Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

Wolfgang Zeiske1, Ilse Smets2, Marcel Ameloot2, Paul Steels2, and Willy van Driessche1

2 Laboratory of Physiology, Limburgs Universitair Centrum, B-3590 Diepenbeek; and 1 Laboratory of Physiology, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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Cell Culture

A6 cells (passages 110-113) obtained from Dr. J. P. Johnson (University of Pittsburgh, PA) were cultured as described earlier (11). Polarized monolayers obtained after 15-30 days of culture on permeable tissue culture inserts (25 mm diameter; Nunc Anopore) were used to perform experiments. For the volume measurements, before seeding cells, the Anopore membranes were coated with fluorescent microspheres of 1 µm diameter (L5081; Molecular Probes, Eugene, OR) embedded in a thin gelatin layer.

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
2&pgr;<IT>f</IT><SUB>c</SUB> = <IT>k</IT><SUB>on</SUB>[B] + <IT>k</IT><SUB>off</SUB> (1)
with kon and koff being blocking and unblocking rate constants, respectively, and [B] the blocker concentration. These parameters, together with INa, the amiloride (0.1 mM)-blockable Isc, served to calculate No and the i using a two-state model for the apical Na+ channel block by apically applied CDPC (35) according to
<IT>i</IT> = <IT>S</IT><SUB>o</SUB>&pgr;<SUP>2</SUP><IT>f</IT> <SUP>2</SUP><SUB>c</SUB>/(<IT>I</IT><SUB>Na</SUB><IT>k</IT><SUB>on</SUB>[B]) (2)
<IT>N</IT><SUB>o</SUB> = <IT>I</IT><SUB>Na</SUB>/<IT>i</IT> (3)
i obeys Ohm's law
<IT>i</IT> = &ggr;(<IT>V</IT><SUB>sc</SUB> − <IT>E</IT><SUB>Na</SUB>) (4)
where gamma  is the single-channel conductance, Vsc is the cellular potential in short-circuit conditions, and ENa is the apical Nernst potential for Na+.

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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Fig. 1.   Calibration curve for cell pH (pHc) in A6 cells. Sixteen in vivo calibrations were done, according to Thomas et al. (36). The ratio, R, of the emitted fluorescent light intensities (F) at 490 and 440 nm (R = F490/F440) is a linear function of pHc. This can be seen for the 2 arbitrarily selected individual tissues ( and triangle ) as well as for the averaged data, with pH = [R + 19(±0.6)]/3.45(±0.08). The other 14 tissues are, for clarity, represented only as dots; the fitted linear relationship for all pooled data is shown as a bold line.

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 Cl--dependent Lorentzian noise (12) that interferes with the CDPC-induced Na+-current noise.

To 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cl-, cell shrinkage will be counteracted by solute influx followed by an obligatory water flow. Even when NaCl or sucrose in the saline is isosmotically replaced with NH4Cl, so-called "isosmotic swelling" due, e.g., to net NH4Cl entrance into the cells, could occur. Such a phenomenon has already been described for A6 cells when basolateral NaCl was replaced by KCl or glycerol (37). Finally, the side of application of the pHc-shifting agent may be of prime importance. Indeed, because NH+4 can enter cells via K+-selective pathways and thus give rise to cell acidification, its basolateral application will have a more distinct effect on pHc decrease than a treatment of the apical barrier that has a negligible PK.

Figure 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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Fig. 2.   Measurements of cell thickness (Tc) during exposure to NH+4-containing NaCl saline. Top trace represents the averaged (N = 5 tissues; total number of beads: 34 ± SE indicated by dots) time course of Tc when 20 mM NH4Cl is exchanged for 40 mM sucrose at constant osmolality. Tc (scale on left) has been normalized to the value recorded just before apical NH+4 addition. Average control Tc was 7.7 ± 0.3 µm for all beads. Bottom trace shows the average percent change in Tc (scale on right) when NH4Cl did not replace sucrose but was added to yield hypertonic solutions. We used 2 tissues with 24 beads. NH4Cl was first introduced apically and, after removal, subsequently in the basolateral Ringer solution. CTR, control.

