Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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P2Y receptor
stimulation increases membrane
Cl permeability in biliary
epithelial cells, but the source of extracellular nucleotides and
physiological relevance of purinergic signaling to biliary secretion
are unknown. Our objectives were to determine whether biliary cells
release ATP under physiological conditions and whether extracellular
ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1
cholangiocarcinoma cells and polarized normal rat cholangiocyte
monolayers. ATP release increased rapidly during cell swelling induced
by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP
(apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole
cell Cl
current activation
and prevented cell volume recovery during hypotonic stress. Moreover,
exposure to apyrase induced cell swelling under isotonic conditions. In
intact normal rat cholangiocyte monolayers, hypotonic perfusion
activated apical Cl
currents, which were inhibited by addition of apyrase and suramin to
bathing media. These findings indicate that modulation of ATP release
by the cellular hydration state represents a potential signal
coordinating cell volume with membrane
Cl
permeability and
transepithelial Cl
secretion.
nucleotide; cholangiocyte; chloride channel; cell volume
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INTRODUCTION |
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PURINERGIC SIGNALING pathways contribute to the
regulation of physiological functions in many, if not all, tissues.
Within the extracellular environment, ATP and UTP act as versatile
autocrine and paracrine effectors that bind selectively to membrane P2
receptors (4). The response to these agonists may be quite complex
inasmuch as de- or transphosphorylation to other nucleotides and
nucleosides produces agonists that recognize different P2 and P1
receptors, and cells often express many receptor subtypes coupled to
distinct biological pathways (14, 15). In neurons and circulating blood cells, ATP stored in intracellular vesicles is released during exocytosis (37, 38). However, epithelial cells are also capable of
releasing ATP and/or UTP in response to physiological stimuli, including changes in cell volume, membrane stress, hypoxia, and receptor stimulation (2, 19, 21, 24). One mechanism for cellular ATP
efflux may be electrodiffusional, with charged
ATP4 molecules permeating
selective membrane channels down an ~10,000-fold concentration
gradient (30, 34). Although not well defined, epithelial release of ATP
would be expected to specifically modify tissue responses by regulating
the local extracellular availability of purinergic agonists.
In liver, purinergic stimulation modulates many fundamental processes, including fatty acid and protein metabolism, glucose availability, and hepatic perfusion (13, 16, 23). Some of these effects appear to require sophisticated paracrine communication among different cell types. In the perfused liver model, for example, ATP released by hepatocytes during adenosine stimulation induces eicosanoid efflux from nonparenchymal cells, which then activate glycogenolysis (26) Recently, ATP in physiological concentrations has been documented in human bile (5). This is intriguing because ATP in bile would be expected to bind to P2Y2 receptors in the apical membrane of cholangiocytes. P2Y2 stimulation enhances secretion across polarized cholangiocyte monolayers, a response analogous in many ways to that observed in other epithelial models (31). Although such a mechanism could contribute directly to bile salt-independent modulation of bile flow, neither the cellular origin of luminal ATP nor the physiological stimuli that regulate ATP release have been identified.
In view of the pivotal role of cholangiocyte secretion in bile
formation, the objectives of these studies were threefold: first, to
characterize ATP release from nonpolarized and polarized biliary
epithelia under basal conditions and to determine whether cell volume
increases stimulate ATP efflux; second, to establish the role of
extracellular ATP as an autocrine regulator of
Cl permeability and cell
volume; finally, to determine whether volume-sensitive ATP release
modulates epithelial Cl
secretion as an endogenous autocrine and paracrine signaling molecule.
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MATERIALS AND METHODS |
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Cells.
