(Received for publication, April 25, 1997)
From the Division of Gastroenterology, Department of
Medicine, University of Colorado Health Sciences Center, Denver,
Colorado 80262, the
Division of Gastroenterology, Department of
Medicine, University of California, San Francisco, California
94143-0538, and the ¶ Division of Gastroenterology, Department of
Medicine, Duke University Medical Center,
Durham, North Carolina 27710
In a model liver cell line, recovery from
swelling is mediated by a sensitive autocrine pathway involving
conductive release of ATP, P2 receptor stimulation,
and opening of membrane Cl channels (Wang, Y., Roman,
R. M., Lidofsky, S. D., and Fitz, J. G. (1996)
Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release
are not known. Based on evidence that certain ATP-binding cassette
(ABC) proteins may function as ATP channels or channel regulators, we
evaluated the potential role of ABC proteins by comparing ATP release
and volume regulation in rat HTC and HTC-R hepatoma cells, the latter
of which overexpress Mdr proteins. In both cell types, Cl
current activation (ICl-swell) and volume
recovery following swelling were dependent on conductive ATP efflux.
The rate of volume recovery was ~6-fold faster in HTC-R cells
compared with HTC cells. This effect is likely due to enhanced ABC
protein-dependent ATP release since (i)
ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 µM verapamil and 15 µM cyclosporin A); (ii)
swelling-induced Cl
current density was similar in
both cell types (approximately
50 pA/pF; not significant); and (iii)
ATP conductance measured by whole-cell techniques was increased
~3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells
exhibited enhanced survival during hypotonic stress. By modulating ATP
release, hepatic ABC proteins may play a key role in the cellular
pathways coupling changes in cell volume to ion permeability and
secretion.
Extracellular ATP is a potent signaling factor that modulates a
variety of cellular functions through the activation of P2 purinergic receptors in the plasma membrane. These receptors are widely
distributed among different liver cell types, including hepatocytes,
cholangiocytes, macrophages, and endothelial cells, but the physiologic
roles have not been fully defined. Cells release ATP in response to
both osmotic and mechanical stimuli, and one mechanism may involve
opening of a channel-like pathway (1, 2). In respiratory epithelia, ATP
release stimulated by cytosolic cAMP activates outwardly rectified
Cl channels coupled to P2U receptors and
enhances Cl
secretion (3). Recent studies in a model
liver cell line support an alternative pathway where increases in cell
volume induce conductive ATP efflux. In these cells, removal of
extracellular ATP or P2 receptor blockade prevents both
Cl
channel activation and volume recovery (1). These
findings suggest functional interactions between ATP release,
P2 receptor stimulation, and Cl
channel
opening in epithelial secretion and volume regulation.
Members of the ATP-binding cassette
(ABC)1 protein family are
likely to be relevant to this volume regulatory pathway for two reasons. First, while the molecular basis for the transmembrane ATP
conductance has not been established, heterologous expression or
up-regulation of ABC family members in some cell models is associated
with enhanced electrodiffusional ATP release. In cystic fibrosis
respiratory epithelia, cAMP fails to stimulate channel-mediated ATP
efflux, a response that is present in native epithelia; CFTR gene
transfer restores the ATP conductance (3, 4). In other cell lines, ATP
release is proportional to the expression of mammalian and
Drosophila Mdr1 P-glycoproteins (5, 6). Second, in some but
not all cell types, Mdr1 P-glycoproteins regulate swelling-activated Cl currents (ICl-swell). Effects
include enhancement of ICl-swell and endowment
of Cl
channel sensitivity to protein kinase C
(mdr1 gene transfer) and increase in
ICl-swell for a given hypotonic stress
(P-glycoprotein overexpression) (7, 8). The cellular mechanisms
involved in these responses and the implications for other cell types
have yet to be clarified.
In hepatocytes, P-glycoproteins transport both amphipathic compounds
and phospholipids across canalicular membranes into bile (9, 10).
However, the functions of multiple other ABC members present in liver
cells are unknown. In light of the putative association of certain ABC
proteins with channel-mediated ATP and Cl transport, we
sought to investigate the role of hepatocellular ABC proteins in these
processes. Findings in rat HTC hepatoma cells were compared with those
in a selected population of HTC cells (HTC-R) that overexpress both
endogenous and novel Mdr proteins (11). These studies demonstrate that
inhibition of P-glycoprotein transport prevents recovery from swelling
and that overexpression of Mdr proteins is associated with enhanced ATP
release, volume recovery, and cell survival during hypotonic stress.
