2 Laboratory of Physiology, We report, for the epithelial
Na+ channel (ENaC) in A6 cells,
the modulation by cell pH (pHc)
of the transepithelial Na+ current
(INa), the
current through the individual Na+
channel (i), the open
Na+ channel density
(No), and the
kinetic parameters of the relationship between
INa and the
apical Na+ concentration. The
i and
No were evaluated
from the Lorentzian INa noise induced
by the apical Na+ channel blocker
6-chloro-3,5-diaminopyrazine-2-carboxamide.
pHc shifts were induced, under
strict and volume-controlled experimental conditions, by
apical/basolateral NH4Cl pulses or
basolateral arrest of the
Na+/H+
exchanger (Na+ removal; block by
ethylisopropylamiloride) and were measured with the pH-sensitive probe
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The
changes in pHc were positively
correlated to changes in
INa and the
apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying
INa was
No. Only the
saturation value of the
INa kinetics was
subject to changes in pHc.
pHc-dependent changes in
No may be caused
by influencing
Po, the ENaC open
probability, or/and the total channel number,
NT = No/Po.
noise analysis; single-channel current; epithelial sodium channel; ammonium; cell volume
CELL PH (pHc) is
under strict control (8, 15). Unexpectedly, cytosolic acidification,
resulting from a lack of oxygen, appeared, at least at short term,
beneficial against major cell damage from ischemia (39). Prior
treatment of several cell types including kidney cells with salines of
pH < 7 reduced or prevented cell damage, such as leak of enzymes or
complete lysis during anoxia. Interestingly, analogous cytoprotective
effects were obtained by treatment with glycine and alanine during
ischemia. So far, no mechanisms of action underlying either
type of cytoprotection could be settled (32, 39).
Recently, pHc has been discussed
to represent another cytosolic second messenger, together with
Ca2+, cAMP, ATP, and other
signaling molecules (18, 19). For instance, in tight epithelia such as
frog skin or the cultured distal kidney cell line A6 from the clawed
toad, Xenopus laevis,
pHc was found to influence apical
and basolateral cation permeabilities such that a concerted up- and
downregulation of apical Na+
(PNa) and
basolateral K+
(PK)
permeabilities (so-called "cross talk") occurred. Within a
comparably narrow range of pHc
(7.4 to 7.0), PNa
as well as PK
became negligible upon cell acidification (18, 19). This might shed
some light on the mechanism of the protective effect of protons. A
closure of epithelial cation channels by cellular acidification could
prevent, after ATP depletion in an anoxic state, the accumulation of
cell Na+, the parallel loss of
cell K+, and a gain in cell NaCl
and, consequently, of water followed by cell disruption.
To study the dependence of plasma membrane ion permeability on
pHc, the so-called
"NH+4 pulse" method has become a
popular means to alter pHc.
Usually, when more than millimolar concentrations of
NH+4 salts are added to the extracellular
saline, an alkalinization of the cytosol due to hydrolysis of the
easily permeant NH3 has been
observed (9, 10, 21). Extracellular NH+4
removal would in turn acidify the cell
["NH+4 prepulse" method (6)]. The rate of the subsequent realkalinization will reflect the activities of pHc-regulating
transporters, such as the
Na+/H+
exchanger or primary active H+
pumps. If otherwise K+-permeable
entrance pathways for NH+4 (10, 23) are in
parallel to the lipid or, as discussed (34), a possible aquaporin
permeation route of NH3, cytosolic
pH drops may also be caused by intracellular
NH+4 hydrolysis, which counteracts the
alkalinization from NH3. When
tissue pH changes are evoked by simple addition (6, 10, 21, 31) of
10-30 mM NH+4 salts to saline, without
being balanced osmotically, cell volume changes may as well influence
ion channel permeabilities (11, 40).
