Uptake of bromosulfophthalein via SO2minus 4/OHminus exchange increases the K+ conductance of rat hepatocytes

Frank Wehner and Hanna Tinel

Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, 44139 Dortmund, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In confluent primary cultures of rat hepatocytes, micromolar concentrations of bromosulfophthalein (BSP) lead to a sizeable hyperpolarization of membrane voltage. The effect is a saturable function of BSP concentration yielding an apparent value of 226 µmol/l and a Vmax of -10.3 mV. The BSP-induced membrane hyperpolarization is inhibited by the K+ channel blocker Ba2+, and in cable-analysis and ion-substitution experiments it becomes evident that the effect is due to a significant increase in cell membrane K+ conductance. Voltage changes were attenuated by the simultaneous administration of SO2-4, succinate, and cholate (cis-inhibition) and increased after preincubation with SO2-4 and succinate (trans-stimulation), suggesting that the effect occurs via BSP uptake through the known SO2-4/OH- exchanger. Microfluorometric measurements reveal that BSP-induced activation of K+ conductance is not mediated by changes in cell pH, cell Ca2+, or cell volume. It is concluded that K+ channel activation by BSP (as well as by DIDS and indocyanine green) may reflect a physiological mechanism linking the sinusoidal uptake of certain anions to their electrogenic canalicular secretion.

liver; anion transport; membrane voltage; cross-talk


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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THE LIVER EXTRACTS a variety of organic anions from portal blood by means of carrier-mediated processes residing in the sinusoidal membrane of hepatocytes. Among these carriers are the Na+-taurocholate cotransporting polypeptide (Ntcp), mediating Na+-dependent transport of conjugated bile salts and, to a smaller extent, of unconjugated bile salts such as cholate (19, 38), and the Na+-independent organic anion transporting polypeptide (oatp1) (23). The latter transporter mediates Na+-independent uptake of cholate and taurocholate as well as of a variety of structurally unrelated substrates, such as bromosulfophthalein (BSP, anionic), used experimentally as a water-soluble substrate of bilirubin transport, ouabain (neutral), and N-(4,4-azo-n-pentyl)-21-ajmalinium (cationic) (6, 23). In addition to oatp1, a variety of other BSP transporters and binding proteins have been described, namely, the bilitranslocase (29, 39, 45), the BSP/bilirubin binding protein (BBBP) (40, 45), and the organic anion binding protein (OABP) (54). Moreover, and most relevant for this study, a low-affinity but high-capacity SO2-4/OH- exchange system has been reported for the sinusoidal membrane of rat hepatocytes that significantly interferes with BSP as well as with the cholephilic drug DIDS (21, 30).

The canalicular membrane voltage of approximately +25 mV when referred to the cytosol (48, 50, 51) was formerly considered the decisive driving force for electrogenic anion secretion into the biliary tract (22, 31, 36). In recent years it has become evident, however, that this voltage is too small to fully account for the observed canalicular concentration of most anionic compounds. Instead, canalicular anion secretion now appears to be achieved to a large extent by a variety of primary active transport processes, including the canalicular bile acid transporter (cBAT) (1, 5, 7), which appears to be identical to the recently cloned sister gene of P-glycoprotein (spgp) (8, 14), and the canalicular multispecific organic anion transporter (cMoat/cMrp/Mrp2) (see Refs. 28 and 33 for review).

It is noteworthy in this respect that, in highly purified canalicular membrane vesicles from rat liver, taurocholate uptake was ATP dependent but not electrogenic, whereas electrogenic but ATP-independent taurocholate transport appeared to reside in the endoplasmic reticulum fraction, frequently contaminating canalicular membrane preparations (26). This finding is in sharp contrast to earlier studies demonstrating voltage-sensitive canalicular bile acid secretion in hepatocyte couplets (53) as well as in the intact liver (46). In bile canalicular membrane vesicles prepared from Mrp2-deficient GY/TR- mutant rats, ATP-dependent uptake of bilirubin glucuronides is not detectable (25), whereas in vivo the biliary output of bilirubin and its conjugates is decreased to only 40% of normal (24). In canalicular membrane vesicles from normal rats, two distinct mechanisms for bilirubin glucuronide transport were identified, one ATP dependent and the other voltage dependent, and the effects of ATP and voltage on transport were additive; in the TR- rat, the ATP-dependent system was absent but voltage-dependent transport was retained (32). This dual dependence of bilirubin glucuronide transport on ATP and voltage, however, does not appear to reflect a voltage dependence of Mrp2 itself (25). Instead, the above results may be interpreted in terms of a yet unidentified low-affinity transport system that is likely to be driven by membrane voltage (25; see also Ref. 33 for review). Taken together, these reports suggest that both ATP- and voltage-dependent transport processes participate in canalicular anion secretion. Besides this differentiation of transporters and driving forces, it is worth mentioning that any form of net anion secretion will, of course, be facilitated by a positive canalicular membrane voltage.

