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Journal of Physiology (2001), 536.3, pp. 703-715
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of Na+, which is then (to a large extent) exchanged for K+ via activation of Na+/K+-ATPase (for reviews, see Lang et al. 1998; Wehner et al. 1998). This cation uptake will be paralleled by a gain of Cl- through the sizeable intrinsic anion conductance of the hepatocyte membrane which, under hypertonic conditions, is probably not changing (Wehner et al. 1995; Wehner & Tinel, 1998). The overall balance of these processes is an increase of cell K+ (and Na+) Cl- compensating for the increase of extracellular osmolarity and thus mediating RVI. In rat hepatocytes, a significant RVI only becomes detectable in confluent monolayer culture (Wehner et al. 1995; Wehner & Tinel, 1998), whereas it is virtually absent in isolated cells (Corasanti et al. 1990), suggestive of a significant role of cell-to-cell contacts in this process. Although the RVI is relatively small and exhibits a rather slow onset when compared with some other systems, it is in excellent agreement with the actual changes of cell Na+ and K+ determined (Wehner & Tinel, 2000).
It was shown recently that, in addition to a stimulation of Na+-H+ antiport and Na+-K+-2Cl- symport, hypertonic stress in rat hepatocytes leads to the activation of a sizeable Na+ conductance as a novel mechanism of Na+ import during RVI (Wehner et al. 1995). Moreover, in a quantitative study performed at 33 % hypertonicity (300 
400 mosmol l-1), it was found that the activation of Na+ conductance was the main mechanism of Na+ uptake with a ratio of 4 : 1 : 1 when referred to the contributions of Na+-H+ antiport and Na+-K+-2Cl- symport (Wehner & Tinel, 1998). When the osmotic sensitivity of the above Na+ importers was tested, however, it became clear that both Na+ conductance and Na+-K+-2Cl- symport did not respond to the lowest level of hypertonicity tested, namely 9 %, and that from 20 % hypertonicity upwards both transporters exhibited almost identical patterns of activation; in sharp contrast, Na+-H+ antiport exhibited some 65 % of its maximal stimulation at 9 % hypertonicity already (Wehner & Tinel, 2000). These results imply a parallel mode of activation for Na+ conductance and Na+-K+-2Cl- symport, but a different mode for Na+-H+ antiport. Because in various systems studied so far, the hypertonicity-induced activation of Na+-K+- 2Cl- symport occurs via PKC (see Hoffmann & Pedersen, 1998, for review), PKC was the prime candidate for the stimulation of this transporter in rat hepatocytes - and for the stimulation of Na+ conductance as well. Of note, PKA appears to be involved in the isotonic activation of Na+-K+-2Cl- symport only (Hoffmann & Pedersen, 1998).
In the present study, the protein kinase inhibitors staurosporine and bis-indolyl-maleimide I were used to test for a role of PKC in the hypertonicity-induced stimulation of the Na+ importers mediating RVI. It was found that, in rat hepatocytes, the hypertonic activation of both Na+ conductance and Na+-K+-2Cl- symport - but not Na+-H+ antiport - probably occurs via PKC.
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METHODS |
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Primary culture of hepatocytes
Isolation and primary culture of hepatocytes were the same as described previously (Wehner & Guth, 1991; Wehner et al. 1992, 1995). Briefly, after heparinization male Wistar rats (210-280 g body weight) were exsanguinated under urethane anaesthesia by in situ perfusion of the liver with nominally Ca2+-free Krebs-Henseleit solution (approved by Regierungspräsident Arnsberg and the Institute Animal Care Committee). After removal, the liver was perfused for 20 min with 0.05 % collagenase A that was dissolved in the same buffer. Following isolation, cells were plated onto collagen-coated gas-permeable dishes (Petriperm; Sartorius, Göttingen, Germany) and cultured in Dulbecco's modified Eagle's medium 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 % CO2-95 % air. Cells formed confluent monolayers within 24 h and were used from days 1 to 3 after preparation except for cable-analysis experiments (see below) that were solely performed at day 2 to limit time-dependent changes of electrical cell-to-cell coupling (cf. Wehner & Guth, 1991).