Therefore, by modulating ATP transport, one or more hepatic ABC
proteins may represent important regulatory elements that directly
influence numerous volume- and ATP-dependent liver cell
functions.
The purinergic agonists used were ATP, UTP, and
ATPS, and the purinergic antagonists were suramin and reactive blue
2. The ATP scavengers used were apyrase and hexokinase. The
P-glycoprotein blockers used were cyclosporin A and verapamil, and the
Cl
channel blocker was
5-nitro-2-(3-phenylpropylamino)benzoic acid. The Ca2+
chelator used was EGTA. The cell viability fluorochrome was propidium iodide. All experimental reagents were obtained from Calbiochem or
Sigma and were exposed to cells or cell suspensions as described.
HTC cells possess ion channels
and purinergic receptors similar to those found in primary hepatocytes
and exhibit rapid recovery from swelling that is mediated by the
opening of Cl channels (12, 13). HTC-R cells represent
specially adapted HTC cells originally selected to resist normally
toxic concentrations of glycocholic acid methyl ester. HTC-R cells have
special advantages for these studies since they overexpress both
endogenous and novel Mdr proteins (11). Both cell types were grown at
37 °C in a 5% CO2 and 95% air atmosphere in minimal
essential medium (Life Technologies, Inc.) containing 5% fetal calf
serum, 2 mM L-glutamine, 100 IU/ml penicillin,
and 100 µg/ml streptomycin. Glycocholic acid methyl ester was
synthesized as described previously (11) and added to the HTC-R cell
medium at concentrations of 300-650 µM. For all
physiologic studies, the culture medium was replaced with standard
isotonic extracellular buffer that contained 140 mM NaCl, 4 mM KCl, 1 mM KH2PO4, 2 mM MgCl2, 1 mM CaCl2,
10 mM glucose, and 10 mM HEPES/NaOH (pH 7.4).
Hypotonic buffer was made by decreasing the NaCl concentration by
20-40% (84-112 mM) as indicated. As determined by a
vapor pressure osmometer, the isotonic extracellular buffer osmolarity
was ~295 mosmol/kg, and the hypotonic buffer (40% decrease in NaCl)
osmolarity was ~205 mosmol/kg.
The mean cell diameter was
measured in cell suspensions at 25 °C by electronic cell sizing
utilizing the Coulter Multisizer, Coulter Sample Stand II, and
Multisizer Accucomp Version 2.0 software (Coulter Electronics, Inc.,
Hialeah, FL) as described previously (14). Briefly, the mean cell
volume of ~10,000 cells/time point was calculated from the mean cell
diameter using the formula 4/3r3. Changes in
size over time are expressed as relative volume by normalizing volume
to the basal period. Any significant alterations in basal cell volume
by experimental reagents are noted. Percent recovery for a given time
point was calculated as follows: ((peak relative volume
relative volume at that time point)/(peak relative volume
1)) × 100.
Whole-cell
currents were measured ~24 h after plating using patch-clamp
recording techniques (15). Cells on a coverslip were placed in a
chamber (~400 µl) and perfused at 4-5 ml/min. Swelling was induced
by switching to a buffer containing 20% less NaCl. The standard
pipette (intracellular) solution contained 130 mM KCl, 10 mM NaCl, 2 mM MgCl2, 10 mM HEPES/KOH (pH ~7.2), and free Ca2+
adjusted to ~100 nM (0.5 mM CaCl2
and 1 mM EGTA) with a total Cl concentration
of 145 mM (16). Cells were viewed through an Olympus IMT-2
inverted phase-contrast microscope using Hoffman optics at a
magnification of ×600. Patch pipettes were pulled from Corning 7052 glass and had a resistance of 3-10 megaohms. Recordings were made with
an Axopatch ID amplifier (Axon Instruments, Inc., Foster City, CA) and
were digitized (1 kHz) for storage on a computer and analyzed using
pCLAMP Version 5.5 programs (Axon Instruments, Inc.). Pipette voltages
refer to the bath. Current-voltage relations were measured by 400-ms
steps to potentials between
120 and +100 mV in 20-mV increments. In
the whole-cell configuration, the pipette voltage corresponds to the
membrane potential, and upward deflections of the current trace
indicate outward membrane current. Whole-cell currents (pA) and current
density (pA/pF) refer to measurements at a pipette voltage of
80 mV
(EK) to minimize any contribution of
K+ currents.