In an earlier study from this laboratory, it was reported that micromolar concentrations of DIDS lead to a considerable hyperpolarization of rat hepatocyte membrane voltage (50). The effect was due to a significant increase in K+ conductance, and, interestingly, it was attenuated in the presence of BSP and was clearly related to the uptake of DIDS. Because DIDS is effectively secreted into bile (2), this raises the question of whether another cholephilic substance, namely BSP, may exert effects comparable to those of DIDS. In addition, it was of interest to further characterize the transport pathway mediating the hyperpolarization of hepatocyte membrane voltage. In the present study, we report a significant increase of rat hepatocyte K+ conductance by micromolar concentrations of BSP. The effect leads to a sizeable hyperpolarization of membrane voltage, and it is mediated by BSP uptake via SO2-4/OH- exchange. The increase of K+ conductance may reflect a physiological mechanism linking sinusoidal anion uptake to canalicular electrogenic anion secretion.


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INTRODUCTION
METHODS
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Primary culture of hepatocytes. Isolation of hepatocytes was the same as described previously (48, 50). After isolation, cells were plated on collagen-coated gas-permeable Petriperm dishes and cultured in DMEM fortified with 10% fetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin-100 µg/ml streptomycin, 1 µmol/l dexamethasone, 10 nmol/l triiodothyronine/thyroxine (T3/T4), and 5 µg/ml bovine insulin at 37°C in 5% CO2 in air. Cells formed confluent monolayers within 24 h and were used on day 1 and day 2 after preparation, except for cable-analysis experiments (see below), which were exclusively conducted on day 2 to minimize culture time-dependent changes in electrical cell-to-cell coupling (48).

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

Electrophysiological techniques. Experimental setup and recording techniques are described in detail in previous reports from this laboratory (48, 50). Briefly, circular sheets of gas-permeable membranes 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, Oberkochen, Germany). The fluid volume above the tissue was 0.1 ml, and cells were continuously superfused at a rate of 4 ml/min. Changes of the superfusate were performed by means of a four-way valve 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).

Two-channel microelectrodes were pulled from 1.5-mm-OD Thick-Septum-Theta borosilicate glass capillaries (World Precision Instruments, New Haven, CT) on a Kopf vertical puller (750, David Kopf Instruments, Tujunga, CA) and had resistances of 80-130 MOmega when filled with 0.5 mol/l KCl and immersed in control Tyrode solution. One channel was used to measure voltage, and the second was used to inject constant current pulses for determination of input resistances. Cell impalements were performed at an angle of 45° under ×320 magnification by use of piezomanipulators (PM 550-20, Frankenberger, Germering, Germany). Criteria for successful impalements have been previously described (48).

In the cable analysis, current pulses 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-fiber borosilicate glass capillaries of 1.5 mm OD (Hilgenberg, Malsfeld, Germany) and had resistances of 70-80 MOmega . The voltage deflections in the second cell (delta Vm2) were plotted against the distance between both electrodes (x). Data were then fitted by use of the form
&dgr;<IT>V</IT><SUB>m2</SUB> = <IT>AK</IT><SUB>o</SUB> <FENCE> <FR><NU><IT>x</IT></NU><DE>&lgr;</DE></FR> </FENCE> (1)
according to Frömter (12), where Ko is a zero-order Bessel function and A and lambda  are constants defining the function. Each set of lambda  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 (Rx) and specific cell membrane resistance (Rz) can be calculated according to
<IT>R</IT><SUB><IT>x</IT></SUB> =  <FR><NU>2&pgr;<IT>A</IT></NU><DE><IT>i</IT><SUB>o</SUB></DE></FR> (2)
and
<IT>R</IT><SUB><IT>z</IT></SUB> = <IT>R</IT><SUB><IT>x</IT></SUB>&lgr;<SUP>2</SUP> (3)
respectively. In Eq. 2, io is the total current applied.

In all measurements, a custom-made 0.5 mol/l KCl flowing junction in series with an Ag-AgCl wire was used as the extracellular reference electrode to avoid liquid junction potentials.