Electrophysiology
The electrophysiological set-up and recording techniques have been described in detail previously (Wehner & Guth, 1991; Wehner et al. 1993, 1995). Briefly, in the cable analysis, a cell was impaled with a two-channel microelectrode pulled from a 1.5 mm o.d. thick septum-theta glass capillaries (WPI, Berlin, Germany) on a horizontal puller (DMZ-Universal Puller; Zeitz-Instrumente, München, Germany) to give a resistance of 80-130 M
when filled with 0.5 mol l-1 KCl. One channel was used to measure voltage, the second to inject constant-current pulses. The changes of membrane voltage resulting from these pulses were recorded with a single-channel electrode in a second cell at 35, 100, 200 and 400 µm from the point of current injection in four separate experiments on different sheets of cell monolayers obtained from a single culture dish. Single-channel electrodes were pulled from inner-fibre capillaries of 1.5 mm o.d. (Hilgenberg, Malsfeld, Germany) and had a resistance of 70-100 M. The voltage deflections in the second cell (
V2) were plotted against the distance between the current injecting and the voltage sensing electrodes (x) and data were fitted by:
V2 = A Ko (x/ ),
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according to Frömter (1972), where Ko is a zero-order Bessel function and A and are constants defining the function. From these constants, cell-to-cell coupling resistance (Rx) and specific membrane resistance (Rz) can be calculated according to:
Rx = (2  A)/io
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and
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Rz = Rx 2,
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respectively, where io is the total current applied (Frömter, 1972; see also Wehner et al. 1992; Wehner & Tinel, 1998).
In the ion-substitution experiments, Na+ was rapidly exchanged for choline by use of an 8-way valve (Valco, Schenkon, Switzerland) close to the experimental chamber and the resultant effects on membrane voltage and input resistance were determined with two-channel microelectrodes. At a superfusion rate of 4 ml min-1 and with a total fluid volume of 0.1 ml above the cells, changes of experimental solutions were achieved within seconds (Wehner & Guth, 1991; Wehner et al. 1992, 1995).
Determination of cell pH
Intracellular pH was monitored as described in earlier reports from this laboratory (Wehner et al. 1995; Wehner & Tinel, 1998, 2000). In brief, experiments were performed on a standard microscope (Diaphot; Nikon, Düsseldorf, Germany) with a 
20 lens that was equipped with a confocal laser-scanning device (MRC-600; BioRad, Hemel Hempstead, UK). Fluorescence was excited by an argon-ion laser (Ion Laser Technology, Salt Lake City, UT, USA) and a helium-cadmium laser (4310N; Liconix, Santa Clara, CA, USA) yielding bands of 488 and 442 nm, respectively. Cells were loaded with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM; Molecular Probes, OR, USA) 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 of 6.4-8.2 (Thomas et al. 1979) bracketing the pH range under consideration here.
Determination of cell volumes
Changes of cell volumes were quantified by use of the 488 nm band of the confocal laser-scanning microscope (see above) and the fluorescent dye calcein as described previously (Wehner & Tinel, 1998, 2000). Briefly, cells were loaded for 45 min with calcein-AM (Molecular Probes) at a final concentration of 10 µmol l-1 and washed subsequently for 5 min in dye-free solution. Calcein is a volume marker of aqueous compartments and exhibits a high degree of fluorescence self-quenching (Kendall & Macdonald, 1983) so that fluorescence decreases when the compound becomes concentrated and increases with its dilution. With the calcein technique, changes of cell volumes can be quantified by the determination of fluorescence in a single confocal plane. The reliability of this technique has been studied extensively (Wehner et al. 1995; Tinel et al. 1996; Wehner & Tinel, 1998, 2000).
Quantification of cell Na+
For the determination of cell Na+, hepatocytes were loaded with 15 µmol l-1 sodium-binding benzofuran isophthalate in its acetoxy-methyl-ester form (SBFI-AM, Molecular Probes) as previously described (Wehner & Tinel, 2000). SBFI fluorescence was measured on an inverted microscope with a 40 oil-immersion objective (Zeiss, Oberkochen, Germany) that was coupled to a ratio-imaging system (PTI, Wedel, Germany). Fluorescence emission at excitation wavelengths of 340 and 375 nm were recorded by an ICCD camera (PTI) and a 510 nm wide-band filter (Omega Optical, Brattleboro, VT, USA). The fluorescence signal was calibrated in high Na+ (135 mmol l-1) and Na+-free solutions with gramicidin (10 µg ml-1) and monensin (40 µg ml-1) as the cationophores (Harootunian et al. 1989; see also Wehner & Tinel, 2000).