Studies in isolated cells were performed using Mz-ChA-1 cells from
human cholangiocarcinoma and in polarized monolayers using normal rat
cholangiocytes (NRCs). Mz-ChA-1 are model biliary cells that express
cytoskeletal proteins, purinergic receptors, and regulated
Cl channels analogous to
primary cholangiocytes (20, 22). After increases in cell volume,
Mz-ChA-1 cells exhibit regulatory volume decrease (RVD) toward basal
values mediated in part by opening of membrane
Cl
channels (29). NRCs are
derived from intrahepatic cholangiocytes and also maintain expression
of differentiated biliary markers in culture. Unlike Mz-ChA-1 cells,
NRCs form polarized monolayers with intercellular tight junctions and
apical microvilli (39); when they are inserted into Ussing chambers,
exposure to secretory agonists leads to an increase in short-circuit
current (Isc) (31). Cells were maintained and passaged as previously described (29, 31).
Solutions.
Unless indicated, the standard extracellular NaCl solution used for all
studies contained (in mM) 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES-NaOH (pH 7.40) with a total
Cl of 150 mM. Solution
osmolarity (vapor-pressure osmometer, model 5500, Wescor, Logan, UT)
was ~295 mosM. Increases in cell volume were induced by exposure to
hypotonic extracellular buffer, which was prepared by
1) lowering the NaCl concentration
[Coulter multisizer, patch clamp (Cl
currents), Ussing
chamber], 2) adding water
[luciferase-luciferin assay, patch clamp (ATP currents)], or
3) exchanging mannitol-containing with mannitol-free extracellular NaCl-rich buffer (Ussing chamber). Exposure to hypotonic stress (15-30% reduction in osmolarity) did
not affect cell viability over 1 h (propidium iodide staining, data not shown).
Reagents.
5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was obtained from
Biomol Research Laboratories (Plymouth Meeting, PA). Amiloride,
adenosine
5'-O-(3-thiotriphosphate)
(ATPS), apyrase, and suramin were obtained from Sigma (St. Louis, MO).
Bioluminescence ATP detection assay.
The methods used are similar to those recently published (8, 35).
Mz-ChA-1 cells grown to confluence in 35-mm dishes (surface area 9.62 cm2) were studied to determine
"bulk" cellular ATP release. To measure "vectorial" ATP
release from cholangiocyte monolayers, NRCs were plated on
collagen-coated semipermeable supports (Millicell HA, diameter 12 mm,
surface area 1.13 cm2,
Millipore-Fisher, Bedford, MA) and were studied when transepithelial resistance exceeded 1,000 · cm2
(EVOHM, World Precision Instruments, Sarasota FL). Before study cells
were washed twice with PBS, and serum-free Optimem-1 (GIBCO BRL, Grand
Island, NY) containing 2 mg/ml firefly luciferase-luciferin (lyophilized reagent, Calbiochem, La Jolla, CA) was added directly to
cells. ATP released from cells into media catalyzes the oxidation of
luciferin, generating bioluminescence. For Mz-ChA-1 cells, bulk ATP
release into media was measured (initial volume 600 µl). Semipermeable supports containing NRC cells were placed on 35-mm culture dishes in a 200-µl volume of media to bath the basolateral side, and 200 µl of media were then added to the apical chamber. Openings in the support allow direct access to basolateral buffer. To
selectively measure ATP efflux from either apical or basolateral NRC
membranes, the luciferase-luciferin reagent was added to medium on one
side of the monolayer only (the side in which ATP is detected), and
medium without reagent was added to the other side (Fig.
1). Dishes containing Mz-ChA-1 cells and
NRC-containing inserts were placed on a platform and lowered directly
into a chamber in complete darkness within a TD-20/20 luminometer
(Turner Designs, Sunnyvale, CA), and cumulative bioluminescence over
15-s intervals was quantified in real time as arbitrary light units
(ALU). Cell volume increases were induced by adding water to dilute
media 20 and 40%; identical volumes of isotonic media were added in
control studies to dissociate the effects of volume changes from
membrane perturbation on ATP release. Luciferase-luciferin
concentrations more than fivefold less did not change detected
luminescence. All solutions (water, media, reagents) that were added to
cells contained identical amounts of luciferase-luciferin so that the
reagent would not be diluted. Addition of apyrase (2 U/ml) to
extracellular media to hydrolyze ATP eliminated bioluminescence. All
studies were performed at room temperature.
