1 Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 2 Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Bombesin, a neuropeptide, stimulates fluid
and HCO3 secretion from
cholangiocytes, but the underlying mechanisms are poorly
understood. In this study, we aimed to examine the effects
of bombesin on ion transport processes involved in the regulation of
intracellular pH (pHi) and
HCO
3 secretion in polarized
cholangiocytes. Isolated bile duct units from normal rat liver were
used to measure pHi by
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 495 nm-to-440 nm dual ratio methods. Bombesin increased
Cl
-HCO
3 exchange
activity but did not affect basal pHi or the activities of
Na+/H+ exchange or
Na+-HCO
3 symport.
Depolarization of cholangiocytes increased basal
pHi and the activity of
Cl
/HCO
3
exchange, suggesting that an electrogenic Na+-HCO
3
symport might function as a counterregulatory pHi mechanism.
Na+-independent acid-extruding
mechanisms were not observed. We conclude that bombesin stimulates
biliary secretion from cholangiocytes by activating luminal
Cl
/HCO
3
exchange, which may be coupled to basolateral electrogenic
Na+-HCO
3 symport.
bicarbonate secretion; ion transport; chloride-bicarbonate exchange; electrogenic sodium-bicarbonate symport
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INTRODUCTION |
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BOMBESIN IS A tetradecapeptide isolated from the skin of the European frog (2, 12) and chemically and biologically homologous to gastrin-releasing peptide (GRP) (25, 35). In the gastrointestinal tracts of various mammals (4, 6, 33, 42), bombesin and GRP are exclusively found in nervous tissue (10, 11, 24) and mediate diverse secretory and motor functions and release numerous peptides, such as neurotensin, motilin, insulin, CCK, secretin, and glucagon (13, 16, 22, 37). The presence of bombesin and GRP in enteric nerves and their potent effects on various gastrointestinal functions strongly suggest a potential role for endogenous GRP (bombesin) in gastrointestinal and liver physiology.
HCO3 secretion from cholangiocytes
plays an important physiological role through counteracting the
increase in acid loads resulting from the cephalic phase of gastric
acid secretion or during food digestion. The ion transport processes responsible for this basal or hormone-stimulated
HCO
3 secretion have been characterized
previously in cholangiocytes isolated from normal and bile
duct-obstructed rats, as well as a cholangiocyte cell line (1, 14, 39).
These studies (1, 14, 39) demonstrated the presence of two acid
extruders in the bile duct epithelium (BDE), an
Na+/H+
exchanger and an electrogenic
Na+-HCO
3 symporter, and
one acid loader, an
Na+-independent
Cl
/HCO
3
exchanger, which is located in the luminal membrane and is functionally
coupled to Cl
channels. It
was also shown (1) that secretin induces an
HCO
3-rich choleresis by stimulating
the activity of this
Cl
/HCO
3
exchanger in cholangiocytes indirectly via activation of
5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB)-sensitive
Cl
channels.
Bombesin and GRP are also known to increase an
HCO3-rich bile secretion in dogs and
pigs (16, 22, 23, 27), but the underlying mechanism or site of action
is poorly understood, partly because of the lack of adequate in vitro
models and complex interactions with other secretagogues.
Recently, we have shown (9) that bombesin stimulates biliary fluid and
HCO
3 secretion by acting at the level
of cholangiocytes, but not hepatocytes, and may mediate the neural
regulation of bile secretion. A dose-dependent fluid secretion in
isolated bile duct units (IBDU) was demonstrated over a wide range of
physiologically relevant bombesin concentrations from 0.1 to 100 nM.
Bombesin (10 nM)-stimulated fluid secretion in IBDU was mediated by
specific bombesin receptors and was almost completely inhibited after
omitting HCO
3 or Cl
from the perfusate,
suggesting a significant dependence on
HCO
3 and
Cl
(9). Moreover, this
stimulated secretion was associated with an increase in luminal pH in
IBDU and biliary HCO
3 secretion in
isolated perfused rat livers, consistent with an increased
HCO
3 secretion at the level of the bile ducts (9).