Measurements of 86Rb+ uptake
Rb+ uptakes were determined as described previously (Wehner et al. 1995; Wehner & Tinel, 1998, 2000). Briefly, circular sheets of confluent monolayers were cut from the bottom of the culture dishes, washed and transferred to 20 ml standard scintillation vials filled with 5 ml of experimental solutions that were gassed with humidified O2 and kept at 36 °C. Solutions were labelled with 1-5 µCi ml-1 86Rb+, and Rb+ uptake was determined after 2, 4, 6 and 8 min of incubation. For determination of Rb+ uptake via Na+/K+-ATPase, experiments were performed in the absence and presence of 2 mmol l-1 ouabain. Rb+ uptake via Na+-K+-2Cl- symport was quantified from the difference in transport rates in the absence and presence of 100 µmol l-1 furosemide. After termination of uptake and cell lysis, aliquots were sampled for liquid scintillation counting and determination of protein content.
Solutions
The normosmotic control solution of 300 mosmol l-1 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 7.4. Osmolarity was increased by addition of 100 mmol l-1 sucrose. Experimental solutions were gassed with humidified O2 and kept at 36.0 ± 0.5 °C. All chemicals were of the highest grade available. Staurosporine and bis-indolyl-maleimide I were purchased from Calbiochem (Bad Soden, Germany).
Statistical analysis
Means ± S.E.M. are presented. In the fluorescence measurements, n denotes the number of cells analysed; in the electrophysiological measurements and in the 86Rb+ uptake studies, n gives the number of monolayers tested. In every set of experiments, measurements were performed on cells/monolayers from at least three different preparations. Student's t tests for paired and unpaired data were applied as appropriate. A value of P < 0.05 was considered significant.
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RESULTS |
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In the first series of measurements, the effects of staurosporine and bis-indolyl-maleimide I on cell volume regulation were quantified. Furosemide (100 µmol l-1) was present throughout these experiments (i.e. Na+-K+- 2Cl- symport was blocked) to establish conditions that are similar to those used for the quantification of cell pH, cell Na+, as well as for the determination of Na+/K+-ATPase activity (and thus for the first differential approach to monitor Na+ conductance; see below and Appendix). In the absence of kinase inhibitors, hypertonic stress led to a rapid decrease of cell volumes to 87.5 ± 0.9 % of control values. This passive behaviour was then followed by a partial volume recovery to 91.3 ± 0.8 % of control within 10 min, equivalent to an RVI value of 30.9 ± 4.5 % when referred to the initial extent of cell shrinkage (n = 22; Fig. 1; cf. Wehner & Tinel, 1998). With both 100 nmol l-1 staurosporine as well as 400 nmol l-1 bis-indolyl-maleimide I, RVI was no longer detectable. Rather, the initial rapid period of cell shrinkage was followed by a slowly developing continuous further decline of cell volumes (see Fig. 1) leading to numerically negative RVI values of -13.6 ± 9.9 % (n = 21) and -19.1 ± 5.5 % (n = 35), respectively, both significantly different from the (furosemide) control with P < 0.001. This behaviour is comparable to results obtained from the same preparation under conditions where all Na+ import pathways involved in RVI were blocked, i.e. in the presence of 10-3 mol l-1 amiloride plus 10-4 mol l-1 furosemide, namely -16.0 ± 1.9 % (cf. Wehner & Tinel, 1998; see Discussion).
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Figure 1. Hypertonicity-induced changes of cell volume Experiments performed under control conditions (CONT; n = 22) and in the presence of 100 nmol l-1 staurosporine (STAU; n = 21) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 35) are shown. See text for further explanations. | ||
We then tested for possible effects of PKC inhibition on the hypertonicity-induced activation of Na+-H+ antiport. All measurements were performed in the continuous presence of 100 µmol l-1 furosemide. Under these control conditions, hypertonic stress (300 400 mosmol l-1) increased cell pH from 7.04 ± 0.04 to 7.47 ± 0.04, i.e. by 0.43 ± 0.03 units (n = 88; P < 0.001; Fig. 2). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, hypertonicity-induced cell alkalinizations were 0.34 ± 0.04 (from 7.07 ± 0.02 to 7.42 ± 0.05; n = 64; P < 0.001) and 0.42 ± 0.04 (from 7.12 ± 0.03 to 7.54 ± 0.03; n = 54; P < 0.001), respectively (Fig. 2). Neither isotonic pH values nor these cell alkalinizations were significantly different among the three experimental groups tested. Likewise, the maximal rates of cell alkalinization (see Fig. 2) which are a measure of the activity of Na+-H+ antiport were 0.088 ± 0.009 and 0.096 ± 0.007 pH units min-1 in the presence of staurosporine and bis-indolyl-maleimide I, respectively. These data are not significantly different from those determined under control conditions, where the maximal change of cell pH amounted to 0.099 ± 0.006 pH units min-1. Taken together these results strongly imply that protein kinase C is not involved in the hypertonicity-induced activation of Na+-H+ antiport. Moreover, the absence of any detectable effect of the rather non-specific kinase inhibitor staurosporine on cell alkalinization renders the contribution of a variety of other protein kinases rather unlikely, including CaM kinase, myosin light chain kinase, protein kinase A, and protein kinase G. From the above increases of cell pH and from the known intracellular pH buffering capacity that equals 25.6 mmol l-1 under these conditions (Wehner & Tinel, 1998) overall H+ extrusion rates via Na+-H+ antiport of 10.9 ± 0.7, 8.8 ± 1.0 and 10.8 ± 0.9 mmol l-1 (10 min)-1 can be computed for control experiments and for the recordings with staurosporine and bis-indolyl-maleimide I, respectively. Because of the known stoichiometry of Na+-H+ antiport of 1 : 1, these data are equivalent to the same amount of Na+ entering the cells under hypertonic conditions.