In the context of using A6 cells as model epithelium for the study of
the consequences of anoxia and the protective effects of protons, we
set out to investigate NH4Cl
addition to, as well as its removal from, NaCl-Ringer solutions without
or with osmotic control. For each method, eventual
alterations in cell volume were recorded.
pHc was monitored using a
membrane-permeant derivative of the pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF;
see Refs. 6 and 9). To inspect transepithelial
Na+ uptake, we monitored
transepithelial conductance
(Gt) and the short-circuit current
(Isc) carried
by Na+. To establish whether
pHc-dependent changes in apical
PNa are related
to changes in single-channel current
(i) or channel density, we conducted
fluctuation analysis with the Na+
channel blocker 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC; see
Ref. 35). Finally, the influence of
pHc changes on
Na+ current
(INa) kinetics
was evaluated and compared with the results obtained from noise analysis.
Depending on the side of application, and the tonicity of the
NH4Cl-containing saline,
pHc and
INa changed in a
complex manner. Under strict conditions, however, changes in
pHc affected only No, the number of
open apical Na+ channels, whereas
the i and also the blocker kinetics
appeared invariant.
Cell Culture
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Cell Volume Measurements
This method was previously described in detail (38). Briefly, cell thickness (Tc) was used as an index for cell volume of confluent monolayers. The apical (upper) side of the monolayer was labeled with fluorescent biotin-coated microbeads. Focussing of the microbeads was automatically performed with a piezoelectric focussing device (PIFOC; Physik Instrumente, Waldbronn, Germany). Tc is defined as the vertical distance between the basolateral and apical beads. Measured Tc values were corrected for the diameter of the fluorescent microbeads by subtracting 1 µm.Electrical Measurements
Transepithelial direct current measurements. Epithelial monolayers were mounted in an Ussing-type chamber (chamber opening 0.7 cm2) designed to eliminate edge damage and were continuously superfused (3-5 ml/min) on both sides with Ringer solutions. The A6 tissues were short-circuited using Ag-AgCl voltage and current electrodes that were connected to the bath solutions with agar bridges containing 3% agar in 1 M KCl medium. We recorded the Isc, as well as the Gt, which corresponds to the Isc deflection induced by a brief voltage deplacement. INa is defined as Isc minus Isc in the presence of 0.1 mM apical amiloride.Noise analysis. Noise analysis methods have been previously described in detail (35). Briefly, Lorentzian noise was induced with the uncharged amiloride analog CDPC (50 µM). Fourier analysis of the fluctuation in current results in power density spectra (PDS; cf. Fig. 6A) normalized to the membrane area. The analysis of the PDS yields the Lorentzian parameters So (plateau) and fc (corner frequency). Assuming that Na+ channel blockage by CDPC fulfills pseudo-first-order kinetics (35) it follows
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Fluorometric Measurement of pHc
Confluent A6 tissues were mounted in an Ussing-type chamber (see Transepithelial direct current measurements) and were loaded from the apical side with BCECF (Molecular Probes) by exposure to a final concentration of 10 µM of the AM form of the dye (stock solution 5 mM in DMSO). Loading was performed for 60 min, at room temperature, in control NaCl Ringer with continuous perfusion at the basolateral side. After loading, excessive dye in the apical solution was washed out for at least 20 min.The fluorescence measurements were performed with an own-built microfluorometer under computer control. We used an inverted microscope (TMD 35; Nikon, Tokyo, Japan) in epifluorescence mode, equipped with a ×32/0.40 objective (Leitz, Wetzlar, Germany). Excitation light of a 100-W Xe lamp (Nikon) was filtered at 440 and 490 nm (440DF20 and 490DF20, Omega Optical). Switching of the interference filters was done with a computer-controlled filter wheel (Lambda-10; Sutter Instrument, Novato, CA). The intensity of the source was reduced by neutral density filters inserted between the microscope and the filter wheel. The fluorescence emission was monitored at 535 nm (interference filter 535DF25; Omega Optical). The fluorescence was detected with a photomultiplier tube (9124A; Thorn-EMI, Middlesex, England) operating in photon counting mode. The pulses were transferred to the computer through a counter/timer board C 660 (Thorn-EMI). The data were collected with a dwell time of 1 s at each wavelength and corrected for the dead time of the counting system. The background due to scattering and autofluorescence was subtracted subsequently from each of the signals.