In this study, we have examined the effect of bombesin on the
H+/HCO3
transport processes involved in intracellular pH
(pHi) regulation, using a novel
functional polarized IBDU. IBDU have been used previously to study the
effects of secretin and bombesin on cholangiocytes and are an ideal
functional polarized cholangiocyte preparation to study bile ductular
secretion (9, 26). The present study is the first to show that the
major underlying ion transport mechanism mediating the
HCO
3-rich choleresis observed in
isolated perfused rat liver and IBDU during bombesin stimulation is the
Cl
/HCO
3
exchanger in cholangiocytes. Bombesin-stimulated increases in
Cl
/HCO
3
exchanger activity result in increased HCO
3 secretion coupled to basolateral
electrogenic Na+-HCO
3
symport. Unlike pig BDE (19, 44), there is no significant
Na+-independent counterregulatory mechanism present in rat
BDE.
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MATERIALS AND METHODS |
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Materials
BSA, penicillin/streptomycin, EDTA, heparin, HEPES, D-(+)-glucose, insulin, soybean trypsin inhibitor (type I-s), DMSO, hyaluronidase, DNase (DN-25), nigericin, amiloride, valinomycin, sodium gluconate, potassium gluconate, and hemicalcium gluconate were purchased from Sigma Chemical (St. Louis, MO). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was obtained from Molecular Probes (Eugene, OR). Bombesin was purchased from Bachem (Torrance, CA). Matrigel was from Collaborative Biomedical (Bedford, MA), collagenase D was from Boehringer Mannheim Biochemicals (Indianapolis, IN), and Pronase was from Calbiochem (San Diego, CA). L-15 medium Leibovitz, MEM,Solutions
The compositions of the Krebs-Ringer bicarbonate (KRB), HEPES, and gluconate buffer solutions have been described previously (1, 39). All peptides were made up in perfusion buffer with 1% (wt/vol) BSA as a carrier.Isolation of Bile Duct Units
Male Sprague-Dawley rats (Camm Laboratory Animals, Wayne, NJ) weighing 200-250 g were housed and allowed free access to water and Purina rodent chow (St. Louis, MO). Intrahepatic bile duct units (IBDU) were isolated as previously described by our laboratory and have been extensively characterized as polarized biliary epithelial cells with a central lumen (26).IBDU were plated on Matrigel-coated small glass coverslips in modified
-MEM medium as previously described (26), incubated at 37°C in
an air-5% CO2 equilibrated
incubator, and used between 18 and 30 h after plating. Viability by
trypan blue exclusion was consistently >95% and was not affected by
preincubations with KRB, HEPES, gluconate,
NH4Cl buffer solutions, and
valinomycin in KRB solution.
pHi Determination
pHi of IBDU was measured as previously described (26), using a SPEX-AR-CM Microsystem microfluorometric method (Spex Industries, Edison, NJ). Previously, a heterogeneous response was observed with respect to the initial luminal size of IBDU (9). IBDU with an initial luminal area <170 µm2 did not show secretory responses to bombesin, possibly due to heterogeneity in receptor distribution; thus these smaller IBDU were excluded from the present study. IBDU, incubated overnight on Matrigel-coated glass coverslips, were loaded with 12 µM BCECF-AM for 20-30 min, washed for 10-20 min with BCECF-free medium, and then transferred into a 37°C thermostated perfusion chamber on the stage of an inverted microscope (IM 35; Carl Zeiss, Thornwood, NY). IBDU easily took up BCECF with a uniform distribution throughout the cytoplasm, and leakage and photobleaching of the dye were negligible. To assess the activities of Na+/H+ exchange, Na+-HCOImmunofluorescent Staining of H+-ATPase in Rat Liver and IBDU
Rat liver and kidney cryosections were fixed for 10 min in 4% formaldehyde and 6% HgCl2 in 140 mM sodium acetate buffer. After a wash in 0.1 M PBS, the sections were blocked with 20% calf serum and 1% polyethylene glycol in PBS for 30 min. The primary antibody (anti-H+-ATPase) was used undiluted for 2 h at room temperature. The section were next washed in PBS and incubated in a goat anti-mouse secondary antibody for 1 h at room temperature, washed in PBS, and mounted in Vectashield (Vector, Burlingame, CA). Sections were viewed and photographed with a Nikon epifluorescence microscope.Statistical Analysis
