Coupling between the intracellular pH and the active transport of sodium in an epithelial cell line from Xenopus laevis

Camp. Biochem. Physiol.Vol. IOZA,No. 1, pp. 7-14, 1992

0300-9629/92 $5.00 + 0.00

0 1992Pergamon Press plc

Printed in Great Britain

COUPLING BETWEEN THE INTRACELLULAR pH AND THE ACTIVE TRANSPORT OF SODIUM IN AN EPITHELIAL CELL LINE FROM XENOPUS LAEKS FRANCOIS CHUARD and JACQUESDURAND* Institute of Physiology, University of Fribourg, Rue du Mu& 5, CH-1700 Fribourg, Switzerland. Telephone: 37-826330; Fax: 37-826519 (Received 11 September 1991)

Abstract-l. The relationship between the rate of active Na + transport and intracellular pH (pH,, measured as the BCECF fluorescence) was studied in A6 monolayers in the presence of CO?. The total buffering power (B,,,,,) and its components (pi and /3c02)were assessed at various pHi. 2. The activitv of Na + IH + and Cl - IHCO, - exchangers were expressed in A6 cells; both antiports were found to particjpate in PH, homeostasis unber stand&d conditions. 3. Alterations in the rate of Na+ transport induced variations in pH,. 4. Na + transport rate was a hyperbolic function of external Na + concentration. The curve was shifted by changing pHi: a mixed inhibition of Na+ transport by pHi was found. 5. The pHi appears as a possible mediator coupling the rate of Na + transport across the apical and the basolateral membrane in tight epithelia.

ithelial transport of Na+, as discussed by Dawson and Richards (1990). The present study was focused on the possible role of H +i as an immediate signal of the regulation. To this end, the investigation was performed under acid-base conditions that enable unaltered pHi regulation, to be sure that the measured ApH, would also occur in vivo. Therefore, A6 cells (a cell line from the distal nephron of Xenopus luevis) were studied in the presence of CO*, an unusual experimental condition for amphibian epithelia. Another rationale for the use of CO, is that the set of conditions chosen in the present study are close to those generally applied to mammalian epithelia, i.e. high Pc,, and HC03concentration and standard pH, of 7.4, thus allowing for a valid generalization of the findings. The buffering power of A6 monolayers was assessed and pH, regulatory processes were verified to be active under conditions suitable for Na+ transport. The observations showed a reciprocal relationship between pHi and the rate of Na + transport, which suggests a role for H + i in mediating the concerted adjustment of the transport processes located at the apical and at the basolateral membrane. A preliminary report of this work has been published in abstract form (Chuard and Durand, 1991).

INTRODUCTION The epithelial cell has the double task of actively transporting salt across the epithelium and at the same time maintaining cellular homeostasis, in other words, the cell must be able to achieve various steady-state levels of transepithelial electrolyte transport, with only minor perturbations in cell volume or composition. Several lines of evidence gathered in the past years in amphibian as well as in mammalian epithelia have led to the hypothesis of the intracellular dialogue between the apical and the basolateral membrane of the epithelial cell (Schultz, 1984; Harvey and Ehrenfeld, 1988). According to this hypothesis, the activity of the transport mechanisms resident in each membrane is coupled via some putative intracellular signal. It is known that hormonal stimulation is achieved via an increase in both the activity of the Na+ pump in the basolateral membrane and the Na+ permeability (PNa) of the apical membrane (Nagel and CrabbC, 1980; SaribanSohraby et al., 1984; Palmer and Speez, 1986). However, the mechanism of spontaneous variations in Na+ transport which are known to occur across tight epithelia as a result of a fluctuating Na+ delivery at the apical membrane, has not been elucidated. Circumstantial evidence suggests that changes in ma+], are linked to regulatory adjustments of P,, and of the Nat pump rate (Schultz, 1984; Harvey et al., 1988; Rick et al., 1988). However, it is not clear whether or not Na + i is the immediate signal. Indeed, several ion species can cross membranes coupled to Na + so that any A[Na ‘Ii would be reflected in variations of other electrolyte concentrations, among which Ca2 + and H + are likely candidates for signaling a fine tuning of membrane processes involved in the active transep*To whom all correspondence