Measurements of intracellular pH. Intracellular pH was monitored by use of a confocal laser scan unit (MRC-600, Bio-Rad, Hemel Hempstead, UK) coupled to a standard microscope (Diaphot, Nikon, Düsseldorf, Germany) with a ×20 objective (Zeiss). For excitation of fluorescence, the device was equipped with an argon ion laser (Ion Laser Technology, Salt Lake City, UT) and a helium-cadmium laser (4310 N, Liconix, Santa Clara, CA), yielding bands of 488 and 442 nm, respectively. Cells were loaded with the dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF, Molecular Probes, Eugene, OR) in its acetoxymethyl ester (AM) form at a final concentration of ~10 µmol/l for 30 min and subsequently bathed in dye-free solution for at least 15 min before the experiment. Cell monolayers were then 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. Images were acquired every 60 s, and data were digitized and analyzed by use of a microcomputer. A calibration was performed at the end of each experiment in 140 mmol/l KCl and 10 µmol/l nigericin at pH values in the range of 6.4-8.2 in 10 mmol/l HEPES-buffered or Tris-buffered solutions, as described by Thomas et al. (41).

Measurements of intracellular Ca2+. Cell Ca2+ was determined as described in earlier reports from this laboratory (43, 44). Briefly, monolayers were loaded for 45 min with 10 µmol/l fura 2-AM (Molecular Probes), dissolved in dimethyl sulfoxide (final concn 0.1%) and pluronic acid (final concn <0.025%). After loading, the cells were rinsed and incubated in standard Tyrode solution for 15 min.

Fura 2 fluorescence was measured by means of a commercially available system that is based on an inverted microscope (IM 10, Zeiss) equipped with a ×20 objective, a photomultiplier (Hamamatsu), a xenon lamp (Osram, Munich, Germany), and a filter wheel, amplifier, and controller unit (Luigs and Neumann, Ratingen, Germany). The fluorescence was excited at 360 and 390 nm, whereas emission was quantified at 510 nm. Calibration of the fura 2 fluorescence signal was performed following addition of the Ca2+ ionophore ionomycin (10 µmol/l), as described by Grynkiewicz et al. (17). Apparent concentrations of free Ca2+ were calculated from the fluorescence ratio, R, according to
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>eff</SUB>(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)&bgr; (4)
where Keff is the effective dissociation constant, assumed to be 224 nmol/l (17), and Rmin and Rmax are the fluorescence ratios at zero Ca2+ and at high (2 mmol/l) Ca2+, respectively; beta  is the ratio of fluorescence intensities at 390 nm in Ca2+-free and Ca2+-containing solutions.

Dimethyl sulfoxide and pluronic acid were obtained from Molecular Probes; ionomycin was purchased from Sigma (Deisenhofen, Germany).

Determination of cell volumes. Changes in cell volume were quantified by use of the fluorescent dye calcein on the confocal laser scan unit. Calcein is a volume marker of aqueous compartments and exhibits a high degree of fluorescence self-quenching (27), so that fluorescence decreases when concentrated and increases when diluted. 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 (42, 51, 52). Cells were loaded for 45 min with calcein-AM (Molecular Probes) at a final concentration of 10 µmol/l and washed thereafter for ~5 min in dye-free solution.

Solutions. The control Tyrode solution contained (in mmol/l) 124 NaCl, 2.7 KCl, 25 NaHCO3, 0.4 NaH2PO4, 1.8 CaCl2, 1.1 MgCl2, and 5.6 glucose. For low-Na+ and low-Cl- conditions, respectively, 95% of Na+ was replaced by choline and 99% of Cl- was replaced by gluconate. In experiments with high-K+ and low-Cl- pulses, K+ was elevated 10-fold in exchange for Na+, and Cl- was 100-fold reduced in exchange with gluconate. The latter experiments were performed in HCO-3-free solutions where HCO-3 was replaced by Cl- or gluconate (24 mmol/l) and HEPES buffer (1 mmol/l). HCO-3-containing and HCO-3-free solutions were gassed with 5% CO2 in O2 and pure O2, respectively. pH was adjusted to 7.4 by addition of 1 mol/l HCl (HCO-3-containing solutions) or 4 mol/l NaOH (HCO-3-free solutions).

NaCl and KCl were purchased from Baker (Deventer, Netherlands) and HEPES from Serva Chemical. All other substances were obtained from Merck (Darmstadt, Germany).

Statistical analysis. Mean values ± SE are presented, 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-tests for paired and unpaired data were applied as appropriate. A value of P < 0.05 was considered significant.


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As depicted in Fig. 1, addition of 300 µmol/l BSP to the superfusate of rat hepatocytes led to a sizeable hyperpolarization of membrane voltage that equaled -5.7 ± 0.5 mV (n = 20; P < 0.001). It occurred in a monoexponential fashion until the new quasi-steady-state voltage was obtained. Upon removal of BSP, membrane voltage slowly returned to the control value. In addition to this effect, the compound slightly increased cell input resistance from 2.9 ± 0.3 MOmega under control conditions to 3.5 ± 0.3 MOmega after 5-min exposure to BSP (P < 0.001).