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Figure 2. Effects of hypertonic stress on the intracellular pH of rat hepatocytes (pHi) Measurements were performed under control conditions (CONT; n = 88) and in the continuous presence of 100 nmol l-1 staurosporine (STAU; n = 64) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 54). The straight lines represent the maximal rates of pH changes that were calculated from the first three data points obtained in 400 mosmol l-1. Under each experimental condition, 100 µmol l-1 furosemide was present. See text for further explanations. | ||
In the next set of experiments, we assessed a possible role of PKC in the hypertonic activation of rat hepatocyte Na+ conductance. In the first (differential) approach, the effects of staurosporine and bis-indolyl-maleimide I on the osmotically induced overall increases of cell Na+ were determined and data were then corrected for Na+ import via Na+-H+ antiport (see above) and Na+ export via Na+/K+-ATPase (see below and Appendix). All experiments of this series were performed in the continuous presence of 100 µmol l-1 furosemide to block Na+-K+-2Cl- symport. Under control conditions, hypertonic stress increased intracellular Na+ concentrations from 14.8 ± 0.3 to 28.3 ± 0.8 mmol l-1, i.e. by 13.5 ± 0.5 mmol l-1 (n = 98; P < 0.001; Fig. 3). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, hypertonicity-induced increases of cell Na+ concentration were 11.3 ± 0.5 mmol l-1 (from 14.0 ± 0.4 to 25.3 ± 0.8 mmol l-1; n = 71; P < 0.001) and 10.2 ± 0.4 mmol l-1 (from 14.4 ± 0.4 to 24.6 ± 0.6 mmol l-1; n = 74; P < 0.001), respectively. These increases of cell Na+ concentrations are significantly different from the control experiments with P < 0.01 and P < 0.001. Likewise, the maximal rate at which cell Na+ concentration was increasing was 1.89 ± 0.11 mmol l-1 min-1 under control conditions; in the presence of staurosporine and bis-indolyl-maleimide I, these rates were significantly decreased to 1.29 ± 0.07 and 1.35 ± 0.06 mmol l-1 min-1, respectively (with P < 0.001 for both; see Fig. 3). With an activity coefficient for Na+ of 0.77 (Horisberger & Giebisch, 1988), the above changes of intracellular Na+ concentrations are equivalent to changes of cell Na+ activities by 10.4 ± 0.4, 8.7 ± 0.4 and 7.9 ± 0.3 mmol l-1, respectively.