MATERIALSAND METHODS A6 cells were grown in amphibian culture medium at 28°C in a water vapor-saturated atmosphere containing 4% CO, and the medium was renewed three times a week. The medium was enriched with 10% fetal calf serum (Flow, Allschwil, Switzerland), penicillin (60 mg/l, Flow) and streptomycin (130 mg/l, Flow). Cells were seeded at a density of 0.5-l .O x lo6 cells/cm2 on polycarbonate transparent filters (pore diameter 1.2pm) or on cellulose nitrate filters (pore diameter 0.45 pm; Millipore, Volketswil, Switzerland). The filters were glued on polycarbonate rings thus forming small

should be addressed. 7

8

FRANCOISCHUARDand JACQUESDIJRAND

cups designed to fit in the experimental chambers. Before use, the filters were coated with rat tail collagen then dried in the presence of NH, vapors, The filter-bottomed cups allowed for diffusion of solutes present in the culture medium so that the cells were bathed in the medium both at the apical and at the basolateral aspects (Sariban-Sohraby er al., 1983). Cells reached confluency in 68 days. However, experiments were performed after at least two weeks; a period required to observe conspicuous electrical values across the monolayers. The pH, was determined by the 2’,7’-biscarboxyethyl5(6’)-carboxyfluoresceine (BCECF, Calbiochem, Luzern, Switzerland) fluorescence technique as described by Aalkjaer and Cragoe (1988). Monolayers were loaded with 2.6 PM BCECF-acetoxymethylester (BCECF-AM, from a stock solution 1 mM in dimethyl sulfoxide) for 1 hr in culture medium. Then, the monolayers were washed three times with a standard physiological saline solution and the filter-bottomed cups were inserted vertically in a cylindrical chamber enabling individual perfusion on the basolateral and on the apical aspects of the cells. This chamber permits the measurement of pH, in a population of polarized cells by means of a routine spectrofluorophotometer, and to examine separately the mechanisms resident in the apical and in the basolateral membrane. A device based on the same principle has recently been described by KrayerPawlowska et al. (1991). The chamber was located vertically inside a spectrofluorimeter (Shimadzu RF WOO).The excitation and the emission beams passed through quartz windows sealed on the chamber. The angle between the monolayer and the incident beam could be varied in order to find out the highest signal to noise ratio. In preliminary tests it was observed that the filter material generated an artefact, probably due to some light dispersion. During the experiments, the data were corrected for this artefact by measuring the ratio I,, 500 nm, J., 525 nm/l,, 450 nm, I,, 475 nm. The ratio eliminated the leakage effect and other possible sources of instability of the fluorescence signal. The monolayer inside the chamber was bathed on both sides with a saline solution that was continuously renewed by means of a peristaltic pump. Experiments for the determination of the buffering power of the intracellular fluid were carried out with isolated cells. For that purpose, cells grown on collagen-coated filters were dissociated by a mild treatment with trypsin, washed, then the viability was assessed by observing the exclusion of the trypan blue. Intact cells represented routinely more than 95% of the isolated cell population. Cells in suspension were then transferred to a quartz vial equipped with a cap and pieces of tubing allowing for fluid circulation. The fluorescence ratio i.,, 500 nm, i.,, 525 nm/l,, 440 nm, A,, 525 nm was recorded. In order to calibrate the fluorescence signal, the pH, was equilibrated with the pH in the incubation solution (pH,) either by the nigericin (20pM)-KC1 technique or by using digitonin (50 PM), then the pH was stepwise decreased by adding successive aliquots of HCl and a straight line of fluorescence ratio vs pH was obtained for each experiment (see Fig. 1A and B). The pH, was continuously measured with a Knick 764 pH meter that corrected the pH values for the temperature automatically. All experiments were carried out at 22°C. The standard incubation solution had the following composition (mM): NaCl 89, NaHCO, 28, KC1 3, CaCl, 2. The solution was equilibrated with a gas mixture containing 6% CO, and 30% C$ in N,. The pH of this standard solution was 7.4. The calibration was carried out at the end of each experiment in a solution containing 20 mM HEPES instead of HCO, - and CO,. Ion substitution was achieved in several experimental protocols; choline chloride (89 mM), or choline sulfate (59 mM), or sodium isethionate (89mM) was substituted for NaCl, K,S0,(2 mM) for KCl, Ca(NO,), (2 mM) for CaCl,. In some experiments, NH&l (2.5 mM) or (NH&SO, (2 mM) was added. The osmolarity of all solutions was close to

