Role of Na⁺ conductance, Na⁺-H⁺ exchange, and Na⁺-K⁺-2Cl⁻ symport in the regulatory volume increase of rat hepatocytes

Abstract

1. In rat hepatocytes under hypertonic stress, the entry of Na⁺ (which is thereafter exchanged for K⁺ via Na⁺-K⁺-ATPase) plays the key role in regulatory volume increase (RVI).

2. In the present study, the contributions of Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport to this process were quantified in confluent primary cultures by means of intracellular microelectrodes and cable analysis, microfluorometric determinations of cell pH and buffer capacity, and measurements of frusemide (furosemide)/bumetanide-sensitive ⁸⁶Rb⁺ uptake, respectively. Osmolarity was increased from 300 to 400 mosmol l⁻¹ by addition of sucrose.

3. The experiments indicate a relative contribution of approximately 4:1:1 to hypertonicity-induced Na⁺ entry for the above-mentioned transporters and the overall Na⁺ yield equalled 51 mmol l⁻¹ (10 min)⁻¹.

4. This Na⁺ gain is in good agreement with the stimulation of Na⁺ extrusion via Na⁺-K⁺-ATPase plus the actual increase in cell Na⁺, namely 55 mmol l⁻¹ (10 min)⁻¹, as was determined on the basis of ouabain-sensitive ⁸⁶Rb⁺ uptake and by means of Na⁺-sensitive microelectrodes, respectively.

5. The overall increase in Na⁺ and K⁺ activity plus the expected concomitant increase in cell Cl⁻ equalled 68 mmol l⁻¹, which fits well with the increase in osmotic activity expected to occur from an initial cell shrinkage to 87.5 % and a RVI to 92.6 % of control, namely 53 mosmol l⁻¹.

6. The prominent role of Na⁺ conductance in the RVI of rat hepatocytes could be confirmed on the basis of the pharmacological profile of this process, which was characterized by means of confocal laser-scanning microscopy.

Under anisotonic conditions, most animal cells, including hepatocytes, initially behave like osmometers and change their volumes according to the extracellular tonicity (Corasanti, Gleeson & Boyer, 1990). In most cell types, however, this passive behaviour is followed by an active readjustment of cell volumes despite continuous hypotonic or hypertonic challenges (see Lang, Ritter, Völkl & Häussinger, 1993; Al-Habori, 1994 for review). These cellular responses are called regulatory volume decrease (RVD) and regulatory volume increase (RVI), respectively.

Hepatocytes in vivo are not thought to experience significant changes in cell volume that are due to alterations of extracellular tonicity. Of considerable physiological relevance, however, are changes in cell volume occurring in the course of substrate transport and metabolism (Häussinger, Gerok & Lang, 1993). In addition, alterations of cell volume due to nutrient transport as well as those elicited by certain hormones (insulin, glucagon) could be shown to function as a control mechanism for hepatocyte metabolism and gene expression (see Häussinger, Lang & Gerok, 1994 for review and references).

In the perfused rat liver, hepatocytes exhibit a partial RVI under hypertonic stress (Haddad, Thalhammer & Graf, 1989; Lang, Stehle & Häussinger, 1989). In contrast, isolated rat hepatocytes remain continuously shrunken when exposed to hypertonic solution and exhibit a post-RVD RVI only, i.e. a significant RVI only occurs after a preperiod of hypotonic stress that was followed by a partial RVD (Bakker-Grunwald, 1983; Corasanti et al. 1990). In a recent report from this laboratory, it could be shown that rat hepatocytes in confluent primary culture are capable of both RVI and a post-RVD RVI (Wehner, Sauer & Kinne, 1995) rendering this preparation a suitable model system for the analysis of these processes.

In most studies reported so far, RVI is mediated via activation of Na⁺-K⁺-2Cl⁻ symport, Na⁺-Cl⁻ symport, Na⁺-H⁺ exchange and/or Cl⁻-HCO₃⁻ exchange leading to an increase in cell salt and water content (Lang et al. 1993). For the RVI of rat hepatocytes the following model was proposed: activation of Na⁺-H⁺ exchange leads to an increase in cell Na⁺; Na⁺ is then exchanged for K⁺ via activation of the Na⁺ pump so that both transporters in concert accomplish a net gain in cell K⁺ (Haddad & Graf, 1989; Lang et al. 1993). This concept is based on the following findings: (1) hypertonic stress stimulates K⁺ uptake (Graf, Haddad, Haeussinger & Lang, 1988), (2) this K⁺ uptake is inhibited by millimolar concentrations of ouabain as well as amiloride (Haddad & Graf, 1989; Haddad et al. 1989; Häussinger, Stehle & Lang, 1990), and (3) hypertonic stress increases cell pH via stimulation of Na⁺-H⁺ exchange (Gleeson, Corasanti & Boyer, 1990). In addition, Häussinger and coworkers provided indirect evidence for a possible contribution of Na⁺-K⁺-2Cl⁻ symport to the RVI of rat hepatocytes: in the isolated perfused liver, bumetanide diminishes the insulin-induced increase of intracellular water space (Häussinger et al. 1991) and frusemide (furosemide) as well as bumetanide partly inhibit insulin-induced K⁺ uptake and volume increase of isolated rat hepatocytes (Hallbrucker, vom Dahl, Lang, Gerok & Häussinger, 1991).

In a recent study on confluent primary cultures of rat hepatocytes, it became evident that in addition to activation of Na⁺-H⁺ exchange hypertonic stress leads to a considerable increase in cell membrane Na⁺ conductance (Wehner et al. 1995). This Na⁺ conductance is inhibited by 10 μmol l⁻¹ amiloride and its contribution to RVI appears to exceed that of Na⁺-H⁺ exchange severalfold. Moreover, at this concentration, amiloride inhibits RVI as well as the volume-induced activation of Na⁺-K⁺-ATPase while leaving the activity of Na⁺-H⁺ exchange virtually unchanged (Wehner et al. 1995).

The aim of the present study was a quantitative analysis of the contributions of Na⁺ conductance, Na⁺-H⁺ exchange, Na⁺-K⁺-2Cl⁻ symport and Na⁺-K⁺-ATPase to the RVI of rat hepatocytes. Experiments were performed on confluent primary cultures by means of confocal laser-scanning microscopy (to determine cell volumes), ion-substitution and cable-analysis experiments (to quantify cell membrane Na⁺ conductance), microfluorometry (to measure intracellular pH as well as pH-buffer capacity), determination of ⁸⁶Rb⁺ uptake (to characterize K⁺ transport pathways), and ion-sensitive microelectrodes (to quantify the actual changes of cell Na⁺ and K⁺ occurring during RVI). Measurements were performed in HCO₃⁻-free solutions to avoid possible interferences with Na⁺-HCO₃⁻ cotransport due to time- dependent changes in intracellular pH-buffer capacity and the electrogenicity of the transporter. The experiments presented here confirm the prominent role of Na⁺ conductance in the RVI of rat hepatocytes and, for the first time, direct evidence for a significant contribution of Na⁺-K⁺-2Cl⁻ symport to this process is obtained. Moreover, the results of our study provide a complete model of Na⁺ (and K⁺) transport during RVI and the overall transport balance fits well with the expected increase of intracellular osmotic activity.