At the end of each experiment, calibration of the BCECF fluorescence
(F) ratio R = F490
nm/F440 nm versus
a given pHc was performed, using
the nigericin-high K+ technique
(36). Cells were clamped at three different pH values (6.6, 7.3, and
8.0) using calibration solutions containing 13 µM nigericin and 137 mM K+. This
K+ concentration closely
approximates the reported cytosolic
K+ concentration in A6 cells (27).
Figure 1 shows the resulting linear fit
through data of 16 in vivo calibrations on different A6 monolayers.
When individual tissue calibration at the end of the experiment failed,
we used the pooled calibration curve {pH = [R + 19.0(±0.6)]/3.45(±0.08)} to
evaluate the experimental data. Figure 1 shows that in A6 cells the
calibration procedure was quite reproducible.
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Statistics
Mean values from N experiments (different monolayers) are given ± SE.Solutions and Chemicals
NaCl Ringer solution had the composition (in mM) 70 NaCl, 3 KCl, 1 CaCl2, 40 sucrose, 5 glucose, and 10 HEPES and was buffered with Tris to a final pH of 7.4 (osmolality 197 mosmol/kgH2O). Under these conditions, the average pHc was 7.34 ± 0.06 (N = 11), which is comparable with the pHc of 7.30 ± 0.02 reported earlier for A6 cells (6). In some cases of a low basal transport rate, 10 mM theophylline was added to the saline. In noise analysis experiments, theophylline was omitted from the solution because it induces additional ClTo investigate pHc effects on INa kinetics from dose-response experiments, the apical Na+ concentration ([Na+]ap) was gradually reduced by substituting Tris+ for Na+. Amiloride, 5-nitro-2-[(3-phenylpropyl)amino]-benzoic acid (NPPB), and ethylisopropylamiloride (EIPA) were purchased from Sigma (St. Louis, MO), and CDPC was obtained from Aldrich (Milwaukee, WI). Final concentrations in the solutions were obtained by adding these blockers from stock in H2O (amiloride) or in DMSO (NPPB, EIPA, and CDPC). Experiments were carried out at room temperature.
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RESULTS |
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Influence of Hyper- and Isosmotic NH+4-Containing Salines on Cell Volume
In many reports in which the influence of NH+4 on cytosolic pH was investigated, high concentrations of the NH+4 salt were added to the bath, giving rise to a noticeable increase in osmolality. In a number of tight epithelia, hyperosmotic cell shrinking strikingly reduces transepithelial Na+ transport as for instance in A6 cells where hypertonic NaCl Ringer abolishes Na+ absorption (40). On the other hand, due to the permeability of the cell membrane for NH3, or NH+4 with ClFigure 2 demonstrates experiments in which
20 mM NH4Cl was isotonically
applied by replacing 40 mM sucrose with 20 mM
NH4Cl (top
trace) or hyperosmotically by simply adding
NH4Cl to the NaCl Ringer solution
(bottom trace). Disturbance of the
cytosolic osmotic condition was assessed by recording the cell height
(Tc) that
reflects cell volume changes as described previously (11). Apical
application of NH4Cl, independent
of the solution osmolality, did not at all influence
Tc. When
NH4Cl was applied basolaterally, Tc remained
unchanged after isosmotic sucrose replacement by
NH4Cl, whereas a remarkable but
reversible decrease in
Tc was observed upon simple hyperosmotic NH4Cl
addition.
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This result confirms previous observations (11) that cell volume does not respond to apical anisotonicity as the apical membrane of A6 cells is known to be quite impermeable to water. We also see that the expected isosmotic volume increase does not occur. This may indicate that there is no massive influx of solute/water or that the volume regulation occurs as fast as the osmotic disturbance (37). Regarding the high osmotic sensitivity of the basolateral side, putatively pHc-related transport changes that result from NH+4 exposure must therefore be studied in the absence of any osmotic imbalance. Moreover, the sidedness of the application of a pHc-shifting agent must be under strict control. Only isosmotic experiments are reported below. Also, a strictly unilateral treatment with pHc-influencing substances was employed.