All data from pH measurements are given as arithmetic means ± SD. Statistical differences were assessed by the unpaired or paired Student's t-test using the INSTAT statistical computer program (GraphPad Software, San Diego, CA). ![]() |
RESULTS |
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Effect of Bombesin on Basal pHi
In a HEPES, nominally HCOEffect of Bombesin on Recovery of pHi From an Acute Acid Load in HEPES
When IBDU were exposed to 20 mM NH4Cl, pHi increased promptly (Fig. 1), as the uncharged NH3 diffused into the cell and combined with intracellular H+. However, this process reversed when NH4Cl was withdrawn from the perfusate and NH3 leaves the cell after releasing H+, causing a rapid fall in pHi to 6.81 ± 0.05 (n = 11). In the absence of HCO
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Effect of Bombesin on Recovery of pHi from an Acute Acid Load in KRB
When isolated cholangiocytes from normal rat livers are maintained in an HCO
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Effect of Bombesin on Activity of
Cl/HCO
3
Exchange
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Study of Counterregulatory pH Mechanism for
Cl/HCO
3
Exchange Activation
Na+/H+
exchanger.
As shown in Fig. 1 and Table 1, bombesin did not have any significant
effect on the
Na+/H+
exchanger, because neither basal
pHi nor recovery rate of
pHi from an acute acid load in
HEPES medium was altered during bombesin stimulation. We also studied
the effect of a specific
Na+/H+
exchanger inhibitor, amiloride (1 mM), on basal
pHi with and without bombesin
stimulation in an HCO3-enriched medium. As shown in Fig. 4, there were no
significant changes in basal pHi
with administration of amiloride in KRB. In addition, the
pHi of IBDU in the presence of
amiloride also remained unaffected during superfusion with bombesin,
which was expected to increase the intracellular acid load from
stimulation of the
Cl
/HCO
3
exchanger activity. Thus, as observed previously with secretin in rat
cholangiocytes and pig BDE (20, 43), these results, in addition to the
findings from Table 1, suggest that the
Na+/H+
exchanger has no significant role in maintaining the
pHi of cholangiocytes either in
the basal state or after bombesin stimulation.
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Na+-HCO3
symporter.
This symporter is a major acid extruder in
HCO
3-containing medium, but, as shown
in Fig. 2 and Table 2, bombesin had no effect on
Na+-HCO
3 symporter
activity as assessed by the recovery rates from an acute acid load with
NH4Cl. However, unlike other pH
regulatory mechanisms, this electrogenic symport can also be activated
secondarily by changes in the membrane potential, as occurs with
Cl
channel activation.
Unfortunately, specific inhibitors for the Na+-HCO
3 symporter are not
available. Thus the role of this symporter as a counterregulatory
pHi mechanism was studied
indirectly by examining the effects of membrane depolarization on basal
pHi and
Cl
/HCO
3
exchange activities using valinomycin (0.5 µM) in KRB solution with
30 mM KCl substituted for equimolar NaCl (1, 46). As shown in Fig.
5 and Table 4,
when the membrane was depolarized by administration of valinomycin and
high K+, the basal
pHi increased by 0.05 ± 0.04 pHU (n = 9, P < 0.05), suggesting stimulation of
an electrogenic
Na+-HCO
3
symport resulting in an increased HCO
3 influx into the cholangiocytes. Furthermore, the increase in
HCO
3 influx that results from this
electrogenic
Na+-HCO
3
symport, in turn, increases
Cl
/HCO
3
exchange activity, as shown in Fig. 5 and Table 4. Together these
findings indicate that a basolateral electrogenic
Na+-HCO
3 symport is most
likely coupled to the luminal
Cl
/HCO
3
exchange by changes in the membrane potential and/or
HCO
3 concentrations and functions to counteract the increased intracellular acid loads resulting from bombesin-stimulated biliary HCO
3
secretion. In addition, these findings also offer a plausible
explanation for the apparent absence of change in the recovery rates
from NH4Cl acid loading during
bombesin stimulation, since this pH maneuver, which involves the same
amount of acid loading, may not adequately account for secondary
effects such as changes in membrane potential and/or
HCO
3 concentration gradients.