METHODS

Primary culture of hepatocytes

Isolation of rat hepatocytes was the same as described previously (Wehner & Guth, 1991; Wehner, Rosin-Steiner, Beetz & Sauer, 1993). Briefly, under urethane anaesthesia (1 g per kg body weight, intraperitoneal) and after heparinization, male Wistar rats (220-280 g body weight) were exsanguinized by in situ perfusion of the liver with HCO₃⁻-containing nominally Ca²⁺-free Krebs-Henseleit solution at 37°C (approved by Regierungspräsident Arnsberg and the Institute Animal Care Committee). After removal, the liver was perfused for 20 min with 0.05 % collagenase A, dissolved in the same buffer. After isolation, cells were plated on collagen-coated gas-permeable Petriperm® dishes and cultured in Dulbecco's modified Eagle medium (DMEM) fortified with 10 % fetal bovine serum, 2 mmol l⁻¹ glutamine, 100 u ml⁻¹ penicillin-100 μg ml⁻¹ streptomycin, 1 μmol l⁻¹ dexamethasone, 10 nmol l⁻¹ triiodothyronine/thyroxine (T₃/T₄), and 5 μg ml⁻¹ bovine insulin at 37°C in 5 % CO₂-air. Cells formed confluent monolayers within 24 h and were used from day 1 to day 3 after preparation, except for cable-analysis experiments (see below), which were exclusively conducted at day 2 to minimize culture time-dependent changes in cell-to-cell coupling (Wehner & Guth, 1991).

Petriperm® dishes were purchased from Bachofer (Reutlingen, Germany), DMEM, penicillin-streptomycin, and glutamine from Flow (Bonn, Germany), collagenase and fetal bovine serum from Boehringer (Mannheim, Germany). All other compounds were obtained from Serva Chemical Company (Heidelberg, Germany).

Determination of cell volumes

Cell volumes were determined by use of a confocal laserscan unit (MRC-600, BioRad, Hemel Hempstead, UK) coupled to a standard microscope (Diaphot, Nikon, Düsseldorf, Germany) with a ×20 objective (Zeiss, Oberkochen, Germany). For excitation of fluorescence, the device was equipped with an argon ion laser (Ion Laser Technology, Salt Lake City, UT, USA) and a helium-cadmium laser (4310 N, Liconix, Santa Clara, CA, USA) yielding bands of 488 and 442 nm, respectively. Images were acquired every 60 s and data were digitized and analysed by use of a microcomputer (RM Nimbus AX/2, Oxford, UK).

Cells were loaded for 45 min with calcein (Molecular Probes, Eugene, OR, USA) in its acetoxymethyl ester form at a final concentration of 10 μmol l⁻¹ and washed, thereafter, for approximately 5 min in dye-free solution. Calcein is a volume marker of aqueous compartments and is, under physiological conditions, pH and Ca²⁺ insensitive (Allen & Cleland, 1980; Kendall & MacDonald, 1983). The compound exhibits a high degree of fluorescence self quenching (Kendall & MacDonald, 1983) so that fluorescence decreases upon its concentration and increases upon its dilution. Thus, determination of calcein fluorescence in a single confocal plane (elicited by the 488 nm band of the argon laser) can be used to quantify changes in cell volumes (i.e. the relative cell volumes with respect to control conditions) at a low rate of photobleaching (Wehner et al. 1995; Tinel, Wehner & Kinne, 1996).

In addition, cell volumes were frequently determined by loading cells with the pH dye 2′,7′-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF; Molecular Probes) and by optically scanning them in the z-axis at an excitaton wavelength of 442 nm, where BCECF is pH insensitive (the isosbestic point). The spatial resolution of this procedure is 0.9 μm and a set of optical sections was acquired within 5 s. Cell volumes were then computed from the measured surface areas of every cross-section (Wehner et al. 1995; Tinel et al. 1996). While this method yields absolute values of cell volumes it induces a significantly higher amount of photobleaching. Therefore, only approximately 30 % of the measurements were performed with the latter technique. Changes in cell volume determined by either method yielded identical results.

In the pharmacological characterization of RVI, experiments were preceded by an incubation period of approximately 40 min to assure diffusion of the respective blocker to the trans side of the monolayers (cf. Discussion). In pilot experiments under normosmotic conditions, there were no detectable changes in cell volumes upon addition of the compounds under consideration for time periods of up to 1 h (data not shown).

Measurements of intracellular pH

Intracellular pH was monitored by use of the confocal laser-scanning microscope (see above) and the fluorescent dye BCECF as previously described (Wehner et al. 1993, 1995). Briefly, after dye loading, cell monolayers were transferred to a Perspex chamber of 0.1 ml volume and continuously superfused. Cell fluorescence was excited by use of the 488 and 442 nm laser bands and cell pH was determined from the fluorescence ratio from both excitation wavelengths. A calibration was performed at the end of each experiment in 140 mmol l⁻¹ KCl and 10 μmol l⁻¹ nigericin at pH values in the range of 6.4-8.2 in 10 mmol l⁻¹ Hepes-buffered or Tris-buffered solutions according to Thomas, Buchsbaum, Zimniak & Racker (1979).

For determination of intracellular pH-buffer capacity, cells were exposed for 10 min to 10 mmol l⁻¹ NH₄Cl in Na⁺-free solutions with choline as the substitute (to prevent pH regulation via Na⁺-H⁺ exchange), which led to a sizeable alkalinization of cell pH. pH-buffer capacity (β_int) was then calculated from the cell acidification following NH₄Cl removal (pH_i(a) - pH_i(b), see Fig. 6) according to the form:

(1)

(Gleeson, Smith & Boyer, 1989; Gleeson et al. 1990; see also Roos & Boron, 1981), where [NH₄Cl]_o and pK are the extracellular concentrations and the pK value (9.30) of NH₄Cl, respectively, and pH_o is the extracellular pH. Total H⁺ efflux (and, thus, total Na⁺ influx) under hypertonic stress occurring via activation of Na⁺-H⁺ exchange can then be calculated as J_H=β_int×ΔpH_i, where ΔpH_i is the hypertonicity-induced alkalinization of intracellular pH (Gleeson et al. 1989, 1990).

Figure 6. Determination of intracellular pH buffer capacity.

In Na⁺-free solutions, 10 mmol l⁻¹ NH₄⁺ was added to the superfusate for 10 min (isosmotically in exchange for choline). Buffer capacity can then be calculated from the cell acidification following NH₄⁺ removal (pH_i(b) - pH_i(a); see Methods). Experiments were performed at external pH values of 7.4 (A; n = 39) and 7.8 (B; n = 41).

Electrophysiological techniques

Experimental set-up and recording techniques are described in detail in previous reports from this laboratory (Wehner & Guth, 1991; Wehner et al. 1993). Briefly, circular sheets of gas-permeable membranes of approximately 1 cm² with confluent cell monolayers were cut from the bottom of the culture dishes and transferred to the superfusion chamber, which was mounted on the stage of an inverted microscope (IM 35; Zeiss). The fluid volume above the tissue was 0.1 ml and cells were continuously superfused at a rate of 4 ml min⁻¹ by means of a multi-channel peristaltic pump (PLG; Desaga, Heidelberg, Germany). Changes of the superfusate were performed by means of a four-way valve (ms-131 D; Whitey, Highland Heights, OH, USA) close to the experimental chamber. All storage vessels, superfusion lines, and the chamber were water-jacketed to achieve a constant temperature of the preparation (36.0 ± 0.5°C).

Conventional two-channel microelectrodes were pulled from 1.5 mm o.d. Thick-Septum-Theta borosilicate glass capillaries (WPI, New Haven, CT, USA) on a Kopf vertical puller (750; David Kopf Instruments, Tujunga, CA, USA) and had resistances of 80-130 MΩ when filled with 0.5 mol l⁻¹ KCl and immersed in control Tyrode solution. One channel was used to measure voltage, the second to inject constant current pulses for determination of input resistances. Cell impalements were performed at an angle of 45 deg under ×320 magnification by use of piezomanipulators (PM 500-20; Frankenberger, Germering, Germany). Criteria for successful impalements have been previously described (Wehner & Guth, 1991).