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Bioluminescence assay standardization. Background luminescence (cells plus media without luciferase-luciferin) was checked before each experiment and was always <0.05 ALU. To approximate ATP released from cells, standard curves of ATP (ATP free acid, Calbiochem) at known concentrations were performed by adding serial dilutions of ATP stock (freshly made at the time of measurements) into 200 µl Optimem-1 medium containing 2 mg/ml luciferase-luciferin. Dilution of media 20-40% with water and addition of isotonic media (all solutions containing luciferase-luciferin) did not significantly affect luminescence detected for given concentrations of ATP. Multiple measurements for each dose of ATP were performed, and luminescence values were stable among all measurements (no evidence of bleaching or signal instability).
Cell volume. Mean Mz-ChA-1 cell volume was measured in cell suspensions by electronic cell sizing (Coulter multisizer, Hialeah, FL) using an aperture of 100 µm as previously described (30). Measurements of ~20,000 cells in suspension at intervals to 30 min after exposure to hypotonic buffer (30% less mosmol) were compared with basal values in isotonic buffer; values are expressed as relative volume normalized to the basal period.
Measurement of Cl and ATP
currents.
Membrane Cl
and ATP
currents were measured and analyzed using whole cell patch-clamp
techniques as previously described (22, 30, 40). Isolated Mz-ChA-1
cells plated on coverslips were studied after ~24 h in a chamber
perfused with extracellular buffer (chamber volume ~400 µl, flow
4-5 ml/min). For measurement of Cl
currents, the
intracellular (pipette) solution contained (in mM) 130 KCl, 10 NaCl, 2 MgCl2, 10 HEPES-KOH, 1 ATP, 0.5 CaCl2, and 1 EGTA (pH 7.3),
corresponding to a free Ca2+
concentration of ~100 nM. To enhance detection of currents carried by
charged ATP molecules, high concentrations of ATP were included in bath
and pipette solutions as the predominant charge carrier (100 mM MgATP,
5 mM MgCl2, 10 mM HEPES, pH ~7.4
TrisOH) (40).
Voltage-clamp measurements of
Isc.
NRCs were grown on collagen-treated polytetrafluoroethylene membrane
filters (Corning Costar, Acton, MA) and used for experiments when the
transmembrane resistance exceeded 1,000 · cm2 (31).
After inserts were mounted in an Ussing chamber (Jim's Instrument
Manufacturing, Iowa City, IA), apical and basolateral membranes were
perfused with standard NaCl-rich buffer that was continuously bubbled
with 100% O2 (~20
ml/reservoir). Buffer temperature was maintained at 37°C using a
recirculating heated water bath (model 800; Fisher Scientific,
Pittsburgh, PA). Transepithelial Isc was measured
under voltage-clamp conditions using an epithelial voltage clamp
amplifier (model EC-825; Warner Instruments, MRA International, Naples,
FL). Cell volume increases were induced by simultaneous reduction of
apical and basolateral buffer osmolarity by 30%.
Statistics. Results are presented as means ± SE, with n representing the number of cells for patch-clamp studies and the number of repetitions for other experiments. All experiments were repeated on at least two study days. For Ussing protocols, each experimental study was paired directly with a control study on the same day. Paired or unpaired Student's t-tests were used to assess statistical significance, and P < 0.05 was considered to be significant.
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RESULTS |
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Increases in Mz-ChA-1 cell volume enhance membrane ATP permeability.
To assess the possibility that ATP in bile is derived from
cholangiocytes, initial studies examined whether human biliary cells
exhibit basal membrane ATP permeability and whether increases in cell
volume enhance cellular ATP efflux. As shown in Fig.
2A, Mz-ChA-1 cells released ATP into extracellular buffer as detected by
the luciferase-luciferin assay (range ~11-43 ALU). In most experiments, addition of isotonic media led to a small but significant increase in ATP release; this effect was most apparent during the
initial compared with subsequent media additions. As shown in Fig.