250 mOsm/l. The HCO; -containing solutions were equihbrated with gas mixtures containing 1.5,6 or 20% CO, and 30% 0, in N,. The intracellular buffering power (8) was determined according to Roos and Boron (1981) in isolated cells in a solution buffered with CO,/HCO;, to which 2.5 mM NH&l were added; this concentration was sufficient to yield an intracellular alkalinization (see Fig. lC), with no appreciable change in the osmolarity of the bathing fluid. B is defined as A[NH,+],/A pH, where A[NH,+], = IOrHe*rH’~H,‘1.. In the presence of CO,, /l total = Bco, + pi, where /?co, = 2.3 [HCO;],. The intracellular bicarbonate concentration was calculated from the HendersonHasselbalch equation, using temperature-corrected values for pK (in a saline solution) and for a (the solubility coefficient of CO,). 8, is the intrinsic buffering power, namely that part of the buffering power due to proteins, phosphates and cellular organelles. Thus, jI,,,,i was measured and /?co, and 1, were calculated. The electrical properties were measured across monolayers grown and treated in a similar fashion to those used for the determination of pHi. To this end, the filter-bottomed cups were inserted into an Ussing chamber; the transepithehal potential difference, AY, and the short-circuit current, I,, were measured by means of thin electrodes made of Ag-AgC1 wires inside pieces of tubing filled with a 1M KC1 solution saturated with AgCl, separated from the physiological incubation solution by porous caps. The electrical resistance of the monolayers, R, was calculated from A

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Fig. I. (AC) Calibration of the fluorescence signal and effect of 2.5 mM NH&l on pH,, (A) Titration of BCECF in cells permeabilized with digitonin. (B) Calibration curve with the data in (A), regression line r = 0.997. C: The horizontal hatched bar shows the period of NH&l application. Note the biphasic alkalinization: initial peak, then slow decline of the fluorescence ratio. The suppression of such a low NH&I concentration induced a mild acidification compared to the initial control period.

pH, and Na+ active transport the change in current corresponding to a voltage deflection of 10 mV using Ohm’s law. The electrodes were connected to an automatic voltage clamp device. The incubation solutions were equilibrated with the gas mixtures described above and continuously renewed by means of a peristaltic pump. The pH, was continuously recorded with a Radiometer pH meter. In both the fluorimeter chamber and the Ussing chamber, the inlets and outlets for the fluid circulation were located in such a way as to enable a rapid and efficient renewal of the incubation solution that would also avoid large differences or jumps in hydrostatic pressure. Results are expressed as the mean + SEM of the number of observations indicated. Statistical analysis was performed, using Student’s f-test for paired or unpaired values, when appropriate. A value of P < 0.05 was considered to indicate statistical significance. Amiloride was given by Merk Sharp and Dohme (Rahway, U.S.A.).

RESULTS Intracellular buffering power The intracellular buffering power measured in a solution equilibrated The experiments were performed at concentrations, chosen between 10

of A6 cells was with 6% CO,. various HCO; and 28 mM, in

order to set different pH, and thus to study /l over a range of pH,. Initial values of pHi between 6.63 and 7.17 were observed. Figure 1C shows that the addition to the incubation medium of a NH&l concentration as low as 2.5 mM caused a fast peak of alkalinization, followed by a slow recovery phase in the continuous presence of NH&l. Removing NH&l induced a mild acidification compared to the initial control value. The mean value of the alkalinization peak was: ApHi = 0.08 + 0.01, N = 14. Such a small perturbation ensured an accurate determination of /Jrotal.In six experiments in which NaCl was replaced by choline-SO, in order to abolish the activity of both the Na+/H+ and the Cl-/HCO; exchangers, /Itotal was not significantly different from the values observed in the presence of the standard concentration of NaCl, indicating that the evaluation of /3,,,, was not affected by the activity of these exchangers. Therefore, the two series of data obtained in the presence of NaCl and in its absence were pooled.