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Figure 3. Effects of hypertonic stress on intracellular Na+ concentrations (Na Experiments were performed under control conditions (CONT; n = 98) and in the presence of 100 nmol l-1 staurosporine (STAU; n = 71) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 74). The straight lines represent the maximal rates of changes calculated from the last data point in 300 mosmol l-1 and the first four points in 400 mosmol l-1. Furosemide (100 µmol l-1) was present throughout the experiments. | ||
The rates of Na+ export via Na+/K+-ATPase were quantified on the basis of ouabain-sensitive 86Rb+ uptake (Wehner et al. 1995; Wehner & Tinel, 1998, 2000). In the control experiments (with furosemide present), ouabain-sensitive 86Rb+ uptake was 3.89 ± 0.56 (i.e. 4.16 ± 0.57 minus 0.27 ± 0.08 in the absence and presence of ouabain) and 5.83 ± 0.75 (i.e. 6.02 ± 0.78 minus 0.20 ± 0.04) nmol (mg protein)-1 min-1 in 300 and 400 mosmol l-1, respectively, equivalent to a hypertonicity-induced increase of this parameter by 1.94 ± 0.40 nmol (mg protein)-1 min-1 (n = 10; P < 0.001; Fig. 4). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, this stimulation of ouabain-sensitive 86Rb+ uptake was completely absent and was 0.06 ± 0.50 and -0.13 ± 0.53 nmol (mg protein)-1 min-1, respectively (4.29 ± 0.39 minus 4.23 ± 0.49 nmol (mg protein)-1 min-1 and 3.17 ± 0.50 minus 3.30 ± 0.43 nmol (mg protein)-1 min-1; n = 5 and P < 0.05 for each). From these data, from the known stoichiometry of Na+/K+-ATPase of 3Na+/2K+, from the ratio of 4 105 cells (mg protein)-1 (Blitzer et al. 1982), from the average cell volume of 5.9 pl (Wehner et al. 1995), and from the osmotically active space of some 69 % of this volume (Wehner & Tinel, 1998, 2000), osmotically induced Na+ export rates of 24.6 ± 5.0, 0.7 ± 6.4 and -1.7 ± 6.7 mmol l-1 (10 min)-1 can be computed for control conditions and for the experiments with staurosporine and bis-indolyl-maleimide I, respectively.
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Figure 4. Quantification of Na+/K+-ATPase activity Hypertonicity-induced changes of ouabain-sensitive 86Rb+ uptake (400 minus 300 mosmol l-1) were determined under control conditions (CONT; n = 10) and in the presence of 100 nmol l-1 staurosporine (STAU; n = 5) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 5). | ||
When corrected for the above rates of Na+ export (as well as for the passive increases of intracellular Na+ caused by cell shrinkage that were calculated to amount to 1.3, 1.4 and 1.4 mmol l-1; cf. Fig. 1), hypertonicity-induced increases of cell Na+ (which under these experimental conditions are supposed to be mediated solely via Na+ conductance and Na+-H+ antiport) were 33.7 ± 0.4 mmol l-1 (10 min)-1 in the control experiments (Fig. 5). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, the increases of cell Na+ were significantly reduced to 8.0 ± 0.4 and 4.8 ± 0.3 mmol l-1 (10 min)-1, respectively (with P < 0.001 for each). After subtraction of the amounts of Na+ uptake via Na+-H+ antiport, hypertonicity-induced Na+ import (via Na+ conductance) under the above experimental conditions was 22.8 ± 0.4, -0.8 ± 0.4 and -6.0 ± 0.3 mmol l-1 (10 min)-1 (Fig. 5). These data strongly suggest that the hypertonic activation of Na+ conductance in rat hepatocytes is mediated by PKC.
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Figure 5. Hypertonicity-induced Na+ import Data obtained under control conditions (CONT) and in the presence of 100 nmol l-1 staurosporine (STAU) or 400 nmol l-1 bis-indolyl-maleimide I (BIM) are depicted. Furosemide (100 µmol l-1) was present throughout the experiments and data were corrected for the amounts of Na+ extrusion via Na+/K+-ATPase (see Fig. 3). Left, Na+ import via Na+ conductance plus Na+-H+ antiport. Right, Na+ import via Na+ conductance (total import minus import via Na+-H+ antiport; cf. Fig. 1). | ||
To obtain additional evidence for the role of PKC in the activation of Na+ conductance cable-analysis experiments were performed. This method is a quantitative and reliable tool for the analysis of specific membrane conductances in confluent monolayer cultures (Wehner et al. 1993, 1995; Wehner & Tinel, 1998, 2000), i.e. under conditions where cells are highly coupled electrically (Wehner & Guth, 1991). Experiments were performed in the continuous presence of 0.5 mmol l-1 quinine to exclude effects of hypertonic stress on K+ conductance (Wehner & Tinel 1998, 2000) so that the decreases of membrane resistance under hypertonic stress reflect the actual increases of cell membrane Na+ conductance (Wehner et al. 1995; Wehner & Tinel, 1998, 2000). Under these conditions and in the absence of kinase inhibitors, hypertonic stress led to a rapid decrease of specific membrane resistance that, in the range of 2 to 10 min in 400 mosmol l-1, amounted to 83.9 ± 1.0 % of the control value (of 9.9 ± 1.9 k cm2; n = 4) on average (Fig. 6). In the presence of 100 nmol l-1 staurosporine (n = 3) and 400 nmol l-1 bis-indolyl-maleimide I (n = 4), this membrane response was completely blocked and specific membrane resistances were 108.6 ± 0.9 and 100.4 ± 0.4 % of isotonic conditions, respectively; for both compounds, this was significantly different from the control measurements with P < 0.001. These data provide strong further evidence for a significant role of PKC in the hypertonic activation of rat hepatocyte Na+ conductance.