pHc and INa Changes During Apical or Basolateral Isovolumetric NH4Cl Exposure
Figure 3, A and B, depicts recordings (from two different epithelia) of pHc and Isc, respectively, when tissues were isosmotically exposed to apical NH4Cl-containing NaCl saline. Typically, a very similar time course in the change of both parameters is observed; apical NH+4, as predicted, alkalinizes the cells, which raises INa. In the presence of apical amiloride, Isc remains close to zero despite the change in pHc (not shown). Figure 3D shows, for eight tissues, the rise in INa induced by apical NH+4, whereas Fig. 3C illustrates (N = 5) the underlying shifts in pHc, demonstrating that an increase in pHc correlates with an increase in INa. With basolateral isosmotic NH4Cl treatment (Fig. 4, A-D), the situation is considerably more complex. Figure 4, A and B, shows simultaneously recorded traces of pHc and Isc, respectively, from the same tissue. In Fig. 4A, during the first phase after NH+4 addition, pHc rises similarly as with apical NH+4. However, this pHc rise is quickly reversed into a marked drop. In addition, another typical feature is observed in the time course of Isc only; right after introduction of basolateral NH+4, before pHc moves, a sharp and immediate current drop occurs followed by a gradual increase that occurs synchronously with pHc, first increasing and then dropping below the control value. Hence, the initial fall in Isc cannot be related to pHc changes, whereas the subsequent changes in INa and pHc are quite similar. Figure 4, C and D, summarizes the late phase drop in pHc (N = 5) and INa (N = 10). At this point, we may state that a probably causal relationship exists between pHc and the magnitude of INa, disregarding for a moment the initial "blip" event in Isc obtained with basolateral NH+4. Cell alkalinization and current rise occur simultaneously with NH+4 on either side (basolaterally only in the beginning). Cell acidification takes place in the late phase with basolateral NH+4. Below we investigate, by means of noise analysis, which apical factor(s) determines the parameters of Na+ transport that are influenced by pHc.
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Evaluation of Na+ Channel Blocker Noise: Influence of pHc on i and No
NH+4
pulses.
To generate a Na+ channel blocker
noise in Isc, we
employed CDPC, a noncharged amiloride analog, rather than amiloride
itself (35). Because simultaneous measurements of
pHc and noise analysis could not
be done, we assume, for the experiment depicted in Fig. 5 and the following ones, that the
respective alterations of pHc due
to isosmotic NH4Cl exposure are
analogous to those reported (e.g., Fig. 3) in the absence of an
inhibitor. A typical protocol for noise analysis is shown in Fig.
5A. In the presence of 50 µM CDPC,
apical sucrose replacement by
NH4Cl led to the already known
rise in INa. A
similar behavior of
Gt, mainly
determined by the apical membrane resistance, is under control of
pHc. Interesting here, but
occurring only in a minority of cases, is the observation that the
alkalinization (causing the
INa rise) seems
subsequently to be counteracted even in the presence of
NH+4, probably by a regulatory mechanism such
as
Cl/HCO
3
exchange (9). This would tend to acidify the cells, just like the
basolateral action of NH+4. After elimination
of apical NH4Cl, we see a slight
undershoot of the parameters before return to control values; this may
be related to an additional and well-known (6, 21) cell acidification after NH+4 removal (see also Fig.
5B). When the agent is applied
basolaterally, we see again the same features for
INa as shown in
Fig. 4B for
Isc, accompanied
by an almost completely proportional behavior of
Gt with the
notable exception of the negative initial blip characteristic for
Isc. After
removal of basolateral NH4Cl, both
Gt and
INa show a marked
negative overshoot. The protocol (Fig.
5A) ends with exposure to apical
amiloride to determine the
Na+-specific part in
Isc (35). The
reason for the current undershoot after basolateral
NH+4 removal becomes clear from Fig.
5B; NH+4
removal leads to a considerable further
pHc drop and
INa decrease
(Fig. 5C), which are followed, like
here when serosal Na+ is present
(Fig. 5, B and
C), by their slow recovery. When
this protocol is repeated, however, in the absence of basolateral
Na+ at the end of the experiment
(Fig. 5C), this
Isc recovery does not occur, which strongly suggests the involvement of a serosal Na+/H+
antiport (6) in the backregulation of
pHc and, consequently, of
INa and
Gt.