2A,
top, the addition of isotonic media
(200 µl added to 600 µl) increased bioluminescence from 31.43 ± 3.12 to 41.71 ± 3.38 ALU (P < 0.05), indicating that mechanical membrane perturbation represents a
stimulus for ATP efflux (n = 8).
However, induction of cell swelling by adding water to decrease media
osmolarity by 20 and 40% led to rapid, sustained increases in
extracellular ATP that were much greater than values in control studies
in which similar volumes of isotonic media were added. Depending on the study day, bioluminescence increased approximately two- to fourfold after a 20% media dilution; ATP levels peaked within 5 min and decreased gradually toward basal values over 30 min. These findings indicate that human biliary epithelial cells release ATP into extracellular media under basal conditions and that both membrane stress (isotonic media addition) and increases in cell volume (hypotonic media addition) enhance ATP efflux independently.
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Mz-ChA-1 cells express volume-sensitive ATP channels.
In detecting extracellular ATP molecules, the bioluminescence assay
does not discriminate between ATP release pathways. Evidence in other
epithelial cells suggests that one mechanism for cellular ATP release
is electrodiffusional, with charged ATP molecules (ATP4) permeating the
cell membrane via selective channel pores (30, 34). Consequently,
modified whole cell patch-clamp techniques (100 mM MgATP on both sides
of the membrane as the principal anionic charge carrier) were utilized
to measure whole cell ATP currents in Mz-ChA-1 cells. As shown in Fig.
2B, in isotonic buffer a basal ATP
conductance was detected, which was nearly linear and reversed near 0 mV, compatible with ATP currents previously described
(n = 7) (27, 30). Similar to
observations using the luciferase-luciferin assay, exposure to
hypotonic buffer (~15% reduction in osmolarity) led to a >10-fold
increase in ATP current density, increasing from
1.34 ± 1.61 to
16.82 ± 4.49 pA/pF at
80 mV
(n = 7, P < 0.001). Thus human biliary cells
possess membrane channels permeable to ATP, and conductive movement of
charged ATP molecules represents one potential pathway for basal and
volume-sensitive ATP efflux.
ATP release contributes to cell volume homeostasis.
In previous studies, both P2 receptor binding and increases in cell
volume have been identified as potent stimuli that increase membrane
Cl permeability (22, 29).
Therefore, additional studies were performed to assess the roles of
volume-sensitive ATP efflux and P2 receptor stimulation in Mz-ChA-1
cell volume recovery (Fig. 3A).
Under control conditions, exposure of cells to hypotonic buffer
(~30% less mosmol) led to a rapid increase in mean cell volume
(n = 6). Peak swelling at 2.5 min
(relative volume 1.20 ± 0.01) was followed by RVD toward basal
values despite continued incubation in hypotonic buffer (relative
volume 1.08 ± 0.01 at 30 min). When the ATP scavenger apyrase (2 U/ml) or the P2 receptor blocker suramin (100 µM) were added to
hypotonic buffer, RVD was significantly inhibited
(n = 6 for each). However, addition of the nonhydrolyzable P2 receptor agonist ATP
S (20 µM) after peak swelling (5.5 min) nearly restored volume recovery in the presence of
apyrase (n = 6) but not suramin
(n = 5).
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Autocrine stimulation of P2 receptors regulates volume-sensitive
Cl channels.