9

100

60

6.6

7.0

6.8

Fig. 2. Buffering power of A6 cells vs pHi, in the presence of CO, (F,,, = 6%). Circles: /ItoLI,;squares: p,; triangles: /Jcol. Different pH, were obtained with various HCO,concentrations. p,,,,, was determined by adding 2.5 mM NH&l to the incubation solution. /Ice, was calculated by the Henderson-Hasselbalch equation. /Ii = /?,,,., - /?co,. Curves were fitted to the data of /I,,,,,, and & by polynomial regression.

Figure 2 showsLLl, ho, and 8, as a function of pHi. In the range of pHi lying between 6.6 and 7.2, the variation of /I,,, was limited between 65 and 85 slykes. However, there were opposed large changes in and /I,: at a more acid pH,, /Ii was the major P czponent to fitotal,whereas at a more alkaline pHi, Pco2 contributed mainly to &,,,. Regulatory mechanisms of pH,

Experiments were performed to verify the existence in A6 cells of the ubiquitous membrane processes involved in pH, regulation, namely the Na+ /H + and the Cl-/HCO,exchangers. For that purpose, monolayers were first incubated in the absence of the Na+ and the effect of Na+ readmission on pH, was recorded. Amiloride was applied as a tool to discriminate between the pH, regulation and an effect of active Na + transport, in order to find out the role of the Na+/H + exchanger solely. Indeed, at a lower concentration (2 p M), amiloride is known to block specifically the Na+ channels (Garty and Benos, 6

A

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Na + Amilo

Na

oNa

Na + Amilo

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Fig. 3. Effect of Na + and of amiloride on pH,. (A) Following a period of incubation in a Na + -free solution (points at left), Na +, 89 mM, and amiloride, 1 mM, were administered concomitantly (intermediate points: amiloride to the apical side solely). A ApH, was observed only when amiloride was withdrawn (points at right). The points corresponding to the data of each monolayer were joined by a line. (B) Similar experiments with 2 PM amiloride. Note that such a low concentration did not prevent the effect of Na+ on pH,.

FRANCOIS CHUARD and JACQIJESDURAND

10 Table I. pH, at an

extracellular Na+

6.98 k 0.03 7.45 * 0.03 8.00 + 0.04

concentration of 0 or 89 mM

pH, (+Na+)

PH, (-Na+)

PH,

6.98 f 0.07 7.14 f 0.06 7.46 f 0.09

7.10 + 0.09 7.22 + 0.08 7.56 + 0.08

N

P

10 8 5


1988) but a higher concentration (1 mM) would also inhibit the Na+ /H + exchanger (Kleyman and Cragoe, 1988). The results are shown in Fig. 3A and B. In the presence of 1 mM amiloride on the apical surface of the monolayers, the readmission of Na+ had no effect on pH, (Fig. 3A); pH, rose to a new plateau value following the removal of amiloride; however, Na + induced an intracellular alkalinization when the amiloride concentration was 2 pM and removing amiloride had no further effect on pHi (Fig. 3B). These results suggest that the cellular alkalinization resulted from an influx of Na + coupled to an efflux of H + via the Na + /H + exchanger. The effect of Na+ was examined at various pHi, the latter being altered by choosing the Fco, and the HCO; concentration so that pH, was 0.5 units lower or higher than that of the standard solution. Table 1 shows the effect of Nat on pH, for three pH,. In the series of experiments under standard conditions (pH, = 7.45, [Nat] = 89 mM), pHi in A6 cells was 7.22 f 0.08 (N = 8; Table 1). In each case the pH, in the presence of Nat was approximately 0.1 units larger than the value observed in the absence of Na + . The effect of Na + was reversible, the removal of that cation following a period of Nat readmission reacidified the cells to the pH, value that prevailed during the first period without Nat. A linear relationship was observed between pH, and pH,, as depicted in Fig. 4 for the two conditions, i.e. absence and presence of Na + . The plain line indicates that an extracellular alkalinization from pH, 7.0 to 8.0 resulted in an intracellular alkalinization of 0.5 units, from pH, 7.0 to 7.5. Because pH, was affected by the presence of Na+ (Table l), the straight line in Fig. 4 was shifted downward in the absence of Na+ relative to control conditions; however, the slope was unaltered. In an attempt to enhance the effect of Na+ on pH, following a period of Na + deprivation, the Clions were also substituted in some experiments with A

.