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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Fig. 3.   Effect of NH+4 on pHc and short-circuit current (Isc). A: typical time course of changes in pHc when 20 mM NH4Cl is added isosmotically (replacement of 40 mM sucrose) to the apical (ap) NaCl saline and after subsequent withdrawal. B: typical time course of changes in (mainly Na+-carried) Isc for another tissue treated as in A. C: comparison (5 tissues) of steady-state pHc before (control) isosmotic apical NH4Cl addition with pHc at maximal point of cell alkalinization. D: comparison of Na+ current (INa) before and at maximal response to apical isotonic treatment with NH4Cl. Shown are the data for 8 tissues.



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Fig. 4.   A: typical time course of changes in pHc when 40 mM basolateral (bl) sucrose is replaced by 20 mM NH4Cl. B: changes in Na+-carried Isc for the tissue shown, with respect to pHc, in Fig. 3A. C: comparison (5 tissues) of steady-state pHc before (control) isosmotic basolateral NH4Cl addition with pHc at its minimum. D: comparison of INa before and at maximal response to basolateral isotonic treatment with NH4Cl. Shown are the data for 10 tissues.

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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Fig. 5.   A: time courses of INa and transepithelial conductance (Gt) during apical or basolateral isotonic treatment (for order of application, see horizontal bar) with 20 mM NH4Cl for a selected tissue. Before NH+4, 50 µM 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) was added to the apical NaCl saline. At the positions indicated by numbers, CDPC-induced INa noise was recorded; the respective values of the Lorentzian plateau (So), the corner frequency ( fc), and, after further evaluation, single-channel current (i) and open-channel density (No) are listed in Table 1. Amiloride (0.1 mM) was added apically for the determination of zero INa. B: time course of pHc during isotonic basolateral NH4Cl addition and subsequent removal in the presence of basolateral NaCl. C: Isc time course during isosmotic basolateral NH4Cl addition and removal, in the presence (left) and in the absence of basolateral NaCl (Tris replacement simultaneous with NH4Cl withdrawal; right).

We used noise analysis of the CDPC-induced fluctuation in Isc at various points in time in which stable current values had been attained. The period of the initial putative pHc rise after serosal NH+4 was too short to perform data recording for noise analysis. The results from this experiment are given in Table 1. pHc had no influence on the fc of the CDPC-induced Lorentzian noise (Fig. 6A). However, the So magnitude was altered dramatically and reflected the direction of changes in Gt and INa (cf. Fig. 5). Analysis on the basis of the two-state channel model for interaction with the blocker clearly revealed (Table 1) that i remained unaffected by changes in pHc. Therefore, pHc exerts a direct control on Na+ channel activity as expressed by No. A rise in pHc augments Gt by means of Na+ channel opening, and vice versa (internal channel pHc "titration"). Figure 6, B and C, summarizes such results for eight tissues. To compare different epithelia with sometimes much different transport capacities, we plotted, in Fig. 6B, the relative magnitude of the So (ratio of So in the presence of NH+4, over So in the absence of NH+4) and the function of the respective relative currents. These parameters turned out to be strictly proportional for a number of different conditions (see legend for Fig. 6). In the framework of the equations used for noise analysis (see MATERIALS AND METHODS), we thus conclude that, as already suggested by the typical experiment depicted in Table 1 and Fig. 5A, only No is responsible for the observed pHc-dependent alterations in INa. Consistently, the corresponding relationship between INa and No is roughly linear (Fig. 6C) for the cumulated data, which impressively underlines that, for all the investigated tissues, the individual i (solid line in Fig. 6C equals averaged i values; see Eq. 3) are of comparable magnitude and remain unchanged during shifts of pHc. Thus, in this sort of experiment, the apical PNa reflects the No, and no attention must be given to i or blocker kinetics as revealed in fc.