Because Mz-ChA-1 cell volume recovery depends on channel-mediated
Cl
efflux, we then explored
the regulatory role of extracellular ATP as a modulator of the
volume-sensitive Cl
conductance using whole cell patch-clamp techniques. These results are
summarized in Fig. 4. In control cells,
exposure to hypotonic buffer (15% decrease in osmolarity) was followed
within 2 min by a large increase in
Cl
current density from
1.41 ± 0.26 to
37.47 ± 5.48 pA/pF (
80 mV,
n = 6, P < 0.001). The characteristics of
the volume-activated Cl
conductance included outward rectification and time-dependent inactivation at positive potentials as previously described. Addition of apyrase (3 U/ml, n = 4) to remove
extracellular ATP or suramin (100 µM,
n = 6) to block P2 receptors nearly
abolished volume-dependent current activation (
2.19 ± 0.71 and
3.42 ± 0.78 pA/pF, respectively). Notably, current inhibition by apyrase and suramin was reversible; after washout of reagent-containing buffer, reexposure of individual cells to hypotonic buffer restored volume-dependent current activation.
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Vectorial ATP release in polarized cholangiocyte monolayers.
Cholangiocytes in vivo are highly polarized, and transepithelial
secretion depends on opening of
Cl channels in the apical
membrane. In contrast to Mz-ChA-1 cells, NRCs more closely resemble
cholangiocytes in vivo, forming polarized monolayers with intercellular
tight junctions. The kinetics of vectorial ATP release from apical and
basolateral membranes was determined separately, and the results are
shown in Fig. 5. Similar to Mz-ChA-1 cells,
basal ATP release was detected from both domains and was notably
greater in apical (8.51 ± 0.64 ALU,
n = 7) compared with basolateral
membranes (2.41 ± 0.05 ALU, n = 8, P < 0.001). In contrast to
Mz-ChA-1 cells, addition of isotonic buffer did not significantly
affect ATP permeability. However, graded buffer dilutions led to a
rapid, sustained increase in the rate of ATP release from both
membranes; bioluminescence peaked at 19.20 ± 1.36 and 26.86 ± 1.47 ALU (apical) and at 11.39 ± 0.89 and 17.83 ± 1.41 ALU
(basolateral) after 20 and 40% decreases in osmolarity, respectively.
Thus polarized cholangiocytes exhibit vectorial basal and
volume-sensitive ATP release.
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Volume-activated transepithelial
Cl currents require extracellular
ATP.
If volume-sensitive Cl
channels are present in the apical membrane, then increases in cell
volume would be anticipated to increase
Isc, the
electrophysiological equivalent of transepithelial secretion.
Therefore, additional experiments were performed to assess the effect
of cell volume on
Isc and the
results are summarized in Fig. 6 and Table
1. Depending on the study day, exposure of monolayers to hyposmolarity (30% less mosmol) led to a 41-69% increase in Isc
that peaked at ~1 min; a representative recording is shown in Fig.
6. Current magnitude and time
course were similar after induction of swelling by exchange of
mannitol-containing with mannitol-free buffer (same electrolyte
composition) or standard buffer with buffer containing less NaCl. In
the first group of control studies, simultaneous reduction of apical
and basolateral buffer osmolarity increased
Isc from basal
values (8.68 ± 1.21 µA/cm2) by 4.52 ± 0.43 µA/cm2 within 1 min
(n = 7, P < 0.01). Volume-activated
transepithelial currents appear to be mediated by an increase in apical
Cl
permeability because
1) amiloride (100 µM) in the
apical buffer to inhibit Na+
absorption via apical membrane Na+
channels had no effect
(
Isc 4.83 ± 0.51 µA/cm2,
n = 5) and
2) addition of the anion channel
blocker NPPB (20 µM) to the apical bathing solution nearly abolished
volume-dependent current activation
(
Isc 0.54 ± 0.27 µA/cm2,
n = 7).
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DISCUSSION |
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Intrahepatic bile ducts play a critical role in controlling the volume
and composition of bile, but their small size and intrahepatic location
have limited efforts to ascertain the cellular mechanisms involved (9).