-

7.5

pHi

I

I,.

I.

7.0

.,

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.

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.

.

.

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Fig. 4. pH, vs pH, in the presence (circles and solid line, r = 0.82) or in the absence of Nat (squares and dashed line, r = 0.79). Each monolayer was incubated in a solution containing 0, then 89, later again 0 mM Na +

1.05 1 0

IO

2;

t [min]

Fig. 5. Effect of 2 PM amiloride on pHi. The vertical arrow indicates the time of addition of amiloride on the apical side. The drug caused a cellular acidification manifested by a decrease of the fluorescence ratio.

the idea to inhibit the Cl-/HCO; exchanger which could have diminished the observed changes in pH,. Indeed, in a medium containing no Cl -, pHi was 7.14 f 0.09 in the absence of Nat, and 7.28 f 0.09 (N = 8) following Na+ readmission; thus, the ApHi was 0.14 units, a figure significantly larger (P < 0.001) than those measured in the presence of Cl -. This observation suggested that the ApHi due to Na+ has been reduced effectively by the activity of the Cl-/HCO; exchanger. A further indication for the existence of a Cll/HCO; exchanger was also provided by examining the effect on pH, of Cl - removal from the incubation solution. Monolayers were first incubated under standard conditions, then isethionate or SO:were substituted for Cl-; a significant elevation of pHi from 7.14 + 0.18 to 7.20 f 0.17 (P c 0.05, N = 4) was observed. These results suggest that the established processes of pH, regulation were expressed in the A6 cell line; they were operative under the standard conditions used here, and contributed to the resting pHi value notwithstanding the large buffering power, especially /Ice,. Active Na+ transport and pHi

The electrical properties of A6 monolayers measured in an Ussing chamber were: AY 38 + 3 mV, I, 16 + 1 PA/cm’ and R 2529 + 142 Rcm2 (N = 48). Preliminary tests have shown that a pretreatment with 0.1 PM dexamethasone for 2 days exalted the transepithelial Na+ transport, resulting in values of AY, I, and R higher by W-100% than untreated monolayers. Therefore, the glucocorticoid was added routinely to the culture medium for 2 days before the experiments. In an attempt to disclose an effect of Nat transport on the pHi, 2 PM amiloride was applied to the apical border of monolayers incubated in a solution containing the standard Na + concentration, equilibrated with various Fc,, the HCOJ- concentration being kept at 28 mM. It should be recalled that 2 PM amiloride blocked the Nat channels resident in the apical membrane, without influencing the Na+/H+ exchanger (Kleyman and Cragoe, 1988). One series of experiments was performed at a constant F, of 6%; in two other series, FCo2 was either lowered to 1.5%