In the cable analysis, current pulses of 3-10 nA were injected into a first cell and the resultant changes in membrane voltage were recorded in a second cell with a single-channel electrode at 35, 100, 200, or 400 μm from the point of current injection in separate experiments. Single-channel electrodes were pulled from inner fibre borosilicate glass capillaries of 1.5 mm o.d. (Hilgenberg, Malsfeld, Germany) and had resistances of 70-80 MΩ. The voltage deflections in the second cell (δV_m2) were normalized to a standard current pulse of 10 nA (Wehner & Guth, 1991; Wehner et al. 1993) and plotted against the distance between both electrodes (x; see Fig. 5). Data were fitted by use of the form:

(2)

according to Frömter (1972), where K_o is a zero-order Bessel function and A and λ are constants defining the function. Each set of λ and A values was obtained in four separate experiments on monolayers from a single culture dish in which impalements were performed at the above-mentioned distances between electrodes. From these constants cell coupling resistance (R_x) and specific cell membrane resistance (R_z) can be calculated according to:

(3)

and

(4)

respectively. i_o is the total current applied.

Figure 5. Effects of an increase in osmolarity on the intracellular pH (pH_i) of rat hepatocy tes.

Experiments were performed on confluent monolayer cultures by means of the fluorescent dye BCECF (n = 11).

Ion-sensitive microelectrodes were constructed as reported in a previous publication (Wehner et al. 1995). Briefly, electrodes were pulled from single-channel borosilicate filament glass capillaries of 1.0 mm o.d. and 0.5 mm i.d. (Hilgenberg) to give resistances of 80-120 MΩ when filled with 0.5 mol l⁻¹ KCl. They were silanized by exposure to dimethyldichlorosilane vapour (Fluka, Neu-Ulm, Germany) at 200°C and baked for 1 h. The electrodes were then back-filled with either Na⁺ ionophore 1-Cocktail A (no. 71176; Fluka) and 0.5 mol l⁻¹ NaCl or K⁺ ionophore 1-Cocktail A (valinomycin; Fluka, no. 00031) and 0.1 mol l⁻¹ KCl. They were calibrated in mixed NaCl/KCl standard solutions at a constant sum of Na⁺ plus K⁺ of 150 mmol l⁻¹. The test solutions for Na⁺- and K⁺-sensitive electrodes contained 150, 50, 15, 5 and 1.5 mmol l⁻¹ NaCl and 150, 100, 10, 2.7 and 1 mmol l⁻¹ KCl, respectively, and all solutions were buffered to pH 7.4 with 5 mmol l⁻¹ Tris. The activity coefficients of Na⁺ and K⁺ were assumed to be constant and equal to 0.77 (Horisberger & Giebisch, 1988). The slope of the Na⁺-sensitive electrodes and the selectivity coefficients of Na⁺ over K⁺ were obtained by fitting the measured potential difference and Na⁺ activities of the standard solutions by use of a non-linear least- square fit routine to the Nicolsky equation (Edelman, Curci, Samarzija & Frömter, 1978; Horisberger & Giebisch, 1988; Wehner et al. 1995) and equalled 60.9 ± 3.2 mV decade⁻¹ and 60 ± 21, respectively (mean values ±s.d., n = 24). The slope of the K⁺-sensitive electrodes was determined accordingly and equalled 55.2 ± 2.0 mV decade⁻¹ (mean ±s.d., n = 12); K_K/Na, however, was found to be virtually zero. In the experiments, a single cell was first impaled with an internal reference electrode pulled from the same capillaries as the ion sensitive one, but filled with 0.5 mol l⁻¹ KCl. Once a stable registration was achieved, the same cell was impaled with the ion-sensitive electrode, which was commonly accompanied by a short transient deflection in membrane voltage (cf. Wehner et al. 1995). Only those experiments were accepted in which the membrane voltage was restored to within 2 mV of the original value. Ion-sensitive electrodes were calibrated before and after use and the intracellular Na⁺ and K⁺ activities were calculated according to Horisberger & Giebisch (1988; see also Wehner et al. 1995).

In all measurements, a custom-made 0.5 mol l⁻¹ KCl flowing junction in series with an Ag-AgCl wire was used as the extracellular reference electrode to avoid liquid junction potentials. This electrode was placed in an additional 1 ml compartment which is connected to the chamber via a hole 1.5 mm in diameter and 15 mm in length and which also contains the suction canula of the superfusion system. Because there is a continuous flow of experimental solutions from the chamber into this compartment any contamination of the preparation via leakage from the reference electrode can be excluded.

Measurements of Rb⁺ uptake

Circular sheets of 16 mm diameter with confluent monolayers were cut from the bottom of the culture dishes, washed and transferred to standard scintillation vials of 20 ml volume filled with 5 ml of experimental solution; solutions were kept at 36°C and continuously gassed with humidified O₂. Rb⁺ uptake was determined by transferring monolayers for 2, 4, 6 and 8 min to identical solutions labelled with 1-5 μCi ml^{−1 86}Rb⁺ (cf. Wehner et al. 1995). Uptake was measured under isotonic conditions (300 mosmol l⁻¹; see Solutions) and 2 min after transfer to 400 mosmol l⁻¹. For determination of Rb⁺ uptake via Na⁺-K⁺-ATPase, experiments were performed in the absence and in the presence of 2 mmol l⁻¹ ouabain. Rb⁺ uptake via Na⁺-K⁺-2Cl⁻ symport was quantified from the difference in transport rates found in the absence and in the presence of 100 μmol l⁻¹ frusemide or 20 μmol l⁻¹ bumetanide. For improvement of resolution, the latter experiments were conducted in the continuous presence of 2 mmol l⁻¹ ouabain. Influxes were terminated by removing the monolayers from the vials and washing them with ice-cold experimental solution of appropriate osmolarity and pH. The lower cell-free surface of the membranes was then carefully blotted on filter paper and the membranes were transferred to 0.5 ml of 2 % sodium dodecyl sulphate in 2 mmol l⁻¹ ethylenedinitrilotetraacetic acid. As pilot experiments with ¹⁴C-labelled sucrose as a second marker revealed, there was no extracellular compartment that was not accessible to the washing and drying procedure used (cf. Wehner et al. 1995). After 60 min of cell lysis, aliquots were sampled for liquid scintillation counting and determination of protein content by the method of Lowry, Rosebrough, Farr & Randall (1951).

Solutions

All experiments were exclusively conducted in HCO₃⁻-free solutions to avoid possible interferences with Na⁺-HCO₃⁻ symport (Fitz, Persico & Scharschmidt, 1989a;Gleeson et al. 1989). Such interferences may arise in the following ways: (1) hypertonicity-induced increases in cell Na⁺ are expected to decrease the driving force for Na⁺-HCO₃⁻ symport leading to a time-dependent change in intracellular pH-buffer capacity. This, in turn, would complicate a determination of Na⁺ transport via Na⁺-H⁺ exchange. (2) In rat liver, Na⁺-HCO₃⁻ symport is electrogenic (Fitz et al. 1989a) and if there were a significant role for this process in hepatocyte RVI, it would be difficult to distinguish its contribution to changes in overall cell membrane conductance from those elicited by an increase in Na⁺ conductance. In principle, the remarkable similarity of hepatocyte RVI in the presence (Wehner et al. 1995) and in the absence of HCO₃⁻ (this study) does not speak in favour of a prominent role of Na⁺-HCO₃⁻ symport in the RVI of rat hepatocytes. However, we cannot exclude a certain contribution of this pathway.