Under physiologic conditions, ATP currents are below the limits of detection (3). To enhance measurements of ATP transport, whole-cell recordings were performed with high ATP concentrations in both the bath and pipette solutions including 100 mM MgATP, 5 mM MgCl2, and 10 mM HEPES/Tris-OH (pH 7.4) as described previously (1, 4, 5). Swelling was induced by 20% dilution of the bath solution (1 ml) with water (200 µl/ml), and currents were measured before and 5 min after dilution.
Cell Viability AssayCellular toxicity was assessed by propidium iodide fluorescence as described previously (17). Propidium iodide (50 µg/ml) is thought to penetrate only damaged membranes to form intercalation complexes with double-stranded DNA with an amplification of nuclear fluorescence (excitation at 530 nm with a 620-nm emission filter). In wells containing subconfluent HTC and HTC-R cell monolayers, the number of positively staining nuclei was quantitated at timed intervals during incubation in isotonic and hypotonic buffers.
StatisticsPooled data are presented as means ± S.E. or S.D., where n represents the number of cell suspensions analyzed by the Coulter Multisizer, the number of cells for patch-clamp studies, and the number of wells for propidium iodide studies. Statistical comparisons were made using the paired or unpaired t test as indicated, and p < 0.05 was considered significant.
In HTC cells, recovery from swelling depends on
channel-mediated ATP release, stimulation of membrane P2
receptors, and activation of Cl currents
(ICl-swell) (Fig.
1) (1). Whole-cell patch-clamp and
cell-sizing studies were initially performed to confirm that a similar
purinergic signaling pathway contributes to volume regulation in HTC-R
cells, a model derived from HTC cells that overexpresses ABC proteins
(11).
In whole-cell recordings of HTC-R cells, basal Cl
currents in isotonic buffer were small. However, incubation in
hypotonic buffer (20% less NaCl) resulted in an increase in
Cl
currents from
219.7 ± 38.8 to
3938.8 ± 389.4 pA (n = 13) (Fig. 2, A and D).
Removal of extracellular ATP by the addition of the ATPase/ADPase
apyrase (1 unit/ml) to hypotonic buffer significantly inhibited
activation of ICl-swell (
201.8 ± 44.8 pA, n = 3; p < 0.001). This effect was
reversible upon return to apyrase-free solutions (Fig. 2,
B-D). The addition of the nonselective P2
blockers suramin (100 µM) and reactive blue 2 (10 µM) to hypotonic buffer also inhibited current activation
(suramin:
380.9 ± 118.6 pA, n = 4; reactive
blue 2:
166.0 ± 48.9 pA, n = 3;
p < 0.001), confirming that the effects of
extracellular ATP involve stimulation of membrane P2
receptors (Fig. 2, C and D). Exposure to ATP
(2.5-10 µM) in the absence of swelling markedly
increased Cl
currents with similar characteristics to
ICl-swell (data not shown). The P2
receptor subtype involved is unknown since UTP (a P2U
agonist) at similar concentrations did not activate Cl
currents, and in previous studies in HTC cells, neither
P2Y-preferring (2-MeSATP) nor P2X-preferring
(
,
-MeATP) agonists stimulated channels (18).
Conductive ATP Release Mediates HTC-R Cell Volume Recovery
Under isotonic conditions, HTC-R mean cell diameter was
23.81 ± 0.164 µm, corresponding to a mean cell volume of
7068 ± 147 µm3 (n = 10). Notably,
the average basal volume for HTC-R cells is 238 ± 6% that of HTC
cells (2958 ± 112 µm3, n = 10;
p < 0.001). As shown in Fig.