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Arrest of the basolateral
Na+/H+
exchanger.
basolateral na+ omission.
Casavola et al. (6) previously reported for A6 cells the existence of
an
Na+/H+
antiport exclusively in the basolateral membrane. Presumably, removal
of Na+ from the basolateral saline
could acidify the cytosol. We tested this by measuring
pHc when serosal
Na+ was replaced by Tris or
choline. In Fig. 7 we show an experiment with Tris (also representative for choline), and it can be seen (A) that, as expected,
pHc drops after
Na+ removal. At the same time, and
tested here (Fig. 7B) with another epithelium, the
INa drops
eventually and thus yields a picture similar to that seen with
basolateral NH+4-induced acidification of the
cytosol. One more salient and, for this sort of maneuver, typical
feature can be discovered in Fig. 7B;
Na+ removal causes a remarkable
initial current overshoot. This might reflect the
PNa of the tight
junctions and a paracellular Na+
flux along the just-established apical to basolateral
Na+ concentration
([Na+]) gradient,
since, in experiments in which the
Na+/proton exchanger was, in the
presence of basolateral Na+,
stopped with EIPA (see below), this phenomenon could not be seen. On
the other hand, control experiments (not shown) in which basolateral
Na+ was omitted in the absence of
apical Na+ still exhibited such an
overshoot that excludes Na+ as
origin. Furthermore, because the apical
Cl channel blocker (29)
NPPB also had no influence on this (not shown), a contribution from
Cl
secretion (12, 29) is
unlikely, and the origin of this phenomenon remains obscure.
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Cytosolic pH and Macroscopic INa Kinetics
Most tight epithelia display a saturating dependence of Na+ uptake on [Na+]ap. This is also the case with A6 cells, and we could recently (35) elucidate that two phenomena are responsible for the saturation of the macroscopic INa; with rising [Na+]ap, the i increases and saturates with an apparent Km of 17 mM. In addition, a saturation-like decrease in No with even faster kinetics was found when raising [Na+]ap. The combined result is the fairly Michaelis-Menten-like saturation of INa with, however, an apparent "macroscopic" Km of ~5 mM. Our present findings from noise analysis suggest No to play the decisive role in determining pHc-regulated INa. In the DISCUSSION, we assume that this conclusion may be extended to conditions in which no channel blocker is present.Because both the pHc-independent
i and the
pHc-dependent
No are a function
(both in hyperbolic but opposite ways) of
[Na+]ap,
the question arises about the manner in which
pHc influences No, i.e., by
changing the channel density or rather the
Km of its
[Na+]ap
dependence (or both). For instance, for a rise in
pHc, an increase of
No should become
visible as an increase in maximal INa when plotting
current-saturationkinetics (Fig. 9). An
alternative would be a shift of the
Km
(KNa), the
half-maximal [Na+].
For many experiments in which pHc
was increased by apical NH+4 or decreased by
basolateral NH+4 or
Na+ withdrawal, we obtained
exactly the same result that is exemplified for the case of 20 mM
basolateral NH+4 in Fig. 9 in which the
INa saturation
function, obtained with apical
Na+-Tris mixtures, is displayed.
As shown in Fig. 9, we could fit a hyperbola (solid lines) to the data
using a Hill coefficient of one, and we did not observe any change of
the apparent macroscopic Km. The cell
acidification after basolateral NH+4 had only
one effect, namely, shifting the maximal current level downward.
Therefore, we do not deal with a
Na+-competitive but rather with an
allosteric block of
INa by cytosolic H+.