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Fig. 1.   Effects of 300 µmol/l bromosulfophthalein (BSP) on membrane voltage (Vm). Voltage deflections result from constant current pulses injected for determination of cell input resistance.

In the tested range of 50-500 µmol/l, the BSP-induced hyperpolarization of membrane voltage was a saturable function of BSP concentration, already suggestive of a transport-related process. Plotting the reciprocal of voltage changes (as -1/delta Vm) vs. the reciprocal of BSP concentrations yielded a straight line with an apparent Km of 226 µmol/l, a Vmax of -10.3 mV, and a correlation coefficient of 0.99 (Fig. 2A).




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Fig. 2.   A: BSP-induced hyperpolarization of membrane voltage (delta Vm) is a saturable function of BSP concentration. Plotted is reciprocal of voltage change (as -1/delta Vm) vs. reciprocal of BSP concentration (500, 300, 200, 100, 67, and 50 µmol/l); n = 6-8 for each data point except for 300 µmol/l BSP (n = 20). B: effects of SO2-4 on BSP-induced membrane hyperpolarization. SO2-4 (25 mmol/l) was either administered at the time when BSP was added (black-diamond ; cis-inhibition), or, after a 10-min incubation period, SO2-4 was removed at time of BSP exposure (; trans-stimulation); n = 4-6 for each data point. For comparison, fit of control data (see A) is given as dashed line. C: same protocols as in B but with 10 mmol/l succinate instead of SO2-4; n = 3-5 for each data point.

The above Km value is comparable to the Ki of BSP that was determined for the SO2-4/OH- exchanger in sinusoidal membrane vesicles of rat liver [i.e., 120 µmol/l (21)]. We therefore tested for a possible cis-inhibition and trans-stimulation of BSP-induced membrane hyperpolarization by SO2-4. For cis-inhibition, 25 mmol/l SO2-4 [Km = 16 mmol/l (21)] was administered at the same time as BSP (and together with 25 mmol/l sucrose, both in exchange for 50 mmol/l gluconate at 82.7 mmol/l constant Cl-). As shown in Fig. 2B, the simultaneous application of SO2-4, in fact, led to a distinct cis-inhibition of BSP-induced voltage changes. To test for trans-stimulation, SO2-4 was first administered during a 10-min preincubation period and was then removed at the time that BSP was added. As is evident from Fig. 2B, this maneuver led to a significant increase of the voltage effects of BSP. Taken together, these results suggest the recruitment of SO2-4/OH- exchange for BSP-induced membrane hyperpolarization as well as a SO2-4/BSP exchange mode of its operation.

We then performed an additional set of experiments with succinate, another substrate of SO2-4/OH- exchange in sinusoidal membrane vesicles (21). As Fig. 2C clearly shows, succinate, like SO2-4, exhibited a pronounced cis-inhibition of the BSP-induced hyperpolarization of membrane voltage, which is comparable in size to that of SO2-4. In addition, preincubation with succinate led to the expected trans-stimulation of membrane hyperpolarization. This latter effect, however, was less pronounced than that of SO2-4, which may be explained in terms of a partial intracellular metabolization of succinate.

In the vesicle system (21), the bile acid cholate exhibited cis-inhibition as well as trans-stimulation of SO2-4/OH- exchange. In contrast, taurocholate did not display any detectable cis-inhibition, although a possible trans-stimulation was not tested (21). In primary cultures of rat hepatocytes, both taurocholate and cholate elicit distinct depolarizations of membrane voltage via activation of cell membrane Na+ conductance (Ref. 47 and Wehner, unpublished results). To test for possible effects of these bile acids on BSP-induced membrane hyperpolarization, we therefore could not administer or remove them at the time when BSP was added and had to follow a modified protocol. Figure 3 summarizes a set of experiments in which either 100 µmol/l cholate or taurocholate was added to the superfusate at 5 min before application of BSP, so that changes in Na+ conductance would be almost complete (47). As is obvious from Fig. 3, in the continuous presence of cholate, BSP-induced voltage changes were significantly reduced, indicative of a sizeable cis-inhibition of transport by this bile acid. This cis-inhibition would of course be falsified by a certain amount of trans-stimulation. In contrast, in the presence of taurocholate, BSP-induced membrane hyperpolarization was considerably increased (Fig. 3). This effect may reflect trans-stimulation of BSP uptake by intracellular taurocholate effectively concentrated via Na+-dependent bile acid transport during the 5-min preincubation period with little if any cis-inhibition (21). Taken together, these results strongly suggest that the BSP-induced hyperpolarization of membrane voltage occurs via BSP transport through an anion-exchange mechanism that accepts SO2-4, succinate, cholate, and possibly taurocholate and that most likely reflects the SO2-4/OH- exchanger of the sinusoidal membrane (21).