pHi and Na+ active transport

or raised to 20% prior to the addition of amiloride. Figure 5 illustrates a typical experiment performed at a FCO, of 1.5%; the addition of amiloride induced a cellular acidification. It was verified that amiloride per se did not influence the fluorescence signal. Table 2 shows that, under each FCo2, amiloride caused a significant acidification, indicating that alteration of Na+ transport modified pHi. For the 26 monolayers used in these experiments, the pH, under standard conditions was 7.18 f 0.04 (pH, = 7.38 _+ 0.01). In another series of experiments in which the I, was measured in an Ussing chamber, the pHi was modified by altering the Fco, and the effect of ApH, on the I,, was studied. In addition, the Na+ concentration in the bathing fluid was changed, so that the relationship between I, and [Na ‘1, was determined at three F,, (1.5, 6 and 20%). The I, declined when the Na + concentration was stepwise diminished, as illustrated in Fig. 6A, B and C: lowering the FCo2 enhanced the I, (Fig. 6B), whereas an increase in FCo2 had the opposing effect (Fig. 6C). The modulation of I, by changing the Fco, was observed for all Na+ concentrations, in other words for all levels of I,. Additional experiments were carried out with the purpose of verifying that pHi, and not pH, or CO, per se could have produced the alterations in I,,. At constant Fcol, a change of the HCO; concentration only on the basolateral side, induced variations in both pHi and I,. Indeed, an acidification (ApHi 0.05) diminished the I, by 56 + 3% (N = 4) and inversely, an alkalinization (ApHi 0.18) yielded a 72 f 17% increase in I,, (N = 4). In contrast with the effect of changing the HCO; concentration on the basolateral side, the same manoeuvre applied to the apical border of monolayers failed to induce significant changes in either pHi or I,. Thus, it can be concluded that the effects of CO2 on the I,, were mediated by changes in pHi. For each Fco2, the normalized values of I, vs [Na’], closely fitted to a hyperbolic curve, as shown in Fig. 7A. A cellular alkalinization produced by lowering the FCOl (cf. Table 2) lifted the curve upward, above the control values, and inversely, a cellular atiidification shifted the curve downward. Figure 7B shows a plot of these data according to Lineweaver-Burk. A mixed inhibition of I, by H+ ions was observed; the concentrations of Na+ yielding a half-maximal saturation of I, were 2.5, 8.5 and 12.5mM at a FCo2 of 1.5, 6 and 20%, respectively. The maximal normalized I, was 120% at an alkaline pH, and 60% at an acid pH, with respect to the standard conditions (100% at 6% CO*).

DISCUSSION

The A6 cell line is generally exploited as a valuable model for a Na + -absorbing tight epithelium (Perkins Table 2. Effect of 2 uM amiloride Fco, (Oh) 1.5 6 20

pH, (-Amiloride) 7.39 * 0.04 7.31 f 0.06 6.79 + 0.07

pH, (+Amiloride) 7.30 f 0.03 7.26 _+0.06 6.74 f 0.08

on DH. N

P

8 9 9


I1

A

6%

FC02

6 In

: t

I

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5

t [min]

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7.01 -

2o t LlO

I 0

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We

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Fig. 6. (A-C) Effect of varying the [Na+] and the Fco2 on I,. The horizontal bars (hatched segments) show the periods during which F,,, and [Na+] (mM) were changed for the values indicated; blank segments indicate control periods. A: Only [Na+] was altered; B: effect of alkalinization at a reduced [Na ‘1; C: effect of acidification at a reduced [Na ‘1. The thick line shows the I, and the thin line depicts the pH,. A, B and C are records from three monolayers.

and Handler, 1981; Sariban-Sohraby et al., 1983; Handler et al., 1984; Asher et al., 1988; Wills and Millinoff, 1990), like amphibian epithelia (Leaf, 1982) or the rabbit colon (Schultz, 1984). However, the acid-base properties of this cell line have not been characterized so far. In the present study the buffering power was measured in the presence of CO,; this condition was chosen because it is closer to the physiological situation than the use of non-volatile buffers (Thomas, 1989) and also in order to mimic the conditions generally used for mammalian epithelia. The standard conditions used in the present study deviate from the in uivo situation that prevails in amphibia, which live with a lower P,,, and a higher pH in the extracellular fluid than mammals; they should allow a generalization with respect to the model of a Na+-absorbing epithelium as, for example, the distal tubule and the collecting duct of the mammalian kidney.

12

FRANCOISCHUARLIand JACQUESDURAND

Or 0

I

I

I

20

I

I

I 60

40

I

I 80

I

I 100

[Na] mM

1/normalized

I

Fig. 7. (A) Normalized I, as a function of [Na+]; (B) Lineweaver-Burk plot of the same data. The three sets of data corresponded to FCo2 1.5% (triangles), 6% (circles) and 20% (squares). In A, the lines

represent hyperbolic curves. Each point is the mean f SEM of four to eight monolayers.