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DISCUSSION |
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A6 cells possess the mechanisms for active and cAMP- and
Ca2+-controlled
Na+ reabsorption (36) and also
Cl secretion (12, 29). The
virtual absence of apical water channels (11) renders the cells
sensitive to basolateral osmotic disturbances only. There is much
evidence (18) that second messengers not only operate on the apical,
rate-limiting barrier for ion transport processes but also are able to
couple properties of apical and basolateral membranes in concert for
optimal transcellular ion movements. A novel coupling agent may be
pHc; in A6 and other tight
epithelia (18, 19, 21), pHc
regulates apical
PNa and basolateral PK in
much the same manner and within a narrow
pHc range, with maximal
permeabilities at pH > 7.5 but vanishing permeabilities at pH < 7.1. In the present paper, we explored which apical parameters are
responsible for the pHc-dependent
modulation of apical Na+ channels.
So far, we may conclude that, whatever means are used to modify pHc, 1) blocker-induced channel fluctuations appear to be unaffected by pHc; 2) with an invariant i, pHc-invariant single-channel conductance and electrochemical apical driving force seem reasonable assumptions; and 3) No available for interaction with apical CDPC is the only parameter that plays a role in the pHc-regulated Na+ transport in the A6 cell line.
Methodical Aspects
The electrical techniques used here, including the simultaneous recording of Tc, have been described and discussed at length previously (11, 35, 38). Especially with respect to the Na+ kinetics, as well as the CDPC noise analysis in A6 cells, using a two-state model, we refer the reader to Smets et al. (35).To evaluate pHc-dependent parameter shifts when using either hyper- or isosmotic NH4Cl-containing solutions, we used Ringer (70 mM NaCl) in which NH4Cl had been added (hyperosmotic) or replaced isosmotic 40 mM sucrose. Anisotonic addition of NH4Cl is commonly used (6, 10, 21) to evoke pHc changes. The inherent dangers are clear, and earlier data show that hypertonic salines as such do already decrease cell volume and No (40). This prompted us to establish conditions of zero volume changes for the use of NH4Cl (Fig. 2).
For unknown reasons, successful cell loading with BCECF-AM could not be achieved sufficiently often; quick washout from the cells after fast dye entrance also occurred frequently. Thus, in many experiments in which individual pHc calibration at the end of an experiment with a given tissue was impossible, calibration had to be performed, as done by others (6, 9), using data from pooled experiments designed for the construction of a calibration curve only (Fig. 1).
Complexities Arising From Attempts to Shift pHc with NH+4 Pulses
With extracellular media containing NH+4, cell alkalinization is a direct consequence of ammonia entry; also, acidification of the cytosol follows withdrawal of external NH+4. It was mostly ignored and only rarely (10, 21) appreciated that NH+4, entering cells in a nonnegligible quantity through normally K+-permeable pathways (24, 50), may lead to direct cell acidification due to intracellular hydrolysis of NH+4. Moreover, because NH+4 will then compete with K+, it may contribute to otherwise typically K+-dependent phenomena (cation transporter fluxes, membrane polarization, or channel currents), or else, impede K+ transporters (42). Therefore, effects additional to those from pHc shifts may be expected, e.g., changes in basolateral membrane K+ channel resistance and ensuing hyper- or depolarization of a normally K+-dependent membrane potential difference. In A6 cells at short circuit, a change so achieved in the negative intracellular electrical potential Vsc (see Eq. 4) would immediately affect the net apical driving force for Na+ entry. If, as in other tissues (10, 21), basolateral permeability of K+ channels for NH+4 is finite, the addition of K+-mimicking NH+4 to the basolateral side could cause a Vsc depolarization that would impede Na+ influx (Eq. 4). Such a mechanism could explain the transient initial bliplike current reduction as seen in Fig. 4B or Fig. 5A, although i, and thus Vsc as part of the driving force, seem unchanged at steady-state conditions (Table 1).There are also hints for cellular pH backregulation after an externally
provoked pHc shift. As can be
seen, e.g., in Fig. 5A, a more
transient
pHc/INa
rise during apical NH+4 may be the
consequence of the activation of the basolateral
Cl/HCO
3
exchanger that has been established in A6 cells (9). On the other hand,
the ubiquitous
Na+/H+
antiport that exists basolaterally in A6 cells (6, 9) must mediate
realkalinization (Fig. 5, B and
C) after
NH+4 removal-induced acidification, an effect
only observed in the presence of basolateral
Na+ and in the absence of EIPA. A
block of the
Na+/K+
antiporter with EIPA excludes that a putative stop of the basolateral Na+/Ca2+
antiport (2) with subsequent rise in cell
Ca2+, which has been discussed to
inhibit PNa (1,
16, 30), is responsible for the
INa drop after
Na+ omission. In contrast,
augmented cell calcium, e.g., after hormones that enhance
Na+ transport such as vasopressin,
has recently been shown to have the dominant function in the
stimulatory hormone action on
INa in A6 cells
(22). In fact, Lyall et al. (25) suspected that a number of
Na+ uptake-activating hormones
exert their effects via cell alkalinization.