3A (Control),
exposure to hypotonic buffer (40% less NaCl) caused a rapid increase
in values by 2 min (mean cell diameter = 25.93 ± 0.21 µm;
mean cell volume = 9132 ± 219 µm3), which
corresponds to a relative cell volume of 1.29 ± 0.03 (n = 10; p < 0.001). Swelling was
followed by a regulatory volume decrease (RVD) to a relative volume of
1.07 ± 0.02 by 30 min. The anion channel blocker
5-nitro-2-(3-phenylpropylamino)benzoic acid (50 µM; no
preincubation) completely inhibited volume recovery (p < 0.001), confirming that RVD is dependent on channel-mediated Cl efflux (Table I).
|
In HTC cells, volume recovery is abolished by (i) removal of
extracellular ATP by the ATP scavengers apyrase and hexokinase, an
effect that is reversed by exposure of the nonhydrolyzable P2 agonist ATPS (25 µM); and (ii)
concentration-dependent P2 receptor blockade
with suramin and reactive blue 2 (Table I) (1). Similarly, in HTC-R
cells, apyrase (3 units/ml; no preincubation) reduced percent RVD at 30 min from 78 ± 7% (controls) to 24 ± 8% (p < 0.001) (Table I and Fig. 3A). In the presence of suramin (100 µM), HTC-R cell swelling was more pronounced
(28 ± 7% increase compared with controls at 2 min;
p < 0.001), and percent volume recovery was
significantly inhibited (15 ± 6% at 30 min; p < 0.001) (Table I and Fig. 3A). These findings suggest that
P2 receptor stimulation by extracellular ATP mediates
Cl
current activation and volume recovery during HTC-R
cell swelling.
Same-day Coulter
studies were performed on both cell populations to assess whether
overexpression of ABC proteins is associated with a difference in
volume recovery rates. As shown in Fig. 3B, volume recovery
was significantly faster for HTC-R cells compared with HTC cells.
Enhanced recovery was most notably early in the volume recovery phase;
percent RVD at 5 min was 32 ± 8% for HTC-R cells
versus 5 ± 9% for HTC cells (p < 0.001). By 30 min, HTC-R cell volume was closer to basal values
(relative volumes of 1.06 ± 0.024 and 1.12 ± 0.023 for
HTC-R and HTC cells, respectively; p < 0.001). These
findings were unexpected since the lower surface/volume ratio should
favor a slower volume recovery phase in the larger cells (HTC-R) and
suggested that factors responsible for RVD, such as ATP and/or
Cl efflux, may be up-regulated in these cells.
To establish whether volume regulation is Mdr protein-dependent, additional Coulter studies were performed in which verapamil, an inhibitor of P-glycoprotein-mediated substrate transport, was added to hypotonic buffer. As shown in Fig. 3C, verapamil (20 µM) markedly attenuated HTC cell volume recovery. The inhibitory effects was apparent at all time points beyond 2 min, and percent volume recovery at 30 min was 19 ± 11% in the presence and 64 ± 6% in the absence of verapamil (p < 0.001) (Table I). These findings suggest that Mdr proteins may contribute directly to the cellular mechanisms that regulate volume recovery.
Inhibition of P-glycoprotein Transport Prevents Activation of ClSince Mdr1 P-glycoproteins have been
implicated in the regulation of ICl-swell in
other cell types, an increase in Cl permeability could
explain the enhanced rate of volume recovery in HTC-R cells. Therefore,
the effects of verapamil and cyclosporin A, another P-glycoprotein
inhibitor, on HTC cell ICl-swell were assessed.
In control studies, exposure of HTC cells to hypotonic buffer (20%
less NaCl) led to a marked increase in whole-cell Cl
currents from
81.1 ± 24.4 to
1697.0 ± 121.2 pA
(n = 13) (Fig. 4,
A and B) as described previously (1, 13). The
addition of verapamil (20 µM) to extracellular buffer
1-2 min prior to hypotonic challenge abolished activation of
ICl-swell (
86.9 ± 15.2 pA,
n = 5; p < 0.001) (Fig. 4,
A and B). This inhibitory effect was reversible
upon removal of verapamil from the bath solution. In similar studies,
cyclosporin A (15 µM) also prevented current activation
(
151.1 ± 62.5 pA, n = 5; p < 0.001) (Fig. 4, A and B).
Examination of Fig. 1 suggests several potential sites for action of
verapamil and cyclosporin A on the Cl conductance,
including direct effects on Cl
channels themselves and/or
indirect effects via inhibition of ATP release. The former possibility
is unlikely since swelling-induced Cl
current density was
not different between HTC and HTC-R cells (approximately
50 pA/pF;
not significant) (Fig. 5B).