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Figure 6. Cable analysis of the hypertonic activation of Na+ conductance The effects of hypertonic stress on specific membrane resistance were determined under control conditions (CONT; n = 4) and in the presence of 100 nmol l-1 staurosporine (STAU; n = 3) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 4). Quinine (0.5 mmol l-1) was present throughout the experiments. See text for details. | ||
As the third line of evidence, changes in cell membrane Na+ conductance were monitored in ion-substitution experiments with low Na+ pulses. Due to the presence of Na+-H+ antiport and the pH dependence of K+ conductance (Henderson et al. 1987; Fitz et al. 1989) a rapid substitution of extracellular Na+, in primary cultures of rat hepatocytes, leads to a slowly developing depolarization that masks the hyperpolarization of membrane voltage via Na+ conductance (Wehner & Guth, 1991; Wehner, 1993; Wehner et al. 1995). Nevertheless, increases in Na+ conductance become readily detectable as a negative shift in the voltage response to low Na+ conditions (Wehner, 1993; Wehner et al. 1995; Wehner & Tinel, 1998). A typical recording exemplifying the experimental protocol used is depicted in Fig. 7A. As is obvious from the figure, hypertonic stress led to a significant shift in the voltage response to low Na+ that was most readily detectable at 20 s after ion substitution and that amounted to -4.0 ± 0.3 mV (n = 24; P < 0.001; Fig. 7B; cf. Wehner et al. 1995; Wehner & Tinel, 1998, 2000). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, this effect was significantly reduced to -1.7 ± 0.5 mV (n = 7; P < 0.001) and -1.5 ± 0.6 mV (n = 7; P < 0.001), respectively. Taken together, three independent lines of experimental evidence strongly suggest that the hypertonic activation of rat hepatocyte Na+ conductance is mediated by PKC.
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Figure 7. Monitoring hypertonic activation of Na+ conductance by means of low Na+ pulses A, effects of hypertonic stress on membrane voltage and on the voltage response to low Na+ solutions, which is most pronounced at 20 s after ion substitution (arrows). Representative experiment. See text for details. B, hypertonicity-induced negative shifts (400 minus 300 mosmol l-1) of the voltage response to low Na+ solutions ( | ||
In the last series of measurements, we assessed the potential role of PKC in the activation of Na+-K+-2Cl- symport. To this end, the rates of this transporter were determined on the basis of furosemide-sensitive 86Rb+ uptake. All experiments were performed in the continuous presence of 2 mmol l-1 ouabain to increase the resolution of the assay (Wehner & Tinel, 1998, 2000). Under these control conditions, furosemide-sensitive 86Rb+ uptake under isotonic conditions and in 400 mosmol l-1 was 0.14 ± 0.08 and 1.20 ± 0.19 nmol (mg protein)-1 min-1, respectively, yielding a hypertonicity-induced increase of this transport by 1.05 ± 0.20 nmol (mg protein)-1 min-1 (n = 10; P < 0.001; Fig. 8). In the presence of 100 nmol l-1 staurosporine and 400 nmol l-1 bis-indolyl-maleimide I, this stimulatory effect was significantly reduced to 0.33 ± 0.21 and 0.35 ± 0.08 nmol l-1 (mg protein)-1 min-1, respectively (n = 5 and P < 0.05 for each). Together, these results clearly show that the hypertonic activation of Na+-K+-2Cl- symport in rat hepatocytes is mediated by PKC.
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Figure 8. Quantification of Na+-K+-2Cl- symport activity Hypertonicity-induced changes of furosemide-sensitive 86Rb+ uptake (400 minus 300 mosmol) were determined under control conditions (CONT; n = 10) and in the presence of 100 nmol l-1 staurosporine (STAU; n = 5) or 400 nmol l-1 bis-indolyl-maleimide I (BIM; n = 5). | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
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In the present study, the role of PKC in the hypertonic activation of rat hepatocyte Na+ conductance, Na+- K+-2Cl- symport, and Na+-H+ antiport was analysed. Block of PKC was achieved by use of (the rather unspecific kinase inhibitor) staurosporine and (the highly specific inhibitor) bis-indolyl-maleimide I (cf. Toullec et al. 1991; Jarvis et al. 1994; Nishimura & Simpson, 1994). Of note, with every experimental approach used, both blockers yielded virtually identical results.