The importance of studying the pHi in the presence of CO, can be readily appreciated from Fig. 2, which shows the total buffering power and its components at varying pHi. Indeed, plotalis relatively stable in a broad range of pHi due to a balance between jIi and Bco*; without CO,, /I would have been at acid values twice as large as at alkaline values. Thus, the experimental perturbations of pHi would have been buffered to a different extent at both extremities of the pH, range explored. In our experimental conditions, there are indications suggesting the activity of Na+ /H + and Cl-/HCO; exchangers in A6 cells. The effect of Na + readmission on pH, observed in the presence as well as in the absence of Cl-, and the effect of a high dose of amiloride, are evidence in support of the existence of a Na + /H + exchange. It should be noted, however, that other types of acid-base transport coupled to Na + , such as electrogenie or electroneutral Na + /HCO; symports, if present, could have participated to the measured ApH, (Frelin et al., 1988; Madshus, 1988). This study was not focused on the latter systems; if they did occur, they certainly contributed little to the pHi homeostasis. The presence of a Cl - /HCO< exchanger is suggested by (1) an increase in pHi (approximately 0.05 units) observed when Cl- in the incubation solution was withdrawn and (2) the enhanced effect of Na+ on pHi in a Cl--free solution compared to the response observed with the standard Clconcentration. The aim of this work was to study the possible reciprocal relationship between pHi and the transep-

ithelial active transport of Na+. The A6 cell line seemed to be a suitable preparation for this purpose, because the cells build tight monolayers, which exhibit an active Na+ absorption that can be measured by the short-circuit technique (Sariban-Sohraby and Benos, 1986). It should be noted that the I, found in the present study (16 pA/cm*) is of similar magnitude to the value of 23 PA/cm* reported by Wills and Millinoff (1990), in contrast to the relatively low I, (less than 7 pA/cm2) observed generally (Handler et al., 1981, 1984; Yanase and Handler, 1986; Granitzer et al., 1991). A conspicuous active flux of Nat was obviously a major requirement for the present purpose. In addition, the transcellular Na+ pathway conforms to the model of Koefoed-Johnsen and Ussing (1958). In this model, Na + enters the cell across the apical membrane, then it is actively extruded across the basolateral membrane by the Nat pump. The apical barrier is thought of as the rate limiting step to the transepithelial Na+ transport. This model, originally put forward to describe the frog skin epithelium, applies also to the rabbit colon and to the mammalian distal nephron. However, the idea that the apical membrane stands as the rate limiting step has been revisited and several lines of evidence have led to the concept of an intracellular dialogue (Schultz, 1984). According to this hypothesis, the intracellular compartment plays a main role in adjusting the properties in both the apical and the basolateral membrane in a concerted manner, in such a way as to maintain cellular homeostasis in the face of large variations of transcellular Nat transport.

pHi and Na+

Several pieces of evidence support the proposal that Na + itself might operate the concerted adjustment in both membranes. Firstly, an increase in [Na+]i was found to induce a decrease in PNa of the apical membrane (Lewis et al., 1976; Turnheim et al., 1978; Schultz, 1984; Sariban-Sohraby and Benos, 1986). Secondly, the activity of the Na+ pump resident in the basolateral membrane is a function of [Na+li; in epithelia, the I,, measured at various levels of [Na+li is an indirect index of this relationship. The latter was established by Rick et al. (1988) for the frog skin; Schultz (1984) reported a sigmoidal function in the rabbit colon. A sigmoidal curve between [Na+]; and the activity of the Na + /K + -ATPase was also demonstrated in cardiac Purkinje fibers (Sejersted et al., 1988). The activity of the Na+ pump is influenced by Na+, as one of its substrates; however, it is far less clear how the Na + channels can be regulated by Na +, itself. Furthermore, the PK in the basolateral membrane is also influenced in a concerted manner, in order to ensure the current flow and the necessary K+ recirculation across that membrane; the putative effect of [Na+li on this process is not understood. Therefore, the concerted adjustments in both membranes required by the “cross-talk” hypothesis cannot easily be explained, based on the idea that Na+, is the immediate vector of information. Several observations have led to the idea that variations of [Na+], may be reflected in changes of [Ca2+], (via the Na + /Ca2 + antiport) and consequently that Ca2+ might act as the putative modulator, especially since Ca*+ was reported to affect Pt.,* (Schultz, 1984; Sariban-Sohraby and Benos, 1986; Garty and Benos, 1988). However, the mechanism of Ca2* action was not convincingly established and thus the role of Ca2+ remains conjectural (Palmer and Frindt, 1987; Harvey and Ehrenfeld, 1988; Ling and Eaton, 1989). The role of Ca2+ as a second messenger mediating various hormonal responses is not dubious, but the concerted adjustment required by the intracellular dialogue is not hormonally governed; moreover, hormonal regulation must continue to operate in the face of different basal steady states of Na+ transport, suggesting that the putative modulator in “cross-talk” might be H + rather than Ca2 + (Harvey and Ehrenfeld, 1988; Dawson and Richards, 1990). When the transepithelial transport of Na+ is altered, the rate of three membrane processes must vary pari passu: (1) Na+ entry through the Na+ channels, (2) the activity of the Na +/K + -ATPase and (3) K + recirculation through K + channels. Each one of these processes has been found to be controlled by pH. Firstly, it has been shown by Palmer (1985) in the toad bladder, by Palmer and Frindt (1987) in the collecting duct of the rat and by Harvey et al. (1988) in the frog skin, that P,, at the apical membrane declined when pH, decreased. Secondly, an effect of pH on the activity of the Na +/K+ -ATPase was reported by Skou (1982) in an enzyme preparation and by Eaton et al. (1984) in the rabbit urinary bladder; they found a reduction in pump rate as the pH was lowered. Finally, the role of pH was also examined on the K + channels or on the permeability of the basolateral membrane. Harvey et al. (1988) in the frog skin and Duffey and Devor (1990) in the