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Fig. 3.   BSP-induced membrane hyperpolarization in continuous presence of 100 µmol/l cholate (black-diamond ) and 100 µmol/l taurocholate (), each administered at 5 min before addition of BSP; n = 4-7 for each data point. Fit of control data is given as dashed line.

To test for a possible contribution of Na+ and Cl- gradients to the voltage effects of BSP, we conducted experiments under low-Na+ and low-Cl- conditions. As Fig. 4 clearly shows, a 95% Na+ substitution by choline slightly decreased membrane hyperpolarizations, leading to a parallel shift of data points to the left. In contrast, a 99% Cl- substitution by gluconate considerably increased the voltage responses to BSP, so that the slope of the regression line was significantly reduced (Fig. 4).


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Fig. 4.   BSP-induced voltage changes in low-Na+ (5% Na+-95% choline; black-diamond ) and low-Cl- solutions (1% Cl--99% gluconate; ); n = 4-7 for each data point. For comparison, fit of control data is given as dashed line.

In principle, the above Na+ and Cl- dependencies could reflect actual properties of the putative BSP-transporting pathway under consideration here. It is equally likely, however, that they may be due to secondary effects of ion substitutions on membrane properties. To discriminate between these hypotheses, we focused on the velocity of BSP-induced membrane hyperpolarizations; i.e., we determined the time constants of the actual voltage changes. These time constants should be rather insensitive to indirect effects of ion substitutions but highly sensitive to any real Na+ or Cl- dependence of BSP transport. Figure 5A exemplifies the way in which the time constants of BSP-induced membrane hyperpolarizations were determined, and data are summarized in Fig. 5B. Unfortunately, it was not possible to obtain data for the lowest BSP concentration (50 µmol/l), due to the limited resolution of our approach at small voltage changes. Nevertheless, Fig. 5B clearly shows that 1/tau is a saturable function of BSP concentration, yielding an apparent Km of 370 µmol/l. This value is 1.6 times higher than the one derived from Fig. 2A, where the amplitudes of voltage changes were considered. A slightly higher value, however, is to be expected because the time constants determined here will include the (constant) time required for superfusate exchange.




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Fig. 5.   A: time course of membrane hyperpolarization by various BSP concentrations (in µmol/l) as indicated. Data points were selected at a rate of 1 per second and fitted to a monoexponential of the form delta Vm = (delta Vm)max(1 - e-t/tau ) where (delta Vm)max is maximum membrane hyperpolarization. Representative experiments are shown. B: time constants of BSP-induced membrane hyperpolarization (tau delta Vm = 1/velocity) vs. reciprocal of BSP concentration. C: tau delta Vm in low-Na+ (black-diamond ) and low-Cl- () solutions. For comparison, fit of control data is given as dashed line.

We then determined the time constants of BSP-induced membrane hyperpolarizations in low-Na+ and low-Cl- solutions. As summarized in Fig. 5C, the tau  values derived under these conditions were not significantly different from those obtained under control conditions. From this we conclude that direct effects of Na+ and Cl- substitution on BSP transport are rather unlikely. Instead, the marked effects of low-Na+ and low-Cl- solutions on the amplitudes of BSP-induced voltage changes (Fig. 4) are probably indirect and may be mediated by inhibition of Na+/H+ exchange and a decrease in overall membrane conductance, respectively (see DISCUSSION).

What is the mechanism of BSP-induced membrane hyperpolarization? In a first attempt to solve this question, we performed experiments in the continuous presence of 1 mmol/l Ba2+, an effective inhibitor of K+ conductance in rat hepatocytes (3, 49). Under these conditions, addition of 300 µmol/l BSP to the superfusate led only to minor changes of membrane voltage that equaled -0.7 ± 0.3 mV (n = 8, not significant; Fig. 6A). These results are indicative of an increase in K+ conductance by BSP and do not support the notion of an electrogenicity of the BSP transport under consideration here. It is also noteworthy in this respect that in sinusoidal membrane vesicles SO2-4/OH- exchange was found to be operating in a strictly electroneutral mode (21).




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Fig. 6.   A: effects of 300 µmol/l BSP on Vm in continuous presence of 1 mmol/l Ba2+. B: effects of high-K+ pulses (increases in K+ from 2.7 to 27 mmol/l in exchange for Na+) on Vm before, during, and after administration of 300 µmol/l BSP as indicated. Representative experiment is shown. C: BSP (300 µmol/l)-induced membrane hyperpolarization (delta Vm), as a function of control voltage (Vm, i.e., voltage before BSP administration). Dashed lines give 95% confidence limits for regression line.