The normosmotic control solution (300 mosmol l⁻¹, pH 7.4) contained (mmol l⁻¹): NaCl, 144; KCl, 2.7; NaH₂PO₄, 0.4; Na⁺-Hepes, 2.5; Hepes, 2.5; CaCl₂, 1.8; MgCl₂, 1.1; glucose, 5.6. pH was adjusted by addition of 4 M NaOH. Increases in osmolarity were achieved by addition of 100 mmol l⁻¹ sucrose. In the ion substitution experiments, Na⁺ was isosmotically reduced 20-fold in exchange with choline. In one series of measurements, Hepes was replaced by Tris and extracellular pH was increased to 7.8. All experimental solutions were continuously gassed with humidified O₂ and kept at 36.0 ± 0.5°C.

Na⁺-Hepes and Hepes were bought from Serva Chemical Co., NaCl and KCl from Baker (Deventer, The Netherlands), and amiloride from Sigma. All other substances were obtained from E. Merck (Darmstadt, Germany).

Statistical analysis

Mean values ±s.e.m. are presented, unless otherwise indicated, with n denoting the number of cell culture dishes. Each series of experiments was performed on primary cultures derived from at least four different animals. Student's t test for paired and unpaired data were applied as appropriate. For the pharmacology of RVI, a one-way ANOVA was carried out, in addition. A value of P < 0.05 was considered significant.

RESULTS

Regulatory volume increase (RVI) and its inhibitors: confocal laser-scanning microscopy

In the first series of experiments, we assessed the relative contributions of Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport to the RVI of rat hepatocytes by pharmacological means. As confocal laser-scanning microscopy revealed, increasing osmolarity from 300-400 mosmol l⁻¹ by addition of sucrose decreased cell volumes to 87.5 ± 1.2 % within 2 min (n = 27; P < 0.001, Fig. 1A). Thereafter, cell volumes gradually increased to 92.6 ± 1.0 % of the control value within 10 min, equivalent to a RVI of 42.5 ± 3.2 % (P < 0.001). These changes in cell volume are virtually identical to those reported for hepatocytes in HCO₃⁻-containing solutions (Wehner et al. 1995). Upon return to 300 mosmol l⁻¹, cell volumes transiently increased to 102.7 ± 0.9 % of control (P < 0.001) and, thereafter, slowly declined to the baseline level.

Figure 1. Rat hepatocyte RVI and its inhibitors.

Effects of an increase in osmolarity on the cell volumes of rat hepatocytes in confluent monolayers, under control conditions (A, n = 27), and in the continuous presence of 10 μmol l⁻¹ amiloride (B, n = 17). For comparison, in B, the hypertonicity-induced changes in cell volumes under control conditions (see A) are depicted as dotted lines. C, RVI of rat hepatocytes under control conditions (see A), in the presence of 10 μmol l⁻¹ amiloride (see B), 1 mmol l⁻¹ amiloride (n = 15), 100 μmol l⁻¹ frusemide (n = 20), and 1 mmol l⁻¹ amiloride plus 100 μmol l⁻¹ frusemide (n = 16). Here, the RVI is defined as the 10 min volume change in a solution of 400 mosmol l⁻¹ referred to the initial 2 min period of rapid cell shrinkage. A negative RVI value means that the initial period of shrinkage is followed by a slow further decrease in cell volume. Values significantly different from control as was determined by unpaired t tests: *P <0.05, **P <0.01 and ***P <0.001. The latter level of significance could be confirmed by means of a variance analysis, yielding F = 60.3. n.s., not significant with P <0.2.

In an earlier report from this laboratory, it could be shown that amiloride at 10 μmol l⁻¹ is an effective blocker of volume-activated Na⁺ conductance in rat hepatocytes with no significant effects on hypertonicity-induced cell alkalinization via Na⁺-H⁺ exchange (Wehner et al. 1995). This provides us with a tool to estimate the contribution of Na⁺ conductance to the overall RVI. As depicted in Fig. 1B and summarized in Fig. 1C, 10 μmol l⁻¹ amiloride significantly reduced the RVI of rat hepatocytes to 3.7 ± 2.3 % (n = 17) already suggesting a prominent role of Na⁺ conductance in the RVI.

In the continuous presence of 1 mmol l⁻¹ amiloride, where Na⁺ conductance is blocked and Na⁺-H⁺ exchange will be reduced to approximately 3 % of control activity (see Tse et al. 1994 for review and references), RVI was not detectable. Moreover, under these conditions, the initial period of rapid cell shrinkage was followed by a continuous further decrease in cell volumes, leading to a negative RVI value of -10.4 ± 3.5 % (n = 15; Fig. 1C).

In the continuous presence of 100 μmol l⁻¹ frusemide, which is an effective blocker of Na⁺-K⁺-2Cl⁻ symport in various tissues (Cabantchik & Greger, 1992), the RVI of rat hepatocytes was reduced to 30.9 ± 4.5 % (n = 22; Fig. 1C) indicative of a sizeable contribution of the cotransporter to this process.

In the presence of 1 mmol l⁻¹ amiloride plus 100 μmol l⁻¹ frusemide, i.e. with Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport blocked, cell shrinkage was most pronounced yielding a negative RVI value of -16.0 ± 1.9 % within 10 min (n = 16; Fig. 1C).

The above experiments suggest a similar participation of Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport in the RVI of rat hepatocytes but they also imply an approximately 3-fold higher contribution of Na⁺ conductance to this process when compared with each cotransporter. A pharmacological approach like the one used here, however, is complicated in two ways: first, if one (or more) of the transporters responsible for RVI is blocked, cell shrinkage under hypertonic stress should be more pronounced and this could result in an increased activation of the others. A comparable cross- talk between different transport mechanisms has also been postulated for the release of organic osmolytes in rat inner medullary collecting duct cells under hypotonic stress (Kinne, Boese, Kinne-Saffran, Ruhfus, Tinel & Wehner, 1996). Second, all three transporters under investigation here are Na⁺ dependent and, thus, a certain cross-talk between them on the basis of changes in cell Na⁺ and driving forces is to be expected. For a quantitative analysis of the contribution of each Na⁺ conductance, Na⁺-H⁺ exchange, and Na⁺-K⁺- 2Cl⁻ symport to the RVI of rat hepatocytes we, therefore, had to use independent strategies solely focusing on the transport pathway under consideration.

Na⁺ conductance: Na⁺ substitutions and cable analysis

In an earlier report from this laboratory (Wehner et al. 1995), it could be shown that, in HCO₃⁻-containing solutions, hypertonic stress leads to a significant shift in the voltage responses to low Na⁺ pulses which was inhibited by 10 μmol l⁻¹ amiloride and which coincided with a pronounced decrease in specific cell membrane resistance. In the present study, we first performed Na⁺ substitution experiments to validate the use of HCO₃⁻-free solutions and, thereafter, quantified the effect of hypertonic stress on specific cell membrane conductance by means of cable analysis.