The identification of cystic fibrosis transmembrane conductor regulator
(CFTR), the protein product of the cystic fibrosis gene, in
cholangiocytes implies that transepithelial transport of
Cl represents one mechanism of secretion; hormonal
stimulation is thought to stimulate bile formation through
cAMP-dependent opening of CFTR-associated
Cl
channels in the apical
membrane (7, 33). More recently, alternative modes for regulation of
secretion through autocrine-paracrine networks within the liver have
been emphasized. The present studies provide important support for
extracellular ATP as such a signal and suggest the presence of dynamic
functional interactions between cholangiocyte cell volume, ATP release,
and purinergic stimulation in the regulation of biliary
Cl
secretion and bile formation.
The principal observations of these studies using human and rat biliary
cell models are that 1)
cholangiocytes exhibit regulated release of ATP into the extracellular
space, 2) increases in cholangiocyte volume represent a potent stimulus for ATP efflux, and
3) the localized increase in
extracellular ATP contributes at the cellular level to recovery from
swelling and at the tissue level to transepithelial Cl secretion though
activation of P2 receptors in the plasma membrane. These findings are
consistent with the recent demonstration of P2Y2 receptors in the apical
membrane of cholangiocytes (31) and with emerging evidence that P2
receptors play an important modulatory role regulating secretion in
many epithelia, including airway, intestine, and pancreas (17, 18, 25).
Although purinergic receptors have been identified in most epithelia,
there is considerable controversy regarding the cellular origins of ATP
and the mechanisms involved in its release. Membrane stress represents
one stimulus for ATP release by hepatocytes (32) and other cell types
(2, 11, 12), but the physiological relevance of mechanical stimulation
to cholangiocytes is not readily apparent. For example, ATP in
concentrations sufficient to bind to P2 receptors is present in human
bile and is released into sinusoidal blood in the isolated perfused
organ in the absence of obvious mechanical changes (5, 26). The present
observations, using a sensitive luminometric assay, indicate that ATP
release by biliary cells occurs independently of membrane perturbation or other stimulation and that extracellular ATP plays a physiological role in cell volume homeostasis. Exposure to apyrase results in an
increase in cell volume. This effect appears specific for extracellular ATP removal because intracellular ATP concentrations are unaffected (unpublished observations) and cell swelling is reversed by
simultaneous exposure to ATPS, a nonhydrolyzable P2 receptor agonist.
In contrast to the modest effects of media addition on ATP release, increases in cell volume resulted in rapid and sustained increases in extracellular ATP. Moreover, changes in membrane ATP permeability are reversible, recovering within 2 min of restoration of extracellular osmolarity. This finding is of great interest because all cells are subject to substantial osmotic challenges as a result of normal transport and metabolism (1) and because volume-sensitive ATP release has been demonstrated recently in several cell types with quite different biological functions including primary hepatocytes, hepatoma cells, fibroblasts, and ciliary epithelia (24, 28, 36, 40). Taken together, these findings suggest that volume-sensitive ATP release pathways may be highly conserved.
Exposure to ATP has been shown previously to increase
Cl permeability of
cholangiocytes and other secretory epithelia. The present studies
suggest two potential physiological roles for volume-sensitive ATP
release. First, at the cellular level, release of ATP into the
extracellular space and P2 receptor stimulation appear to be necessary
for cell volume recovery during hypotonic exposure. Because a similar
role for ATP release in cell volume regulation has been identified in
both model and primary hepatocytes (28, 40), this might represent a
mechanism shared by multiple liver cell types.