This increase in K+ conductance was further characterized in ion-substitution experiments in which the extracellular K+ concentration was transiently increased from 2.7 to 27 mmol/l (Fig. 6B). Under control conditions, this maneuver depolarized membrane voltage by 8.1 ± 0.5 mV (n = 11). In the presence of 300 µmol/l BSP, this voltage response was increased to 12.0 ± 0.6 mV (P < 0.001). After washout of BSP, high K+ depolarized membrane voltage by 3.0 ± 0.5 mV, which is significantly less than under control conditions (P < 0.001).

If, in fact, BSP increases the K+ conductance of rat hepatocytes, the amplitude of BSP-induced membrane hyperpolarization should be a function of control membrane voltage. In other words, if basal K+ conductance is high and control voltage is relatively negative, the increase in K+ conductance by BSP and the resultant shift of membrane voltage toward EK, the K+ equilibrium potential, should be relatively small; if, on the other hand, basal K+ conductance is low and membrane voltage is further away from EK, addition of BSP should lead to a more pronounced hyperpolarization of cell membranes. As shown in Fig. 6C, this is clearly the case. Plotting the delta Vm values induced by 300 µmol/l BSP derived from 42 single experiments as a function of control voltages and fitting these data by means of linear regression yields a correlation coefficient of 0.79, and with P < 0.0001 the slope of the regression line is different from zero. The Vm intercept is at -57.2 mV, which is 16.3 mV more negative than the average control voltage, i.e., -40.9 ± 0.8 mV. Taken together, the results summarized in Fig. 6, A-C, strongly support the notion that the BSP-induced hyperpolarization of membrane voltage reflects a distinct increase of K+ conductance.

To elucidate a possible role of Cl- conductance in the effect of BSP on membrane voltage, Cl- substitution experiments were performed in which 99% of Cl- was replaced with gluconate. These measurements were performed in HCO-3-free solutions to avoid secondary effects of Cl- substitution via Cl-/HCO-3 exchange (4) and the dependence of K+ conductance on cell pH (3, 11, 20, 49). Under these conditions, low-Cl- pulses depolarized membrane voltage by 18.6 ± 1.0 mV (n = 6; Fig. 7). With 300 µmol/l BSP present, this voltage response was decreased to 6.3 ± 0.8 mV (P < 0.001). After removal of BSP, low Cl- depolarized the cells by 16.2 ± 1.6 mV, which is not significantly different from control. This effect of BSP on the voltage response to low Cl- could reflect an actual decrease in Cl- conductance. It is equally likely, however, that it is indirect and due to the prominent increase in K+ conductance outlined above because from ion-substitution experiments only relative ion conductances can be obtained. This latter hypothesis is, in fact, corroborated by the cable analysis experiments outlined below. Moreover, it is noteworthy that, in rat hepatocytes, Cl- could be shown to be in electrochemical equilibrium across the cell membrane over a wide range of membrane voltages (16) so that the contribution of Cl- conductance to this parameter will in most instances be expected to be minor.


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Fig. 7.   Effects of low-Cl- pulses (decreases in Cl- from 156.5 to 1.57 mmol/l with gluconate as substitute) on Vm before, during, and after administration of BSP. HCO-3-free solutions. Representative experiment is shown.

At first sight, the increase of cell input resistance by 300 µmol/l BSP to 120% of the control value is contradictory to the proposed increase of K+ conductance. In confluent primary cultures of rat hepatocytes, however, cells are tightly coupled electrically, and cell input resistance could be shown to be mainly determined by cell-coupling resistance rather than by the actual cell membrane resistance (48, 51). To quantify both parameters, we therefore performed a cable analysis with pairs of intracellular microelectrodes (see METHODS for details). Addition of 300 µmol/l BSP to the superfusate of rat hepatocytes decreased Rz from 4.3 ± 0.6 to 2.8 ± 0.3 kOmega · cm2 (n = 4, P < 0.02; Fig. 8A). After washout of BSP, Rz increased to 5.6 ± 0.8 kOmega · cm2, which is significantly higher than control (P < 0.05). Thus the BSP-induced changes in Rz exactly match the changes in K+ conductance determined by means of high-K+ pulses (Fig. 6B). In addition to the above effects on Rz, BSP increased Rx from 2.6 ± 0.2 to 3.1 ± 0.4 MOmega (P < 0.05; Fig. 8B). This change of Rx readily explains the observed increase of cell input resistance. The effect may be interpreted in terms of a decreased cytosolic conductivity after an increase of conductive K+ release. Taken together, our results strongly suggest that the BSP-induced hyperpolarization of membrane voltage reflects a distinct increase of cell membrane K+ conductance.