Figure 2 illustrates the effects of increasing osmolarity on membrane voltage and on the voltage reponses to low Na⁺ solutions. Upon change to 400 mosmol l⁻¹, membrane voltages transiently depolarized from -33.0 ± 1.2 mV to -26.1 ± 1.5 mV (n = 12; P < 0.001). In addition, there was a sustained decrease in cell input resistances from 7.4 ± 0.9 MΩ to 6.9 ± 1.0 MΩ (P < 0.001). In rat hepatocytes, a quantitative analysis of Na⁺ conductance by means of Na⁺ substitutions is complicated by the presence of Na⁺-H⁺ exchange and the pH dependence of K⁺ conductance (Henderson, Graf & Boyer, 1987; Fitz, Trouillot & Scharschmidt, 1989b) which in concert lead to slowly developing membrane depolarizations (Wehner & Guth, 1991; Wehner, 1993; Wehner et al. 1995) and which mask the expected hyperpolarizations of membrane voltage. Accordingly, in normosmotic solutions, a 20-fold reduction of extracellular Na⁺ led to depolarizations which equalled 2.1 ± 0.4 mV at 30 s in low Na⁺ solution (which is the most sensitive time point for this kind of analysis; Wehner et al. 1995). Increasing osmolarity to 400 mosmol l⁻¹ clearly shifted this membrane response to more negative values: taking the above time point in low Na⁺ solution as reference, membrane voltages changed by -2.0 ± 0.4 mV and by -0.8 ± 0.4 mV after 200 s and 500 s in 400 mosmol l⁻¹, respectively (n = 12; cf. Fig. 2). This is equivalent to negative shifts in the voltage responses to low Na⁺ by -4.0 ± 0.6 mV (P < 0.001) and -2.8 ± 0.5 mV (P < 0.001). In the continuous presence of 10 μmol l⁻¹ amiloride, these voltage shifts were reduced to -0.8 ± 0.3 (P < 0.005) and -0.4 ± 0.3 mV (P < 0.005; n = 8; data not shown). Taken together, these results already strongly suggest that, also in HCO₃⁻-free solutions, hypertonic stress leads to a significant increase in cell membrane Na⁺ conductance.

Figure 2. Effects of an increase in osmolarity on membrane voltage and on the voltage response to low Na⁺ solutions.

Vertical deflections result from injected current pulses of 1 nA. For the times indicated, Na⁺ was reduced 20-fold in exchange with choline. Note the negative voltage shift in the responses to low Na⁺ upon change to 400 mosmol l⁻¹, which is most pronounced at 30 s after ion substitutions (arrows).

In the following series of experiments, changes in cell-to-cell coupling resistance and specific cell membrane resistance were analysed by means of cable analysis. Figure 3 depicts a typical data set (n = 1) derived from four separate measurements (performed at 4 distinct distances between a current injecting and a voltage sensing microelectrode) on four confluent monolayers from a single culture dish. As summarized in Fig. 4A, increasing osmolarity from 300 to 400 mosmol l⁻¹ elicited an immediate and sustained decrease in cell coupling resistance (from 5.6 ± 1.7 MΩ under normosmotic conditions) which equalled 87.9 ± 3.0 % of control at 10 min in hypertonic solution (n = 6; P < 0.05). Upon return to 300 mosmol l⁻¹, cell coupling resistance transiently increased to 107.3 ± 3.5 % (P < 0.05) and, thereafter, returned towards control values. These changes in cell coupling resistance closely resemble the passive changes of cell volumes under hypertonic stress (see Fig. 1A) and, thus, are likely to reflect alterations of cytosolic conductivity rather than actual changes of gap junctional permeability. Most interestingly, hypertonic stress also led to a slowly developing decrease in specific membrane resistance (from 4.5 ± 0.6 kΩ cm²) to 77.2 ± 4.2 % of control within 10 min (P < 0.05; Fig. 4B). With respect to the observed shifts in the voltage responses to low Na⁺ solutions (see above) which were more pronounced at 200 s rather than 500 s in a solution of 400 mosmol l⁻¹, the slow time course of this membrane response was somewhat surprising. We, therefore, argued that a transient decrease in K⁺ conductance upon hypertonic stress may participate in the overall changes in specific membrane resistance thus masking a more rapid increase in Na⁺ conductance. A decrease in K⁺ conductance appears to be the main mechanism of RVI in mouse hepatocytes (Graf et al. 1988; Khalbuss & Wondergem, 1990; Wang & Wondergem, 1991) and a transient K⁺ conductance decrease has also been proposed to be part of the RVI of rat hepatocytes (Wehner et al. 1995). Figure 4C depicts a cable analysis of specific membrane resistance performed in the continuous presence of 500 μmol l⁻¹ quinine, which is an effective blocker of K⁺ conductance in hepatocytes (Bear, Davison & Shaffer, 1988; Wehner, Beetz & Rosin-Steiner, 1992). In control experiments under isotonic conditions, there were no detectable changes in cell volumes upon addition of quinine over time periods of some 30-40 min (n = 13; data not shown). Under these conditions, hypertonic stress led to a significantly accelerated decrease of specific membrane resistance (from 5.5 ± 0.8 kΩ cm²), which was virtually complete after 3 min and which equalled 78.7 ± 2.4 % of control at 10 min in 400 mosmol l⁻¹ (P < 0.02; n = 4). This value is very close to the maximum decrease obtained in the absence of quinine.

Figure 3. Cable analysis of electric cell-to-cell coupling and specific cell membrane resistance.

Monolayers were impaled with 2 microelectrodes, the 1st for current injection and the 2nd to monitor the resultant deflections in membrane voltage (δV_m2) at 35, 100, 200, or 400 μm from the first impalement in 4 separate experiments conducted on sheets from the same culture dish. Measurements were performed in continuous intracellular recordings under normosmotic conditions (t = 0) and at various times after change to 400 mosmol l⁻¹ (from which t = 200 s and t = 500 s are exemplified). Experimental data were fitted to zero-order Bessel functions. The above procedure yields a single but continuous data set for λ and A (n = 1) from which electric cell-to-cell couplings and specific cell membrane resistances can be calculated (see Methods).

Figure 4. Effects of hypertonic stress on electric cell-to-cell coupling (A) and specific membrane resistance (B and C).

Experiments depicted in A and B were performed under control conditions (n = 6 for each data point; cf. Fig. 5). In C, specific membrane resistances were determined in the continuous presence of 500 μmol l⁻¹ quinine (n = 4).

On the basis of the experiments depicted in Fig. 4C, and because Cl⁻ conductance is most probably not changing during RVI (see Discussion), hypertonicity-induced increases in cell Na⁺ expected to occur via activation of Na⁺ conductance can be estimated. It has to be considered, however, that part of the observed increase in membrane conductance will be due to an increase in cytosolic conductivity following cell shrinkage, which also becomes evident as apparent decrease in cell coupling resistance (see Fig. 4A). We, therefore, calculated changes in cell membrane (Na⁺) conductance with reference to the value at 1 min in 400 mosmol l⁻¹ where cell shrinkage is almost complete (see Fig. 1A and B) and from where on there are virtually no further changes in cell coupling resistance (Fig. 4A). Accordingly, in the presence of quinine, specific cell membrane resistances equalled 5.0 kΩ cm² at 1 min and (on the average) 4.5 kΩ cm² between 3 and 10 min of hypertonic stress (cf. Fig. 4C). With an average surface area of a single cell of 19.2 × 10⁻⁶ cm² ((17.5 × 10⁻⁴ cm)²×π× 2; calculated from a mean cell diameter of 35 μm (Wehner & Guth, 1991; Wehner et al. 1995), which could be confirmed by means of confocal laser-scanning microscopy, and because for this calculation the cell surface on both sides of the monolayer has to be considered) this is equivalent to membrane resistances of 260 and 234 MΩ. From this increase in membrane conductance by 0.43 nS and with a driving force of -75.5 mV for conductive Na⁺ entry (V_m - E_Na= -33.0 mV - 42.5 mV; where E_Na is calculated on the basis of the average intracellular Na⁺ activity between 3 and 10 min in 400 mosmol l⁻¹, namely 22.8 mmol l⁻¹; cf. Fig. 9A) an increase in inward current by 32.5 pA cell⁻¹ can be computed. Under the assumptions made, this current increase is equivalent to a Na⁺ influx of 202 fmol (10 min)⁻¹ (32.5 × 10⁻¹² C s⁻¹× (96500 C mol⁻¹) × 600 s (10 min)⁻¹). Because the average cell volume in 300 mosmol l⁻¹ is 5.9 pl, as was determined by confocal laser-scanning microscopy, this Na⁺ influx will tend to increase cell Na⁺ by 34.3 mmol l⁻¹ (10 min)⁻¹.