                              
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Table 1.   Lorentzian parameters, i, and No from the CDPC Isc noise at the time periods indicated by numbers for the experiment shown in Fig. 4A




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Fig. 6.   A: power density spectrum of the Isc noise in the presence of 50 µM apical CDPC. The control spectrum (black-diamond ) is calculated for position 1, and the lower spectrum (bl + NH4Cl; ) is calculated for position 6 as indicated in Fig. 5A. Fitted and calculated parameters are listed in Table 1. B: ratio (normalized to control) of the CDPC-Isc noise Lorentzian So at 50 µM CDPC plotted vs. the analogous relative INa for different tissues and various treatments. According to theoretical considerations, this function must intercept both abscissa and ordinate at 0; the finding of a linear relationship So/SCTRo = (i/iCTR) · (INa/ICTRNa) suggests that the relative i (i/iCTR) remains fairly constant over all tissues and pHc conditions. ×, Control; , NaCl in the presence of apical NH+4 (cell alkaline); , after removal of apical NH+4; , in the presence of basolateral NH+4 (cell acidic); open circle , after removal of basolateral NH+4; triangle , removal of basolateral NH+4 combined with basolateral Na+ replacement by Tris+. C: INa plotted as a function of No for the same tissues and for the same conditions (cf. symbols) depicted in B. According to theory (cf. MATERIALS AND METHODS), the slope of the resulting linear relationship for the pooled data (solid line) equals the average i (mean i = 0.196 ± 0.005 pA).

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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Fig. 7.   A: effect of basolateral replacement of Na+ by Tris+ on pHc. B: same experiment as in A for another tissue; shown, however, is the time course of INa. C: time course of INa and Gt when 50 µM CDPC was first added apically followed by basolateral Na+ replacement by Tris+ (CDPC present), plus 0.1 mM apical amiloride, and basolateral cation reversal (presence of amiloride). Parameters from CDPC noise at times indicated: arrow 1: fc = 93.4 Hz, So = 4.23 × 10-20 A2 · s/cm2, i = 0.17 pA, No = 38.17 × 106 channels/cm2; arrow 2: fc = 97.9 Hz, So = 0.91 × 10-20 A2 · s/cm2, i = 0.16 pA, No = 10.42 × 106 channels/cm2.

Noise analysis (a typical experiment is shown in Fig. 7C, see legend for details) led for the case of serosal Na+ substitution by Tris to exactly the same finding as for the cell acidification during the late phase of basolateral NH+4 treatment (Fig. 5A); only channel density was reduced by Na+ removal, whereas i and CDPC kinetics remained unaffected. Figure 8 summarizes for eight tissues the outcome of this type of experiment. Again, as outlined in Fig. 6 for the effects of apical or basolateral NH+4, a linear relationship was found not only between the relative So and the relative INa (Fig. 8A) but also for the dependence of INa on No (Fig. 8B).


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Fig. 8.   Relationship between the relative So magnitudes vs. the relative INa (A) and linear function INa vs. No (B) from experiments in which basolateral Na+ (CTR, ×) was replaced by Tris+ (). For further details, see Fig. 6, B and C. From the slope in B we obtain a mean pHc-independent i = 0.178 ± 0.003 pA.

NA+/H+ EXCHANGE BLOCKING BY EIPA. To check our above hypothesis that omission of basolateral Na+ brings the serosal Na+/H+ exchanger to a halt, we tried to inhibit it directly (6), in the presence of Na+, with 50 µM EIPA in the serosal bath. In Table 2, we show results from noise analysis of three tissues that were studied under these conditions, before and after at least 15 min treatment with basolateral EIPA. Again, the results fully mirror those obtained above with Na+ removal in that No was the sole parameter affected by the EIPA-dependent arrest of the exchanger; these results point to an EIPA-induced drop in pHc.

                              
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Table 2.   Effect of 50 µM EIPA in the basolateral NaCl-saline on INa, i, and No for 3 tissues

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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Fig. 9.   Typical example of apical INa kinetics vs. apical Na+ concentration ([Na+]ap) in control conditions (black-diamond ) or when basolateral NH4Cl (20 mM) was introduced isosmotically (). [Na+]ap was varied by substitution for Tris+. Solid lines represent hyperbolic fits to the data. The so-obtained maximal INa is 10.1 µA/cm2 for control and 4.3 µA/cm2 after the pHc drop induced by basolateral NH+4. The corresponding apparent Km for Na+ are 5.7 (black-diamond ) and 5.0 () mM, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 the alpha -ENaC has been discussed by Chalfant et al. (7) to be a proton target during intracellular titration.

Summary 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).


    ACKNOWLEDGEMENTS

We thank E. Larivière, J. Simaels, R. Van Werde, M. Ieven, G. Raskin, J. Vanderhallen, W. Leyssens, and P. Pirotte for technical assistance.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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