The effect of extracellular acidification was tested on the native epithelial Na+ channel (ENaC) in A6 epithelia and on the cloned ENaC expressed in Xenopusoocytes. 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 (I Na) by 68.4 ± 11% (n = 9) at 60 min. This increase of I Na was attributed to an increase of open channel and total channel (N T) 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 pHostimulate the native ENaC by increasing N T 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
- noise analysis
- channel density
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 (P o) is inhibited by pHi in excised membrane patches. Zeiske et al. (25) have also recently reported that open channel density (N o) 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 pHicause a rapid (within minutes) inhibition of ENaC expressed inXenopus 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 ofP o 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 (I sc) in the Xenopus kidney cell line A6. This stimulation was not a direct effect of the pH change in that the increase of I sc 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 I sc observed by small apical acidification in polarized A6 epithelia is due to increases of total channel density (N T). A small decrease of the single channel current (i Na) was also observed and is likely due to apical membrane depolarization. This decrease of i Na slightly underestimated the stimulation of the macroscopic I sc. The changes of N T and I sc 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 ofN T with an acidic luminal environment. We speculate that the increase of N T 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
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 (V oc) and appreciable transepithelial resistance (R T). With the use of the solutions described below, monolayers exhibited V oc in the range of −40 to −60 mV and R T in the range of 7–12 kΩ · 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 α-, β-, and γ-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 (g m) 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.
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 I scwas 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).
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 pHimeasurements 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'st-test where appropriate. Significance was determined at the 95% confidence level (P < 0.05).
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,I sc 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. 1along with a continuous recording of the I sc 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 I sc and presumably the apical Na+ channels.
The time course of the effects of decreasing pHo is summarized in Fig. 2. These data summarize the changes of the amiloride-sensitive currents (I Na). Extracellular acidification with HEPES-based buffer caused a gradual stimulation of theI Na 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.
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 g m. As evident from Fig. 3, the majority of the g m is amiloride sensitive and is attributed to ENaC.
The effects of a small decrease of pHo with HEPES-buffered solution on the g m are summarized in Fig.4. Within the resolution of the first measurement (30 s), pHo of 6.4 caused an increase ofg m. 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).
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. 5 A). 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. 5 B). The corner frequency of the Lorentzian function exhibits a linear relationship with CDPC concentration, as expected from a first-order reaction (Fig. 5 C). 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.
Figure 6 shows the effects of decreasing pHo on the blocker equilibrium constant (K B). This constant is calculated from the ratio of the blocker off and on rates and is therefore independent ofP o, 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 ofK B are not due to simple changes of the net charge on this blocker. As evident from Fig. 6,K B 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 K B 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.
To determine the mechanisms of stimulation of theI Na, 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 N o. These changes were accompanied by a compensatory decrease of i Na. These effects on i Na are likely due to a decrease of the electrochemical gradient across the channel rather than a change of the single channel conductance (seediscussion).
The mechanisms underlying the stimulation ofI Na 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 N o and a compensatory decrease ofi Na. Consistent with the slightly faster changes of I Na with pHo 5.4, these changes of N o 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.
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. Figure9 is a representative effect of BAPTA followed by pHo 6.4 on the I sc. 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 I sc with pHo 6.4.
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 I Na. This trend was not altered after treatment with pHo 6.4, andI Na showed no appreciable evidence of stimulation (Fig. 10 A). Similarly, no significant effects of pHo were observed on i Na,N o, and P o in BAPTA-pretreated cells within the first 30 min. A significant increase of P o 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 onN o appear to involve [Ca2+]i.
A major advantage of noise analysis is that this technique allows the calculation of single channel parameters and N T. This circumvents problems with variable channel gating, as observed with ENaC, and allows a more accurate estimate ofP o and N T. The effects onN T 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 ofN T via mechanisms that involve increases of [Ca2+]i. As observed from Figs. 1-10, the changes of N T with pHo 5.4 were more rapid than those observed with pHo 6.4.
Table 1 summarizes our findings with extracellular acidification. The only significant changes observed at both decreased pHo were those involvingi Na and N T. No significant changes were observed in the BAPTA-pretreated cells. In both cases, and in the absence of BAPTA, decreasing pHocaused an ∼60% increase of I Na mediated via an approximately twofold increase of N T and a small ∼20% decrease of i Na.
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.
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 N T and a small compensatory decrease of i Na. These effects were dependent on [Ca2+]i, as pretreatment with BAPTA prevented the changes of I Na, i Na, and N T.
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), pHois 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 N o 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 P o, which is shown to be rapidly inhibited by small intracellular acidification (4, 19,20). Second, a decrease of pHi is expected to decrease N o in A6 cells (25), which is opposite to our observed effects with decreasing pHo. Third, similar experiments in toad bladder found that a pHodown 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 N Twere 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 N T.
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 I sc; 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 N T 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 ofN T. The increase of N Twas [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.
We thank Dr. Willy Van Driessche (K.U. Leuven) for helpful discussions regarding the pKa of CDPC.
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:).
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.
- Copyright © 2000 the American Physiological Society