Figure 9. Effects of hypertonic stress on the intracellular activities of Na⁺ and K⁺.

n = 7 and n = 6 in A and B, respectively.

Na⁺-H⁺ exchange: cell alkalinization and intracellular pH buffer capacity

It is well established that in rat hepatocytes, hypertonic stress increases the rate of Na⁺-H⁺ exchange, which leads to a sizeable increase in intracellular pH (Gleeson et al. 1990; Häussinger et al. 1990; Wehner et al. 1995). To quantify the contribution of Na⁺-H⁺ exchange to the hypertonicity-induced increase in cell Na⁺ we performed microfluorometric measurements with the fluorescent dye BCECF. In the first series of experiments, we found that increasing osmolarity from 300 to 400 mosmol l⁻¹ led to a reversible increase in cell pH from 7.13 ± 0.02 to 7.49 ± 0.04 within 10 min (n = 30; P < 0.001; Fig. 5).

In the second series, we determined the intracellular pH buffer capacity of rat hepatocytes in confluent monolayers following the approach of Gleeson et al. (1989; see also Roos & Boron, 1981). To this end, cells were exposed for 10 min to 10 mmol l⁻¹ NH₄Cl in Na⁺-free solutions. Experiments were performed at external pH values (pH_o) of 7.4 and 7.8 at which internal pH (pH_i) equalled 6.90 ± 0.03 (n = 39) and 7.41 ± 0.01 (n = 41), respectively. Under both conditions, addition of NH₄Cl led to a sizeable and slowly stabilizing alkalinization of intracellular pH (Fig. 6A and B). Upon withdrawal of NH₄Cl, pH_i then declined from 7.12 ± 0.02 to 6.44 ± 0.03 and from 7.58 ± 0.03 to 6.87 ± 0.02 at a pH_o of 7.4 and 7.8, respectively, from which pH buffer capacities (β_int) of 28.5 ± 1.2 mmol l⁻¹ and 22.7 ± 1.0 mmol l⁻¹ can be calculated (see Methods). This is in good agreement with data reported by Gleeson et al. for subconfluent primary cultures of rat hepatocytes (i.e. 23.4 and 17.6 at a pH_o of 7.4 and 7.8, respectively; Gleeson et al. 1990).

From both sets of measurements and with an average β_int of 25.6 mmol l⁻¹, a total hypertonicity-induced H⁺ efflux of 9.2 mmol l⁻¹ (10 min)⁻¹ can be computed (J_H=ΔpH_iβ_int= 0.36 × 25.6 mmol l⁻¹), which will be equivalent to the same amount of Na⁺ entering the cell via Na⁺-H⁺ exchange.

Na⁺-K⁺-2Cl⁻ symport: frusemide- and bumetanide-sensitive ⁸⁶Rb⁺ uptake

The role of Na⁺-K⁺-2Cl⁻ symport in the RVI of rat hepatocytes was assessed by determination of time-dependent ⁸⁶Rb⁺ uptake. All experiments of this series were performed in the continuous presence of 2 mmol l⁻¹ ouabain (i.e. under conditions where Na⁺-K⁺-ATPase is blocked; see below, Na⁺-K⁺-ATPase, cell Na⁺ and cell K⁺: ouabain-sensitive ⁸⁶Rb⁺ uptake and ion-sensitive electrodes) to improve the resolution of measurements. In 300 mosmol l⁻¹, Rb⁺ uptake equalled 2.76 ± 0.14 and 0.59 ± 0.15 nmol (mg protein)⁻¹ min⁻¹ in the absence and presence of 100 μmol l⁻¹ frusemide, respectively, yielding a frusemide-sensitive fraction of 2.02 ± 0.27 nmol (mg protein)⁻¹ min⁻¹ (n = 4; Fig. 7A). In 400 mosmol l⁻¹, this frusemide-sensitive Rb⁺ uptake was significantly increased to 2.93 ± 0.12 nmol (mg protein)⁻¹ min⁻¹ (n = 4; P < 0.01; Fig. 7B), i.e. by 0.91 ± 0.22 nmol (mg protein)⁻¹ min⁻¹.

Figure 7. Stimulation of Na⁺-K⁺-2Cl⁻ symport.

⁸⁶Rb⁺ uptake of rat hepatocyte monolayers was determined in the presence of 2 mmol l⁻¹ ouabain (^) and 2 mmol l⁻¹ ouabain plus 100 μmol l⁻¹ frusemide (□), in 300 mosmol l⁻¹ (A) and 2 min after change to 400 mosmol l⁻¹ (B). n = 4 for each experimental condition.

Experiments performed with 20 μmol l⁻¹ bumetanide yielded very similar results. In 300 mosmol l⁻¹ Rb⁺ uptake in the absence and in the presence of bumetanide equalled 1.98 ± 0.10 and 0.55 ± 0.14 nmol (mg protein)⁻¹ min⁻¹, so that the bumetanide-sensitive fraction amounted to 1.43 ± 0.22 nmol (mg protein)⁻¹ min⁻¹ (n = 4; data not shown). In 400 mosmol l⁻¹, bumetanide-sensitive Rb⁺ uptake was increased to 2.28 ± 0.08 nmol (mg protein)⁻¹ min⁻¹, i.e. by 0.85 ± 0.32 nmol (mg protein)⁻¹ min⁻¹, (P < 0.05).

The above experiments yield a volume-activated Rb⁺ uptake via Na⁺-K⁺-2Cl⁻ symport of 0.88 nmol (mg protein)⁻¹ min⁻¹ on the average. One has to consider, however, that in confluent monolayers grown on gas-permeable membranes, only approximately one-half of the total cell surface will be readily accessible for the uptake solution (see Discussion) and, thus, activation of Na⁺-K⁺-2Cl⁻ cotransport (as well as activation of Na⁺-K⁺-ATPase; see below) will be underestimated by a factor of 2. Accordingly, and because for rat hepatocytes 1 mg protein is equivalent to 4 × 10⁵ cells (Blitzer, Ratoosh, Donovan & Boyer, 1982) and the average cell volume equals 5.9 pl (Wehner et al. 1995) we can calculate that hypertonicity- induced activation of Na⁺-K⁺-2Cl⁻ cotransport will tend to increase cell Na⁺ by approximately 7.5 mmol l⁻¹ (10 min)⁻¹ (2 × 8.8 nmol (mg protein)⁻¹ (10 min)⁻¹ × (4 × 10⁵ cells (mg protein)⁻¹)⁻¹ × (5.9 pl)⁻¹).