Complementary studies in NRC monolayers support a second potential
physiological role for ATP release in the regulation of transepithelial
Cl secretion. In intact rat
liver, hypotonic perfusion to induce cell swelling leads to an increase
in bile acid secretion and bile formation (3). The present studies
suggest that endogenous volume-sensitive release of ATP from
cholangiocytes into bile may stimulate ductular secretion through
binding of P2 receptors and opening of
Cl
channels in the apical
membrane (Fig. 7). It is notable that the inhibitory effects of ATP
removal and P2 receptor blockade on volume-sensitive increases in
Isc are
incomplete, implying the presence of additional P2 receptor-independent
anion conductance pathways as well. Hepatocytes, the principal liver
parenchymal cell, also invest considerable cellular resources in the
release and metabolism of ATP at the apical (canalicular) membrane (6). In a similar fashion, any ATP derived from hepatocytes that escapes luminal degradation would have direct access to the apical membrane of
cholangiocytes located downstream as bile flows away from its canalicular origins within the network of intrahepatic ducts. Because
volume-sensitive ATP release has recently been demonstrated from
primary human hepatocytes (28), ATP may play a role in coordinating the
separate hepatocyte and ductular components of bile formation, a
process referred to as hepatobiliary coupling (Fig. 7). Such a
mechanism is analogous to that involving the peptide hormone
endothelin, which also exerts local control over ductular secretion by
binding to specific receptors in the apical membrane of cholangiocytes
(10).
Assuming that biliary ATP functions in this manner as a local signal
regulating cholangiocyte Cl
secretion and bile formation, several critical questions remain to be
answered. First, the molecular basis of membrane ATP permeability and
the cellular signals that control the permeability pathway(s) have not
been defined. Cytoplasmic ATP molecules are present inside cells in
concentrations much greater than those observed extracellularly, and
cytoplasmic ATP molecules exist largely in anionic forms. Thus opening
of an ATP pore or channel represents an attractive option and is
consistent with the presence of volume-sensitive currents carried by
ATP detected by whole cell recordings (Fig. 1). Permeation of
ATP molecules through selective membrane channels could account for the
rapid rise in bioluminescence observed after hypotonic challenge.
However, because of the high concentrations of ATP required to detect
these currents (100 mM), the relevance of these findings to
physiological cellular pathways is still conjectural. The specific
advantages of the luciferase-luciferin assay are an increased
sensitivity, an ability to measure ATP release from intact cells, and
the detection of both charged and electroneutral ATP molecules released
by any transport mechanism.
Second, it is not clear from these studies how much ATP is required to elicit cellular responses. Generation of ATP standard curves indicates that the amount of ATP present in extracellular media during cholangiocyte swelling is ~150-240 nM (Mz-ChA-1 cells) and ~90-115 nM (apical NRC membranes), values below the IC50 of ~300 nM observed for activation of P2Y2 receptors. These luminometric measurements, however, are likely to underestimate the local availability of ATP molecules at the membrane surface, where there is a dynamic interplay among release, degradation, and receptor binding. Third, changes in extracellular ATP levels detected by bioluminescence are dependent on the rates of ATP transport out of the cell and subsequent dephosphorylation by ecto-ATPases. At present, the relative contributions of these pathways are not established. Finally, whether P2 receptors in addition to the P2Y2 subtype contribute to volume-dependent current activation is unknown. Notably, the luciferase-luciferin assay does not detect ADP nor UTP, both of which may be enzymatically generated from ATP and contribute to cellular responses by stimulating distinct P2 receptor subtypes.
These and other studies suggest that regulated release of ATP may be a
general property of epithelial cells. Evidence herein using biliary
cell models supports the presence of dynamic functional interactions
among cell volume, ATP release, and membrane
Cl permeability that
contribute importantly to local regulation of
Cl
secretion and bile
formation. Thus characterization of the mechanisms involved in ATP
release and metabolism may offer new strategies for the pharmacological
manipulation of biliary secretion in genetic and acquired cholestatic
liver diseases, where ductular secretion is deficient and therapeutic
options are few. However, the universal presence and functional
diversity of purinergic receptors in many different epithelial cell
types suggest that alternative modes of ATP release are likely to exist
in other cells, with different cellular strategies tuned to match local
environmental cues and specific cellular functions.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46082, DK-43278, and K08-DK-02539-01, and by the Waterman Family Fund for Liver Research.
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FOOTNOTES |
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This study was presented at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, IL, November 1997.
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: R. M. Roman, Campus Box B-158, UCHSC, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: rick.roman{at}UCHSC.edu).
Received 3 December 1998; accepted in final form 5 February 1999.
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