As it is generally assumed (6) that the Na+/H+ antiport starts to work only after a certain degree of cell acidification, the question of why our methods that putatively stop the exchanger cause an immediate fall in pHc and INa arises. One reason could be that metabolism produces enough protons, and another reason could be that ion channels allow a constant "leak" of protons into the cells (24), so that the exchanger is permanently active. This is the case, e.g., in frog skin (20).
In some reports on A6 cells, the above discussed points and problems arising from the choice of ill-defined experimental conditions [e.g., simple bilateral NH4Cl exposure, including bilateral isosmotic NaCl replacement by NH4Cl to study the Na+/H+ exchanger (6) or INa kinetics (9)] have been ignored. Such studies of pHc-related transport activities are then suited to yield erroneous interpretations, such as claiming a "mixed competition" (6) of intracellular protons with extracellular Na+.
Parameters of Apical Na+ Transfer
Our data provide strong evidence that the kinetic parameters, i.e., KNa with respect to apical Na+ (see Fig. 9) and fc with respect to CDPC block (Figs. 5 and 7), are unaffected by maneuvers that change pHc. With a pHc-independent i, both single-channel conductance and the net apical driving force (see Eq. 4) are virtually pHc independent: 1) at Ringer-[Na+]ap, the apical Nernst potential for Na+ is stable as the rate of the basolateral Na+-K+-ATPase is not a function of pHc, at least not above pH 6.9 (13); and 2) the practically indistinguishable pHc-titration curves of apical PNa and basolateral PK (18, 19) ensure that pHc changes both permeabilities always by the same factor, therefore yielding constant fractional membrane resistances and constant Vsc.With respect to "spontaneous" open-closed conformational changes
and our inference that
No is
pHc dependent whether CDPC is present or not, the "inherent" (when blocker is absent) ENaC open probability
(Po) could be
subject to pHc, which would result
in a change in
No, being the
product of Po and
the total number
(NT) of
Na+ channels (open plus closed).
Indeed, a most recent report (7) demonstrated for the -subunit of
ENaC, expressed in Xenopus oocytes or
reconstituted in planar lipid bilayers, that cytosolic-side acidification reduced
Po (approaching 0 at pHc < 7) and mean open time
while increasing the mean closed time, with unaffected single-channel conductance. In addition,
NT could vary if
a fraction of channels, as a consequence of acidification, disappeared,
either by becoming permanently closed or by endocytotic removal.
Indeed, a drop in pHc
is known to result in apical exocytosis of
H+ pumps in some tight epithelia
(4, 17). Moreover, influences of
pHc on vesicle traffic have been
described (14), and the role of exocytotic events underlying the
stimulatory action of several hormones on
Na+ transport in tight epithelia
is heavily discussed (16) as is the
INa stimulation
by cell volume increase, which can be prevented by interaction with
cytoskeleton-directed drugs (28). It has been reported that
cytoskeletal elements, such as small actin filaments, induce
Na+ channel activity in A6 cells
(5). We are presently unable to decide whether
pHc affects only
Po or also
NT.