Na⁺-K⁺-ATPase, cell Na⁺ and cell K⁺: ouabain-sensitive ⁸⁶Rb⁺ uptake and ion-sensitive electrodes

Na⁺ entering hepatocytes via activation of Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport will finally be extruded via Na⁺-K⁺-ATPase. In this respect, it is of interest to know the extent by which Na⁺-K⁺-ATPase is activated because this will be of considerable importance for the overall (inorganic) osmolyte balance. The same holds for the hypertonicity-induced changes in cell Na⁺ and cell K⁺.

Transport rates of hepatocyte Na⁺-K⁺-ATPase were monitored by determination of ouabain-sensitive ⁸⁶Rb⁺ uptake. In 300 mosmol l⁻¹, Rb⁺ uptake equalled 7.5 ± 0.7 and 2.5 ± 0.2 nmol (mg protein)⁻¹ min⁻¹ in the absence and in the presence of 2 mmol l⁻¹ ouabain, respectively, yielding an ouabain-sensitive fraction of 5.0 ± 0.7 nmol (mg protein)⁻¹ min⁻¹ (n = 5; Fig. 8A). In 400 mosmol l⁻¹, Rb⁺ uptake was increased to 11.7 ± 1.5 in the absence (n = 5; P < 0.01) and to 3.3 ± 0.2 nmol (mg protein)⁻¹ min⁻¹ in the presence of 2 mmol l⁻¹ ouabain (P < 0.01; Fig. 8B), from which an increase in ouabain-sensitive Rb⁺ uptake to 8.6 ± 1.6 nmol (mg protein)⁻¹ min⁻¹ can be computed (P < 0.01). This stimulation of Na⁺-K⁺-ATPase will tend to increase cell K⁺ by approximately 30.5 mmol l⁻¹ (10 min)⁻¹ (2 × 36.0 nmol (mg protein)⁻¹ (10 min)⁻¹× (4 × 10⁵ cells (mg protein)⁻¹)⁻¹× (5.9 pl)⁻¹) and, due to the coupling ratio of 3 Na⁺:2 K⁺, will tend to decrease cell Na⁺ by 45.8 mmol l⁻¹ (10 min)⁻¹.

Figure 8. Stimulation of Na⁺-K⁺-ATPase.

⁸⁶Rb⁺ uptake was determined under control conditions (•) and in the presence of 2 mmol l⁻¹ ouabain (^), in 300 mosmol l⁻¹ (A) and 3 min after change to 400 mosmol l⁻¹ (B). n = 5 for each experimental condition.

Figure 9A summarizes experiments in which intracellular Na⁺ activities were determined by means of ion-sensitive microelectrodes. Increasing osmolarity from 300 to 400 mosmol l⁻¹ augmented cell Na⁺ activity from 16.1 ± 2.1 mmol l⁻¹ to a new steady state level of 23.0 ± 3.1 mmol l⁻¹ (n = 6; P < 0.01). Changes in cell K⁺ upon hypertonic stress are depicted in Fig. 9B. In 300 mosmol l⁻¹, the intracellular K⁺ activity equalled 76.3 ± 7.6 mmol l⁻¹ and was significantly increased to 103.4 ± 10.4 mmol l⁻¹ at 10 min after change to 400 mosmol l⁻¹ (n = 7; P < 0.005). Because the above changes in cell Na⁺ and cell K⁺ will be accompanied by equivalent changes in cell Cl⁻, this will lead to an increase in total osmotic activity by 68 mosmol l⁻¹. This value is in good agreement with the increase in osmotic activity expected to occur on the basis of the RVI in our experiments (which led to an increase in cell volume from 87.5 to 92.6 % of control within 10 min), namely 53 mosmol l⁻¹ as was determined by use of a Boyle-van't Hoff plot of relative cell volume vs. 1/osmolarity. The hypertonicity-induced changes in cation activities are equivalent to increases in intracellular Na⁺ and K⁺ concentrations of 9.0 mmol l⁻¹ and 35.2 mmol l⁻¹, respectively, if one assumes an activity coefficient of 0.77 for both Na⁺ and K⁺ (Horisberger & Giebisch, 1988).

DISCUSSION

In the intact liver, rat hepatocytes under hypertonic stress are capable of a partial RVI (Haddad et al. 1989; Lang et al. 1989). In sharp contrast, isolated rat hepatocytes undergo a RVI only when transferred back from hypoosmotic to normosmotic conditions (post-RVD RVI) (Corasanti et al. 1990), i.e. in hypertonic solutions, cells remain continuously shrunken for time periods of up to 30 min (Bakker-Grunwald, 1983; Corasanti et al. 1990). In an earlier report from this laboratory (Wehner et al. 1995), it became evident that rat hepatocytes in primary culture that are grown to confluency on collagen-coated gas-permeable membranes also exhibit a significant RVI and, thus, appear to be a suitable in vitro model for the analysis of this process. In the above-mentioned study (Wehner et al. 1995), it became evident that in addition to activation of Na⁺-H⁺ exchange (Haddad & Graf, 1989; Gleeson et al. 1990; Häussinger et al. 1990) hypertonic stress leads to a prominent increase in Na⁺ conductance. Moreover, indirect evidence for a possible contribution of Na⁺-K⁺-2Cl⁻ symport to the RVI of rat hepatocytes has also been reported (Häussinger et al. 1991; Hallbrucker et al. 1991). This raised the question to what extent Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport may contribute to the hypertonicity-induced Na⁺ influx and, consequently, to the RVI of rat hepatocytes.

We followed two different strategies to answer this question. (1) Na⁺ influxes via Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport were quantified by means of cable analysis and measurements of intracellular Na⁺ activities, determination of cell alkalinization and pH-buffer capacity, and measurements of frusemide- and bumetanide-sensitive ⁸⁶Rb⁺ uptake, respectively. These experiments yield a ratio of approximately 4:1:1 for the contribution of these transporters to the hypertonicity-induced changes in cell Na⁺ and approximately 4:1:2 if the overall primary cation influx is considered. Given the high intrinsic cell membrane Cl⁻ conductance in rat hepatocytes we do not think that Cl⁻ influx via Na⁺-K⁺-2Cl⁻ symport is of significant relevance for the RVI of rat hepatocytes (see below). These independent transport measurements do not impair cross-talk phenomena between different transporters on the basis of activation set-points, nor does the analysis of one transporter alter the driving forces for the others. For the quantification of ion movements, a parallel, simultaneous and constant activation of transport pathways over a time period of 10 min was assumed as a first approximation. Figure 10 summarizes the contributions of the various Na⁺ pathways participating in the RVI of rat hepatocytes to overall Na⁺ transport. All three transporters together would tend to increase cell Na⁺ by 51.0 mmol l⁻¹ (10 min)⁻¹. This value is in excellent agreement with the measured sum of Na⁺ extrusion via Na⁺-K⁺-ATPase plus the actual increase in Na⁺ concentration, namely 54.8 mmol l⁻¹ (10 min)⁻¹.

Figure 10. Cell model of Na⁺ transport during RVI.

Depicted are the changes in cell Na⁺ (in mmol l⁻¹ (10 min)⁻¹) expected to occur via Na⁺ conductance (left), Na⁺-H⁺ exchange (top), Na⁺-K⁺-2Cl⁻ symport (right), as well as Na⁺-K⁺-ATPase (bottom). The actual increase in cell Na⁺ during 10 min of hypertonic stress is given in square brackets.