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Fig. 8.   Cable analysis of effects of 300 µmol/l BSP on specific cell membrane resistance (Rz; A) and cell coupling resistance (Rx; B); n = 4 for each data point. See METHODS for details.

What is the mediator of the BSP effect on K+ conductance? In our analysis, we first focused on intracellular pH (pHi). Figure 9 summarizes experiments in which pHi was determined by use of the fluorescent dye BCECF. Addition of 300 µmol/l BSP to the superfusate led to a small but significant decrease of pHi from 7.02 ± 0.07 to 6.95 ± 0.06 (n = 11, P < 0.001), most probably mediated via SO2-4/OH- exchange. With a stoichiometry of 1:2 for this transporter and a total pHi buffer capacity of 60 mmol/l in HCO-3-containing solutions (15), this intracellular acidification would be equivalent to an intracellular concentration of BSP to ~2 mmol/l, i.e., sevenfold above chemical equilibrium. In any case, the BSP-induced decrease of pHi would lead to a reduced rather than to an increased K+ conductance (3, 11, 20), and, consequently, it is not the mediator of BSP-induced membrane hyperpolarization. At the end of each experiment, a 25 mmol/l NH+4 pulse was administered to validate our recording techniques.


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Fig. 9.   Effects of 300 µmol/l BSP on intracellular pH (pHi); note small but significant transient decrease of pHi on addition of compound. At end of each experiment, a 25 mmol/l NH+4 pulse was applied as a control; n = 11 for each data point.

In the next series of experiments, we determined intracellular Ca2+ concentrations ([Ca2+]i) by means of the fluorescent dye fura 2. As exemplified in Fig. 10A, 300 µmol/l BSP led to a small transient increase of [Ca2+]i that equaled 16 ± 5 nmol/l (n = 7, P < 0.02). This response is too weak to mediate any changes in membrane conductances and most likely reflects an indirect effect of BSP-induced cell acidification. Figure 10B depicts an experiment in which [Ca2+]i was elevated by use of 100 µmol/l ATP as a positive control (10, 34, 37).



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Fig. 10.   Effects of 300 µmol/l BSP (A) and 100 µmol/l ATP (B) on intracellular Ca2+ (Ca2+i). Each experiment is representative of 4 others.

Given the high turnover rate of SO2-4/OH- exchange (21), BSP-induced cell swelling and a resultant activation of volume-sensitive K+ channels (9, 18, 49) could be a plausible explanation for the observed membrane effects of the compound. As Fig. 11A clearly shows, however, 300 µmol/l BSP did not significantly change cell volumes. In Fig. 11B, the extracellular osmolarity was increased from 300 to 400 mosmol/l as a positive control. This maneuver led to an initial decrease of cell volumes to 88.4 ± 0.5% of control, followed by a partial regulatory volume increase to 94.1 ± 0.9% within 10 min (51, 52). Taken together, our results clearly show that the BSP-induced increase of rat hepatocyte K+ conductance is mediated neither by cell pH, cell Ca2+, nor cell volume.



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Fig. 11.   A: BSP (300 µmol/l) has no significant effect on hepatocyte cell volume; n = 7. B: for times indicated, 100 mosmol/l sucrose was added to superfusate as a positive control; note pronounced cell shrinkage and partial regulatory volume increase; n = 18.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we describe a dose-dependent and reversible increase of rat hepatocyte K+ conductance by micromolar concentrations of BSP. This increase in K+ conductance leads to a sizeable hyperpolarization of membrane voltage that, with 200 µmol/l BSP, equaled 5.9 mV. In an earlier report from this laboratory, it was found that the anion transport inhibitor DIDS hyperpolarized rat hepatocyte membrane voltage from -44.0 to -73.1 mV at 200 µmol/l (50). Upon removal of the drug, membrane voltages stabilized at -58.5 mV, so that the reversible portion of the effect equaled 14.6 mV. As with BSP, these DIDS-induced voltage changes were exactly paralleled by significant (but more pronounced) changes in cell membrane K+ conductance. The DIDS effects were competitively inhibited by BSP, and they were clearly mediated by hepatocellular uptake of the stilbene (50). In addition, preliminary results with indocyanine green [another cholephilic organic substance (35)] reveal that 100 µmol/l of the compound reversibly hyperpolarizes rat hepatocyte membrane voltage by 5.3 ± 1.1 mV (n = 6; Wehner, unpublished observations). This raises the question of how these chemically distinct substrates of bilirubin/BSP transport may exert their action on K+ conductance and if the observed effects could reflect a physiological mechanism.