By use of three independent experimental protocols we clearly showed that the hypertonic activation of rat hepatocyte Na+ conductance is, in fact, mediated by PKC: (i) the differential approach where the overall changes of cell Na+ were determined and corrected for the contributions of Na+-H+ antiport and Na+/K+-ATPase so that (with Na+-K+-2Cl- symport blocked) this increase of cell Na+ is very likely to reflect the actual activation of Na+ conductance, (ii) the analysis of specific membrane resistance by means of cable analysis which yields a direct and quantitative measure of changes of Na+ conductance (Wehner et al. 1995; Wehner & Tinel, 1998, 2000), and (iii) the semi-quantitative analysis of changes of Na+ conductance by use of low Na+ pulses (Wehner et al. 1995; Böhmer et al. 2000). All three procedures demonstrated a significant block of the hypertonicity-induced activation of Na+ conductance by both staurosporine and bis-indolyl-maleimide I.
In a recent report from this laboratory, it was shown that rat hepatocytes express all three subunits of the epithelial Na+ channel (
-, 
- and 
-rENaC) (Böhmer et al. 2000). Moreover, -rENaC was found to be a functional component of the hypertonicity-induced Na+ conductance of rat hepatocytes (Böhmer & Wehner, 2001). At first sight, together with these findings the distinct role of PKC in the hypertonic stimulation of rat hepatocyte Na+ conductance reported here appears to be contradictory to a recent study on Xenopus oocytes heterologously expressing hENaC and rENaC (Awayda, 2000) where PKC (stimulated by phorbol-myristate-acetate, PMA) appeared to (unspecifically) inhibit ENaC trafficking and, in addition, to (specifically) inhibit the activity of both ENaC isoforms once inserted into the membrane. Likewise, in the same expression system, staurosporine as well as chelerythrine (a selective inhibitor of PKC) were found to block hENaC currents, whereas PMA was found to increase currents in some but not all oocytes (Volk et al. 2000). It should be taken into account, however, that on one hand, the Xenopus oocyte while commonly being used as an expression system may differ considerably from a native preparation with respect to its intracellular signalling cascades (e.g. to the PKC isoforms employed). The role of PKC in the hypertonic activation of rat hepatocyte Na+ conductance, in contrast, was studied in the native system. On the other hand, we cannot tell at present about an actual contribution of - and -rENaC to the Na+ channel in rat hepatocytes and, likewise, possible accessory proteins cannot be excluded that may well modify the channel's properties. Of note in this respect, the hypertonicity-induced Na+ conductance of rat hepatocytes exhibits a rather low affinity to amiloride (Wehner et al. 1997b; Wehner & Tinel, 1998), a sensitivity profile of EIPA > amiloride > benzamil = phenamil (Wehner et al. 1997b; Böhmer et al. 2000), and a PNa/PK of approximately 1.4 only (Böhmer & Wehner, 2001), which is distinct from the properties of -, - and -rENaC, at least under isotonic conditions (cf. Böhmer et al. 2000). Nevertheless, the results given in the present study clearly indicate that, in rat hepatocytes, PKC mediates the hypertonic activation of Na+ conductance.
As could be shown on the basis of furosemide-sensitive 86Rb+ uptake, PKC plays a significant role in the hypertonicity-induced activation of Na+-K+-2Cl- symport in rat hepatocytes as well. This finding is similar to reports from a variety (but not all) of the systems studied so far; from some preparations significant contributions of other kinases (e.g. Ca2+/calmodulin-dependent kinase and myosin light chain kinase, but not protein kinase A) to the hypertonic activation of the transporter have been reported (see Hoffmann & Pedersen, 1998, for review). Our results also prove the hypothesis of a common mechanism of intracellular signalling for Na+-K+-2Cl- symport and Na+ conductance in rat hepatocytes that was based on the parallel activation patterns of these transporters as a function of hypertonic stress (Wehner & Tinel, 2000). As a matter of fact, with nothing known so far about the signalling mechanisms involved in the hypertonicity-induced activation of Na+ conductance but with a prominent role of PKC in the hypertonic activation of Na+-K+-2Cl- symport in various systems (see above), these parallel activation patterns let us to propose PKC as the most likely candidate for the activation of conductive Na+ entry in hepatocytes as well.