active transport

13

rabbit colon observed that the conductance of the basolateral membrane, which is predominantly permeable to K+ ,, was diminished as a result of an intracellular actdification, as discussed also by Dawson and Richards (1990) and by Kolb (1990). Thus, an increase of [Hfli was reported to cause a diminution in the three membrane mechanisms involved in the transepithelial transport of Na + . These findings led Dawson and Richards (1990) to propose a conceptual model to explain the coupling between the apical and the basolateral membrane, in which [H ‘Ii plays a pivotal role. The present study and that of Harvey and Ehrenfeld (1988) provided experimental support for this model under physiological conditions. Indeed, both Harvey and Ehrenfeld (1988) and the present study described a concomitant variation of pH, and Na+ transport. The former authors found a cellular acidification following ouabain administration; however, this manoeuvre was likely to affect diverse cellular components, especially [Na +li, thus the activity of the Na+/H + and that of the Na + /Ca2 + exchangers. We found a cellular acidification following the administration of amiloride at a concentration which had no effect on the Na+ /H + exchanger, suggesting that pHi regulatory processes were not involved. Thus, the adjustment of the membrane mechanisms to any given level of Na+ transport did not appear merely as a by-product of pH, regulation. The relationship between pH, and Na+ transport appeared to be reciprocal: the inhibition of Na+ transport influenced the pH, and conversely, modifications of pH, altered the transport of Na+. This effect was noticed on the apparent values of both K, and V,,,, the two parameters defining kinetics of the Michaelis-Menten type (Fig. 7A), revealing a mixed inhibition of the rate of Na + transport by H +!, as can be expected if several membrane processes were affected by H +,. These observations in A6 monolayers are in agreement with the effect of pHi on the transport properties in amphibian as well as in mammalian epithelia, as discussed above. The findings do not seem to be restricted to special experimental conditions, since they were made in the presence of C02, in cells exhibiting a large intracellular buffering power and an operational regulation of pH,. Therefore, this study supports the hypothesis that the intracellular dialogue between the apical and the basolateral membrane of the epithelial cell is mediated by pH,.

Acknowledgemenfs-We wish to thank Dr W. DurandArczynska for fruitful discussion. The technical assistance of Mr J. Ruffieux is gratefully acknowledged. The A6 cells were generously given by B. C. Rossier, Institute of Pharmacology, University of Lausanne, Switzerland. The work was supported by the Swiss RRR-Finanzpool, grant no. 7-G3/88 and by the Swiss National Foundation, grant no. 32-027825.89.

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14

FRANCOIS CHUARDand JACQUES DURAND

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