In pilot experiments, we did not observe any significant effects of SO2-4 and succinate per se on membrane voltage (data not shown). From this we can exclude a direct cross-talk between SO2-4/OH- exchange and K+ conductance as well as a parallel mode of activation. Moreover, our microfluorometric determinations of cell pH, cell Ca2+, and cell volume clearly show that none of these parameters is the mediator of BSP-induced K+ channel activation. This leaves us with the notion of a sensory mechanism linking transport rate or intracellular anion activity to K+ conductance; alternatively, BSP (as well as DIDS and indocyanine green) may interact directly with the K+ channel molecule itself. Additional experiments are necessary to differentiate between these hypotheses, namely, single-channel recordings on excised membrane patches.

Although the mechanism by which BSP increases K+ channel activity remains to be elucidated, the effect is clearly related to BSP uptake via SO2-4/OH- exchange. 1) SO2-4 and succinate, both substrates of the above pathway (21), exhibit pronounced cis-inhibition as well as trans-stimulation of BSP-induced membrane hyperpolarization. In addition, cis-inhibition was also found for the bile acid cholate (21). The apparent trans-stimulation by taurocholate may be interpreted in terms of a concentrative uptake of the bile acid in the preperiod of the experiments. 2) Application of 300 µmol/l BSP led to a significant decrease of intracellular pH by 0.07 units, expected to occur from SO2-4/OH- exchange and a BSP/OH- exchange mode of operation. From this effect one can calculate a sevenfold concentrative uptake of BSP above chemical equilibrium. Most likely, the driving force for this transport is provided by its coupling to Na+/H+ exchange via cell pH because, in the vesicle system, an inwardly directed Na+ gradient stimulated SO2-4/OH- exchange approximately threefold, and this effect was inhibited by both DIDS and amiloride (21). This indirect Na+ dependence of SO2-4/OH- exchange also readily explains the observed effects of low-Na+ conditions on the amplitudes of BSP-induced membrane hyperpolarizations (Fig. 4). 3) Although most other (putative) sinusoidal anion transporters exhibit Km values for BSP of a few micromoles per liter, or less (23, 40, 45, 54), the apparent Ki of BSP for SO2-4 transport via SO2-4/OH- exchange was found to be as high as 120 µmol/l (21). This value is comparable to the apparent Km for BSP-induced membrane hyperpolarization reported here, namely, 226 µmol/l (Fig. 2A).

Under all experimental conditions used in this study, 1/delta Vm was a strictly linear function of 1/BSP with no detectable deviation at low BSP concentrations. Moreover, at 50 µmol/l BSP, which is the lowest concentration used in our experiments, all high-affinity (but comparably low-capacity) BSP transporters studied so far are fully saturated. From this we conclude that the concentration dependence of the BSP effect on membrane voltage shown here, as well as its interaction with the known substrates of SO2-4/OH- exchange, will almost exclusively reflect the actual properties of this low-affinity but high-capacity transporter.

The control membrane voltage of rat hepatocytes in primary culture equals approximately -41 mV (this study). For the same system, canalicular voltages of -14 mV on the average were reported (48), so that the canaliculus will be 26 mV positive with respect to the cytosol. Although canalicular secretion in many cases involves primary active transport processes (see Refs. 13 and 28 for review), this voltage across the canalicular membrane per se will favor any form of electrogenic anion secretion (see introduction). A hyperpolarization of sinusoidal membrane voltage by 5-6 mV (200 µmol/l BSP, 100 µmol/l indocyanine green; this study) or 15 mV [the reversible portion of the effect of 200 µmol/l DIDS (50)] will increase this canalicular membrane voltage to 33 and 42 mV, respectively, i.e., to 120 and 156% of control. We therefore hypothesize that BSP-, indocyanine green-, and DIDS-induced activation of rat hepatocyte K+ conductance may reflect a physiological cross-talk mechanism linking sinusoidal uptake of these compounds to canalicular electrogenic secretion.

In conclusion, we observe a dose-dependent hyperpolarization of rat hepatocyte membrane voltage by BSP. The effect is due to a significant increase of cell membrane K+ conductance, and it is mediated by BSP uptake via SO2-4/OH- exchange. The activation of K+ conductance may reflect a physiological mechanism facilitating the canalicular electrogenic secretion of certain anions.


    ACKNOWLEDGEMENTS

We thank R. K. H. Kinne for helpful discussion and for continuous support of the project. This work would not have been possible without the invaluable technical assistance of G. Beetz, A. Giffey, and S. Rosin-Steiner. We are also grateful to U. Kirschner and C. Böhmer for critical reading of the manuscript. The competent and enthusiastic secretarial help of D. Mägdefessel is also gratefully acknowledged.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: F. Wehner, Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Postfach 10 26 64, 44026 Dortmund, Germany (E-mail: frank.wehner{at}mpi-dortmund.mpg.de).

Received 10 December 1998; accepted in final form 15 February 1999.


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