty and colleagues (7). Using toad bladder vesicles, these investigators found that pHi affects the interaction of the Na+ channel with [Ca2+]i. In these experiments, the ability of Ca2+ to inhibit Na+ uptake was greatly reduced by decreasing pHi from 7.4 to 7.0. In this case, it would be expected that a decrease of pHi may relieve the inhibition of the channel by Ca2+ and cause its stimulation. It is unclear if a similar process occurs with changes of pHo, as this requires that protonation of an externally accessible site on the channel or associated protein leads to changes in the interaction with internal Ca2+. The above hypotheses await further experimental testing.

Potential physiological significance. We demonstrated that extracellular acidification caused a more than twofold increase of channel density. In a native Na+-absorbing epithelium, such as the cortical collecting duct, this could cause major changes in Na+ reabsorption. If the present findings can be extended to native epithelia, it is possible that this mechanism may prime the principal cells to increase their capacity for Na+ transport and allow for a better recovery after inhibition by a large acid load. A second hypothesis was proposed by Leaf et al. (16) who made the original observation of stimulation of Na+ transport by pHo in the toad bladder. They remarked that many clinically encountered conditions associated with excess plasma acidosis and increased urinary H+ secretion are also accompanied by the need for Na+ conservation. In this case, the stimulation of Na+ transport by luminal acidity would constitute an intrinsic mechanism that conserves Na+. This mechanism may also serve to prevent excess Na+ loss through Na+/H+ exchangers in the presence of increased luminal H+.

pHo was found to activate Na+ transport in A6 epithelia. This activation was primarily due to an increase of NT. The increase of NT was [Ca2+]i dependent and was prevented by buffering [Ca2+]i with BAPTA. This stimulation may represent an intrinsic mechanism of channel regulation leading to increased Na+ reabsorption. It is unclear if the cloned channel behaves in a similar manner, since the currents in oocytes expressing the cloned ENaC were only transiently stimulated by pHo.


    ACKNOWLEDGEMENTS

We thank Dr. Willy Van Driessche (K.U. Leuven) for helpful discussions regarding the pKa of CDPC.


    FOOTNOTES

This work was supported by a Grant-In-Aid from the Louisiana American Heart Association and by a Louisiana Education Quality Support Fund grant from the Louisiana Board of Regents to M. S. Awayda.

Address for reprint requests and other correspondence: M. S. Awayda, Dept. of Medicine, SL 35, Tulane Univ. School of Medicine, New Orleans, LA 70112 (E-mail: mawayda{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 20 January 2000; accepted in final form 6 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arthur, JM, Collinsworth GP, Gettys TW, Quarles LD, and Raymond JR. Specific coupling of a cation-sensing receptor to G protein alpha -subunits in MDCK cells. Am J Physiol Renal Physiol 273: F129-F135, 1997[Abstract/Free Full Text].

2.   Awayda, MS, Ismailov II, Berdiev BK, Fuller CM, and Benos DJ. Protein kinase regulation of a cloned epithelial Na+ channel. J Gen Physiol 108: 49-65, 1996[Abstract].

3.   Awayda, MS, and Subramanyam M. Regulation of the epithelial Na+ channel by membrane tension. J Gen Physiol 112: 97-111, 1998[Abstract/Free Full Text].

4.   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].

5.   Chase, HS, Jr, and Awqati Q. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. J Gen Physiol 77: 643-665, 1983.

6.   Fischbarg, J, and Whittembury G. The effect of external pH on osmotic permeability, ion and fluid transport across isolated frog skin. J Physiol (Lond) 275: 403-417, 1978[Abstract].

7.   Garty, H, Asher C, and Yeger O. Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions. J Membr Biol 95: 151-162, 1987[ISI][Medline].

8.   Garty, H, Civan ED, and Civan MM. Effects of internal and external pH on amiloride-blockable Na+ transport across toad urinary bladder vesicles. J Membr Biol 87: 67-75, 1985[ISI][Medline].

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

10.   Harvey, BJ, Thomas SR, and Ehrenfeld J. Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium. J Gen Physiol 92: 767-791, 1988[Abstract].

11.   Hayslett, JP, Macala LJ, Smallwood JI, Kalghatgi L, Gasalla-Herraiz J, and Isales C. Adenosine stimulation of Na+ transport is mediated by an A1 receptor and a [Ca2+]i-dependent mechanism. Kidney Int 47: 1576-1584, 1995[ISI][Medline].

12.   Hayslett, JP, Macala LJ, Smallwood JI, Kalghatgi L, Gassala-Herraiz J, and Isales C. Vasopressin-stimulated electrogenic sodium transport in A6 cells is linked to a Ca(2+)-mobilizing signal mechanism. J Biol Chem 270: 16082-16088, 1995[Abstract/Free Full Text].

13.   Helman, SI, Liu X, Baldwin K, Blazer-Yost BL, and Els W. Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia. Am J Physiol Cell Physiol 274: C947-C957, 1998[Abstract/Free Full Text].

14.   Hering-Smith, KS, Cragoe EJ, Jr, Weiner ID, and Hamm LL. Inner medullary collecting duct Na+-H+ exchanger. Am J Physiol Cell Physiol 260: C1300-C1307, 1991[Abstract/Free Full Text].

15.   Jans, D, Simaels J, Cucu D, Zeiske W, and Van Driessche W. Effects of extracellular Mg++ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions. Pflügers Arch 439: 504-512, 2000[ISI][Medline].

16.   Leaf, A, Keller A, and Dempsey EF. Stimulation of sodium transport in toad bladder by acidification of mucosal medium. Am J Physiol 207: 547-552, 1964[ISI].

17.   Montrose, MH, Friedrich T, and Murer H. Measurements of intracellular pH in single LLC-PK1 cells: recovery from an acid load via basolateral Na+/H+ exchange. J Membr Biol 97: 63-78, 1987[ISI][Medline].

18.   Palmer, LG. Voltage-dependent block by amiloride and other monovalent cations of apical Na channels in the toad urinary bladder. J Membr Biol 80: 153-165, 1984[ISI][Medline].

19.   Palmer, LG. Modulation of apical Na permeability of the toad urinary bladder by intracellular Na, Ca, and H. J Membr Biol 83: 57-69, 1985[ISI][Medline].

20.   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].

21.   Riccardi, D, Park J, W-S, Lee Gamba G, Brown EM, and Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92: 131-135, 1995[Abstract].

22.   Ussing, HH, and Zehran K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 23: 110-127, 1951[ISI].

23.   Van Driessche, W, De Smet P, and De Smedt H. Poorly selective cation channels in the apical membrane of A6 cells. Pfluegers Arch 426: 387-395, 1994[ISI][Medline].

24.   Warncke, J, and Lindemann B. Voltage dependence of Na channel blockage by amiloride: relaxation effects in admittance spectra. J Membr Biol 86: 255-265, 1985[ISI][Medline].

25.   Zeiske, W, Mets 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 Cell Physiol 279(6):C1896-C1905
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society