In some systems such as Ehrlich cells (Pedersen et al. 1996), Na+-H+ antiport appears to be regulated by PKC, but in a variety of other preparations it clearly is not (see Hoffmann & Pedersen, 1998, for review). Our results clearly show that, in rat hepatocytes, PKC is not involved in the hypertonicity-induced activation of Na+-H+ antiport. Likewise, the activation pattern of this transporter is significantly different from those of Na+ conductance and Na+-K+-2Cl- symport (Wehner & Tinel, 2000). Given the high efficiency of conductive Na+ entry in the RVI of rat hepatocytes (Wehner & Tinel, 1998), the question remains as to why these cells employ a pH-regulatory mechanism (the activation of which leads to significant changes of one of the most delicately regulated intracellular parameters) as an additional Na+ import pathway in this process (cf. Wehner & Tinel, 2000). Because Na+-H+ antiport is clearly the most sensitive (but low capacitance) Na+ import mechanism involved in RVI (Wehner & Tinel, 2000), however, it may be part of the signalling cascade employed further upstream in the co-ordination of this process. Interestingly, in a recent study on rat cardiac myocytes, it became evident that Na+ influx via stimulation of Na+-H+ antiport activates the 
- and 
-isoforms of PKC, with both enzymes being involved in the hypertrophic response of these cells (Hayasaki-Kajiwara et al. 1999). Whereas the possible signal transduced by Na+-H+ antiport in rat hepatocytes is clearly not cell alkalinization (Wehner et al. 1997a), an increase in cell Na+ as a possible triggering mechanism has not yet been tested.
Given the high efficiency of staurosporine and bis-indolyl-maleimide I in blocking the activation of both Na+ conductance and Na+-K+-2Cl- symport, significant effects of these compounds on cell volume regulation were to be expected. In fact, inhibition of PKC led to a distinct inhibition of RVI that was comparable to the one observed in the presence of 10-3 mol l-1 amiloride plus 10-4 mol l-1 furosemide (Wehner & Tinel, 1998, 2000), i.e. under conditions where all Na+ import mechanisms involved in the RVI process are blocked. In addition, Na+/K+-ATPase in rat hepatocytes could be shown to be stimulated by diacylglycerol (DAG) as well as by the tumour promotor PMA (Lynch et al. 1986), both activators of the c- and n-isoforms of PKC (see Liu & Heckman, 1998, for review). In line with these observations, in the present study, staurosporine and bis-indolyl-maleimide I reduced the hypertonicity-induced increase of ouabain-sensitive and time-dependent 86Rb+ uptake to virtually zero (Fig. 4). Given the known activity of rat hepatocyte Na+/K+-ATPase as a function of cell Na+ (van Dyke & Scharschmidt, 1983; Wehner & Tinel, 2000) and taking into account the ongoing activity of Na+-H+ antiport as well as the only partial inhibition of Na+-K+-2Cl- symport by these compounds, this effect is too pronounced to be solely explained in terms of a complete block of the hypertonicity-induced increase of cell Na+ (see also Fig. 5).
In conclusion, in rat hepatocytes, the hypertonicity-induced activation of Na+ conductance and Na+-K+- 2Cl- symport - but not Na+-H+ antiport - is mediated by PKC. The actual isoforms of PKC involved in this process, the signalling mechanisms further upstream of PKC, as well as the regulators of Na+-H+ antiport remain to be defined.
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APPENDIX |
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TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
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In the differential quantification of Na+ conductance it is assumed that Na+ import under hypertonic conditions occurs via Na+ conductance, Na+-H+ antiport, and Na+-K+-2Cl- symport; Na+ is then to a large extent exchanged for K+ via activation of Na+/K+-ATPase (Wehner & Tinel, 1998, 2000). With Na+-K+-2Cl- symport blocked by furosemide, conductive Na+ entry can be determined as total Na+ entry minus Na+ import via Na+-H+ antiport plus Na+ export via Na+/K+-ATPase.
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REFERENCES |
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Acknowledgements
We wish to thank Dr R. K. H. Kinne for helpful discussion and for his continuous support of the project. The critical reading of the manuscript by Dr P. Lawonn, Mrs K. Bierhals and Mrs H. Olsen is also gratefully acknowledged. This work would not have been possible without the excellent technical assistance of Mrs G. Beetz and Mr A. Giffey.
Corresponding author
F. Wehner: Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Postfach 500247, 44202 Dortmund, Germany.
Email: frank.wehner{at}mpi-dortmund.mpg.de
Authors' present address
H. Heinzinger and F. van den Boom: Institut für Physiologische Chemie, Abteilung Biochemie Supramolekularer Systeme, Medizinische Fakultät, Ruhr-Universität, Bochum, Germany.
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)
Significantly different from control with P < 0.05. Furosemide (100 µmol l-1) was present throughout the experiments.