Moreover, in the presence of verapamil (20 µM), the
addition of 10 µM ATP to the bath solution led to a
marked increase in HTC whole-cell Cl
currents, which were
indistinguishable from those activated by ATP in the absence of
verapamil (Fig. 4C). These findings strongly suggest that
verapamil is not functioning as a Cl
channel blocker and
that Mdr proteins may therefore regulate a more proximal component in
the signaling cascade, such as ATP release (Fig. 1).
Swelling-induced ATP Conductance Is Enhanced in HTC-R Cells
Enhanced HTC-R cell volume recovery may be related to an
up-regulation of purinergic signaling processes secondary to increased availability of ATP. To characterize the ATP conductance, whole-cell studies were performed with ATP4 as the major charge
carrier (1). Compared with HTC cells, the basal ATP conductance was
greater in HTC-R cells (
2.7 ± 0.5 versus
0.6 ± 0.21 pA/pF; p < 0.01). Exposure to hypotonic buffer (20% less NaCl) markedly increased HTC-R cell ATP currents to
46.2 ± 8.3 pA/pF. Notably, swelling-induced ATP current density was ~3-fold greater in HTC-R cells compared with HTC cells
(
16.1 ± 10.4 pA/pF; p < 0.001) (Fig.
5A).
These findings indicate that HTC-R cells exhibit
enhanced ATP release and more efficient volume homeostasis. To address
the physiologic significance of this response, additional studies examined HTC and HTC-R cell viability with propidium iodide (50 µg/ml) at timed intervals during a 24-h exposure to either isotonic (control) or hypotonic buffer (Fig. 6).
HTC and HTC-R cell death during incubation in isotonic buffer did not
differ (<1% at 0-12 h and <3% at 24 h; not significant), and
viability during hypotonic exposures 6 h was similar to control
values. In contrast, longer hypotonic exposures increased cell death
compared with controls (87 and 79% increases for HTC and HTC-R cells
at 24 h, respectively). In addition, HTC cell death during
incubation in hypotonic buffer for 12 and 24 h was significantly
greater compared with HTC-R cells (cell death at 12 h: 3.2 ± 0.55% for HTC cells and 1.14 ± 0.48% for HTC-R cells; at
24 h: 19 ± 2.20% for HTC cells and 11.5 ± 2.32% for
HTC-R cells; p < 0.001). These findings suggest that
enhanced ATP release confers a selective survival advantage during
prolonged periods of osmotic stress.
Hepatocytes release ATP in response to multiple stimuli, including
increases in cell volume, extracellular adenosine, and mechanical
stress (1, 2, 19). Transmembrane ATP efflux occurs through a
channel-like pathway in accordance with an electrochemical gradient
that favors movement into the extracellular space. In rat hepatoma
cells, ATP functions as a local autocrine agonist that regulates cell
volume by opening Cl channels coupled to membrane
P2 receptors (1). The present studies provide evidence that
ABC proteins play a important role in volume-sensitive ATP transport.
First, overexpression of Mdr proteins is associated with an increase in
ATP conductance. Second, inhibition of P-glycoprotein transport
eliminates volume recovery and activation of
ICl-swell. Finally, the effects of
P-glycoprotein inhibition are bypassed by exogenous ATP. Enhanced ATP
efflux is associated with more efficient volume regulation and enhanced cell survival during hypotonic stress.
Liver cells are localized in a critical anatomic site where they
receive dual perfusion from the systemic and portal circulations. Consequently, they are subject to unusually large osmotic demands related to high rates of protein synthesis, gluconeogenesis, and organic solute transport (20). Moreover, varying exposure to nutrients,
hormones, and oxidative stress between the fed and fasted states leads
to dramatic changes in ion transport at rates up to 1010
ions/cell/s. Despite this, conductive efflux of solutes such as
Cl, K+, and taurine maintains volume within a
narrow physiologic range (13, 21, 22). Extracellular ATP, which is
present in liver circulation, stimulates membrane Cl
channels similar to those activated by swelling. Furthermore, both ATP
and decreases in cell volume have catabolic effects, presumably related
to modulation of gene and protein expression (19, 20, 23, 24). Based on
recent studies implicating volume-sensitive ATP release in recovery
from cell swelling, identification of the cellular mechanisms involved
in ATP efflux represents an important site for regulation of liver cell
and organ function.
