Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, 44139 Dortmund, Germany
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ABSTRACT |
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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
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INTRODUCTION |
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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
SO24/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
SO24/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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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 M
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(1) |
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(2) |
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(3) |
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
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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).
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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RESULTS |
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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 M
under control
conditions to 3.5 ± 0.3 M
after 5-min exposure to BSP
(P < 0.001).
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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/
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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The above Km value is comparable to the
Ki of BSP that was determined for the
SO24/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
SO24/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
SO24/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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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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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/
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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We then determined the time constants of BSP-induced membrane
hyperpolarizations in low-Na+ and low-Cl
solutions. As summarized in Fig. 5C, the
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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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
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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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 k · cm2 (n = 4,
P < 0.02; Fig. 8A).
After washout of BSP, Rz increased to 5.6 ± 0.8 k
· 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 M
(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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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 SO24/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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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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Given the high turnover rate of
SO24/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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DISCUSSION |
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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
SO24 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 SO24/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/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
SO24/OH
exchange. The
activation of K+ conductance may reflect a physiological
mechanism facilitating the canalicular electrogenic secretion of
certain anions.
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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.
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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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