Modeling of the pHc Dependence of PNa
Figure 9 suggests that we deal with an allosteric site where the interaction of internal protons should be noncompetitive with external Na+. This may also explain, even when pHc changes, the constancy of the parameter i (and thus single-channel conductance; see Eq. 4), which is under dominant influence of external Na+. With respect to the findings on the pHc sensitivity of patch-clamped ENaC-type epithelial Na+ channels (18, 19), reversible vesicle fusion may seem less likely than reversible allosteric opening-closing [by (de)protonation] of permanently resident apical Na+ channels, e.g., by affecting Po. At present, most recent publications about presumable structures of the ENaC (26, 33) do not yet provide conclusive hints for a tentative identification of the titrated allosteric intracellular site(s). However, according to the published pHc-titration curve of the A6 cells (18, 19) as well as for the cloned (7) Na+ channel, the apparent pKa range (7.2-7.5) may point to a histidine as titrated group. For instance, His-94 in theSummary and Perspectives
With respect to our data, we arrive at the following conclusions. 1) The apical A6 cell membrane permits little, if any, apical entry of NH+4 but rather NH3, which causes a cytosolic alkalinization (sometimes followed by pHc "backregulation"). The concomitant rise in Gt and INa is exclusively due to a rise of No, probably caused by allosteric opening of apical Na+ channels. All other parameters of apical Na+ transfer remain unaffected. 2) Basolateral NH+4 first increases pHc (and therefore Gt and INa) due to effects identical to those discussed for apical NH+4 exposure. Secondarily, however, NH+4, as pointed out also by other groups for other tissues (10, 21), enters the cells. This occurs probably (21, 23, 41) via otherwise K+-permeable conductive pathways, since we see an immediate rise in Gt in parallel to a drop in INa. This can easily be understood if, initially, NH+4, as imitator of K+, depolarizes the cell negative short-circuit potential that reduces the net driving force for apical Na+ entry. Subsequent H+ release from entered NH+4 ions would decrease apical PNa, which is possibly due to a shortened open time and a prolonged closure [Chalfant et al. (7)], thus leading to a decrease in time-averaged No. Subsequent NH+4 withdrawal will tend to further acidify the cell, and, depending on the activity of basolateral Na+/H+ exchange, pHc will recover.A variety of messenger roles for intracellular protons have been proposed to date, including the concerted cross-talk modulation of apical Na+ and basolateral K+ permeabilities in tight, Na+-transporting epithelia. Another possible consequence of a rise in cell H+ concentration could be a liberation of cell Ca2+ from storage proteins or vesicles, so that Ca2+ would be the final messenger (30), although cell Ca2+ has recently been shown to be stimulatory rather than inhibitory (20).
Alkali ion channels deliver protons to the cytosol (25) that will pile
up in the cell when energy supply is short as in the condition of
ischemia or anoxia, and glycolysis and ATP splitting without
regeneration will add even more to cell acidification which, in the
end, would tend to deregulate cell life. This adverse, positive-feedback reaction chain may, however, be brought to a halt if
the influx pathway for protons, the alkali ion channels, is closed down
in a negative-feedback loop by cytosolic protons so that the cells
would neither lose K+
basolaterally nor gain Na+
apically and, consequently,
Cl and water. That may
prevent cell volume increase and rupture. Such mechanisms may, at least
in part, account for the observed protective effects of internal
H+ when they are derived from the
"therapeutic" acidification of the extracellular bath. It is most
interesting that EIPA and similar drugs that stop the
Na+/H+
exchanger have been shown to be protective in conditions of cardiac ischemia (3, 34).
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ACKNOWLEDGEMENTS |
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We thank E. Larivière, J. Simaels, R. Van Werde, M. Ieven, G. Raskin, J. Vanderhallen, W. Leyssens, and P. Pirotte for technical assistance.
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FOOTNOTES |
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W. Zeiske and I. Smets contributed equally to the present report.
This project was supported by research grants from the "Fonds voor wetenschappelijk onderzoek" (G.0179.99) and the Interuniversity Poles of Attraction Programme (IUAP, P4/23), Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs.
Present address of W. Zeiske: Section of Animal Physiology, Dept. of Biology, Univ. of Osnabrück, D-49069 Osnabrück, Germany.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. Van Driessche, Laboratory of Physiology, K. U. Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium (E-mail: Willy.VanDriessche{at}med.KULeuven.ac.be).
Received 9 December 1998; accepted in final form 27 May 1999.
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