Several observations indicate that volume-sensitive ATP efflux is
mediated by ABC proteins. First, both verapamil and cyclosporin A
abolish swelling-induced Cl current activation, and
verapamil inhibits volume recovery during hypotonic stress. It is
notable that the effects of verapamil on Cl
currents are
reversed by exogenous ATP. Second, overexpression of ABC proteins in
HTC-R cells is associated with an exaggerated rate of volume recovery.
Finally, volume-activated ATP conductance is markedly enhanced in HTC-R
cells.
Both verapamil and cyclosporin A have been shown to inhibit P-glycoprotein-mediated transport across hepatocyte membranes in multiple model systems (25-33). The effects of verapamil are not likely secondary to changes in intracellular Ca2+ since verapamil at the doses used does not change intracellular Ca2+ in isolated hepatocytes or alter passive Ca2+ transport across membrane vesicles (34, 35). Cyclosporin A also inhibits P-glycoprotein transport, but through a different mechanism by binding to canalicular membrane P-glycoproteins; induction of intrahepatic cholestasis in patients taking this drug may be related to attenuation of canalicular P-glycoprotein-mediated substrate transport (31, 36).
The specific ABC proteins involved in hepatic ATP trafficking are not known. The functions of several hepatocellular P-glycoproteins have recently been elucidated, including transport of amphipathic compounds (rodent mdr1a/1b and human MDR1) and phospholipids (murine mdr2 and human MDR3) across canalicular membranes into bile (9, 10). However, hepatocytes and other cells appear to possess multiple additional ABC proteins. In the liver, these novel proteins have been implicated indirectly in the transport of bile acids and hormones (11, 30). Consequently, it will be important to further evaluate the role of ABC proteins whose endogenous functions have not yet been determined.
Characterization of other important aspects of this signaling pathway will require further study. Most important, the mechanism(s) whereby ABC proteins such as P-glycoproteins and CFTR modulate ATP release, such as directly conducting ATP or regulating other as yet unidentified ATP efflux pathways, is presently unclear (37, 38). In addition, studies in isolated cells do not allow direct conclusions regarding polarity of membrane receptors and channels involved. Finally, the intracellular signals that regulate P-glycoprotein function and ATP transport have not yet been determined.
Since physiologic changes in liver cell volume are commonplace, it is
interesting that these cells literally invest energy, in the form of
ATP, on such an elaborate regulatory mechanism. In addition to
activating conductive efflux pathways that maintain cellular integrity,
the hydration state in itself directly alters cellular metabolism,
solute transport, and gene expression (20). While these effects target
the metabolic balance of hepatocytes, local release of ATP may also act
as a paracrine signaling factor by modulating membrane transport in
other cell types within the liver. For example, while increased bile
formation during hepatocellular swelling has been attributed as least
in part to enhanced canalicular bile acid transport, it is attractive
to speculate that volume-sensitive ATP release into the bile duct lumen
also contributes by stimulating secretion at the ductular level.
Support for this model includes evidence that (i) ATP is present in
physiologic concentrations in human bile; (ii) hypotonic perfusion of
rat livers increases bile ATP levels; and (iii) stimulation of apical
P2 receptors in cholangiocytes (IC50 ~ 2 µM) induces transepithelial Cl and fluid
secretion across biliary cell monolayers (39, 40).
ATP transport also represents a potential pathway that couples the cellular metabolic state to membrane ion permeability. Swelling during anoxic depletion of cytosolic ATP is associated with loss of hepatocyte viability (41, 42). Volume recovery mediated by conductive ion efflux, which depends on ATP release, reduces cell death during these conditions (43, 44). The survival advantage of HTC-R cells during prolonged hypotonic stress may be related to enhanced ATP-dependent volume regulation. Admittedly, the role of other factors, such as larger cell size, cannot be discounted.
In conclusion, extracellular ATP has emerged as an important signaling
factor capable of influencing volume-related and other cellular
functions. ATP transport represents an exciting potential addition to
the expanding list of substrate transport pathways regulated by ABC
proteins in liver. Moreover, these findings may be relevant to other
epithelia that possess ABC proteins and ATP-sensitive Cl
channels as well. Further characterization of the cellular and molecular mechanisms involved in ATP transport will hopefully aid in
the development of pharmacological approaches that favorably impact on
bile formation and hepatocellular function.