(2) The RVI of rat hepatocytes was quantified by means of confocal laser-scanning microscopy and the role of the different Na⁺ transporters involved was investigated on the basis of a pharmacological protocol. From this approach secondary effects on transport activation and driving forces are to be expected, but these experiments prove the physiological significance of each transporter for the RVI of rat hepatocytes. Moreover, they provide us with a semiquantitative estimate of transport rates: if one considers the RVI under control conditions and the overall 10 min cell shrinkage in the presence of 1 mmol l⁻¹ amiloride plus 100 μmol l⁻¹ frusemide as 0 and 100 % inhibition, respectively, then the inhibitory potency in blocking RVI would equal 56 % for 10 μmol l⁻¹ amiloride, an additional 22 % for 1 mmol l⁻¹ amiloride, and approximately 20 % for 100 μmol l⁻¹ frusemide. This would imply a relative contribution of Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport to the RVI of rat hepatocytes of approximately 3:1:1.

In the present study, cable analysis of specific cell membrane resistances was carried out in the absence and in the presence of quinine (Fig. 4B and C). The differences in time course by which hypertonic stress increases membrane conductance under both experimental conditions support the notion that in addition to activation of Na⁺ conductance a transient decrease in K⁺ conductance may occur. This additional effect was already supposed in an earlier report from this laboratory (Wehner et al. 1995).

In our appraisal of conductive Na⁺ entry during RVI, we assumed that changes in Cl⁻ conductance are not involved in the observed increase of specific cell membrane resistance under hypertonic stress. This assumption is based on Cl⁻ substitution experiments, in which Cl⁻ was reduced 100-fold in exchange for gluconate. Under normosmotic conditions, this manoeuvre depolarized membrane voltages by 16.5 ± 2.6 mV (n = 8; data not shown). Although there was a clear tendency towards reduced voltage responses in 400 mosmol l⁻¹, most probably reflecting activation of Na⁺ conductance, these changes remained short of significance: voltage changes averaged 14.3 ± 2.7 mV and 14.2 ± 2.5 mV at 200 and 500 s after osmolarity increase, respectively. These data are very similar to those obtained earlier under identical experimental conditions (Wehner et al. 1995). From this we conclude that an increase in Cl⁻ conductance does not contribute to the observed increase in specific cell membrane resistance under hypertonic stress. It is noteworthy that the intrinsic basal Cl⁻ conductance of rat hepatocytes appears to fulfil the required Cl⁻ transport expected to occur concomitant to the increase in cell Na⁺ and K⁺. A comparable mechanism was also proposed for the RVD of mouse (Wondergem & Wang, 1991) as well as rat hepatocytes (Wehner et al. 1992).

In our calculations of Na⁺ movement via Na⁺-K⁺-2Cl⁻ symport and Na⁺-K⁺-ATPase we assumed that, in confluent monolayers on gas-permeable membranes, only approximately one-half of the total cell surface will be readily accessible for the uptake solution. This estimate is based on the following evidence. (1) In K⁺ and Cl⁻ substitution experiments, confluent monolayers of rat hepatocytes exhibit K⁺ and Cl⁻ transference numbers that equal approximately 50 % of those determined in isolated rat hepatocytes (Graf, Henderson, Krumpholz & Boyer, 1987; Wehner & Guth, 1991). (2) As confocal laser-scanning microscopy reveals, the average height of confluent cell monolayers and the thickness of gas-permeable membranes in the culture dishes used equals some 7 and 25 μm, respectively. In between both structures, there is a fluid compartment of approximately 7 μm in height, most probably maintained by the collagen matrix of our preparation. (3) In the determination of time-dependent ⁸⁶Rb⁺ uptake, there were virtually no significant intercepts on the y-axis that would be expected to occur if, within the experimental time frame, there were a sizeable diffusion of ⁸⁶Rb⁺ to the trans side of the monolayers. (4) In the preperiod of a few cable-analysis experiments, the effects of quinine on membrane voltage were monitored. Addition of quinine to the superfusate led to an initial depolarization of some 8-12 mV within approximately 5-10 s, probably reflecting inhibition of K⁺ channels on the cis side of the cell layers. Thereafter, there was a continuous further depolarization of comparable size that slowly stabilized after some 30-40 min. We interpreted this latter behaviour to reflect diffusion of quinine to the trans side of the monolayers and, consequently, introduced an appropriate preincubation period prior to the measurements. Nevertheless, this finding also supports the notion that within an experimental time frame of some 8 min the trans side of confluent rat hepatocyte monolayers will not be accessible to experimental manoeuvres performed on the cis side.

In the ion substitution experiments, we observe a maximal hypertonicity-induced shift in the voltage response to low Na⁺ of -4.0 mV. Because in these measurements, Na⁺ was reduced 20-fold and due to the geometry of our system outlined above, this effect most probably reflects an actual increase in Na⁺ transference number by 0.1 (2ΔV_m/ΔE_Na= 2 × 4.0 mV/79.3 mV). This apparent increase in Na⁺ conductance is remarkably similar to the 10 % increase in specific cell membrane resistance determined in the cable-analysis experiments.

The relatively low contribution of Na⁺-H⁺ exchange to overall cation influx raises the question of whether activation of Na⁺-H⁺ exchange may fulfil additional assignments, namely, if cell alkalinization may serve as a second messenger involved in the activation of Na⁺ conductance and/or Na⁺-K⁺-ATPase. Cell acidification decreases the apical Na⁺ permeability in toad urinary bladder (Palmer, 1985) and decreases the open probability of single Na⁺ channels in rat cortical collecting duct (Palmer & Frindt, 1987). In frog skin epithelium, cell acidification decreases and cell alkalinization increases apical Na⁺ conductance (Harvey, Thomas & Ehrenfeld, 1988; Ehrenfeld, Lacoste & Harvey, 1992) and the same holds for the open probability of Na⁺ channels in A6 cells (Harvey, 1995). With respect to the Na⁺-K⁺-ATPase, Eaton, Hamilton & Johnson (1984) analysed in detail the effects of cell pH on the activity of this transporter in rabbit urinary bladder and reported a strong positive correlation of both parameters between pH 6.5 and 7.5. In recent experiments, possible effects of cell alkalinization on rat hepatocyte Na⁺ conductance and Na⁺-K⁺-ATPase were investigated and compared with those elicited by hypertonic stress (Wehner, Kinne, Tinel, 1997b). To this end, osmolarity was increased from 300 to 400 mosmol l⁻¹ at constant extracellular pH (7.4), whereas an osmotically induced cell alkalinization by 0.3 units was mimicked by increasing extracellular pH from 7.4 to 7.8 under normosmotic conditions. The results indicate, however, that a cell alkalinization comparable in size to that following stimulation of Na⁺-H⁺ exchange in hypertonic solutions is neither responsible for activation of Na⁺ conductance nor Na⁺-K⁺-ATPase.

In summary, we employed electrophysiological and microfluorometric techniques, as well as measurements of time-dependent ⁸⁶Rb⁺ uptake, to quantify the contribution of Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport to the RVI of rat hepatocytes. It is found that the relative contribution of these transporters is approximately 4:1:1 for the hypertonicity-induced increase in cell Na⁺ and 4:1:2 if overall primary cation influx is considered. The sum of Na⁺ influx via Na⁺ conductance, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport is in excellent agreement with the amount of Na⁺ extrusion via Na⁺-K⁺-ATPase plus the actual increase in cell Na⁺. Moreover, the calculated overall osmotic balance fits well to the increase in intracellular osmotic activity expected to occur from the actual amount of RVI. The prominent role of Na⁺ conductance in the RVI of rat hepatocytes as well as the physiological relevance of conductive Na⁺ entry, Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl⁻ symport could be confirmed by use of confocal laser-scanning microscopy and a pharmacological protocol.