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Na+-independent
acid-extruding mechanism(s).
A vesicular H+-ATPase has been
proposed as another potential counterregulatory pH mechanism after
secretin-stimulated HCO3 secretion in
pig BDE (19, 44). However, its presence in rat or human cholangiocytes
has not been previously demonstrated (8, 40). As shown in Fig.
6, a rapid acidification of the
pHi of IBDU, which was observed by
substituting Na+ with choline
either in HCO
3-enriched or -free medium (data not shown) for 30-60 min, could not be overcome by administration of bombesin and is only reversed with readmission of
Na+. Moreover, as shown in Fig.
7, immunofluorescent staining of rat liver
and IBDU with H+-ATPase antibody
was also negative in contrast to the positive control in rat kidney, in
which H+-ATPase has been
previously demonstrated (50). These findings strongly suggest that an
Na+-independent acid-extruding
mechanism such as H+-ATPase is not
present in rat cholangiocytes.
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DISCUSSION |
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Although secretin has been thought to be the major regulator of biliary
HCO3 secretion, early physiological studies in dogs showed that an
HCO
3-rich choleresis increased not
only with feeding (28) but also with sham feeding (34), suggesting that
neural regulation may be involved in postprandial bile formation.
Recently, we have shown (9) that the neuropeptide bombesin can
stimulate biliary HCO
3 secretion in
the rat. These studies (9, 34) as a whole suggested that neuropeptides
such as bombesin stimulate biliary
HCO
3 secretion at physiological
concentrations and thus may play a significant role in a neurally
mediated HCO
3-rich choleresis.
The present study demonstrates for the first time that the
bombesin-stimulated HCO3 and fluid
secretion in BDE is mediated by an increase in
Cl
/HCO
3
exchanger activity, as assessed by rates of
pHi recovery in IBDU after
established Cl
removal and
readmission protocols (1, 26, 39). This increase in the
Cl
/HCO
3
exchanger activity occurred without affecting basal
pHi. In cholangiocytes (1, 39) and
in many other cell types, including hepatocytes (3, 47) and pancreatic
ducts (18, 30),
Cl
/HCO
3
exchange is the major acid-loading mechanism, and it mediates
HCO
3 secretion/absorption. Thus stimulation of this exchanger by bombesin can account for the increases
in biliary HCO
3 secretion previously demonstrated in isolated perfused rat livers and IBDU (9). Together,
these findings suggest that
Cl
/HCO
3
exchange is most likely to be the primary mechanism for
bombesin-stimulated HCO
3 and fluid secretion in cholangiocytes (7). This conclusion is further supported
by previous ion substitution studies demonstrating near complete
dependence of the bombesin response on the presence of both
Cl
and
HCO
3 in the medium (9).
In pancreatic duct cells (18, 30) and isolated cholangiocytes (1), an
increased HCO3 secretion via the
Cl
/HCO
3 exchanger
is thought to be coupled to an activation of
Cl
channels. Thus the
bombesin-stimulated secretion in cholangiocytes also likely involves an
activation of Cl
channels
that provides a favorable
Cl
gradient and electrical
membrane potential changes necessary for the stimulation of
Cl
/HCO
3
exchange and electrogenic
Na+-HCO
3
symport, respectively. Furthermore, this coupling of
Cl
channels with the
Cl
/HCO
3
exchanger at the luminal membrane, in turn, can provide a
"Cl
recycling"
mechanism whereby Cl
exits
cholangiocytes via Cl
channels on the apical side and recycles via the
Cl
/HCO
3
exchanger after bombesin stimulation. This mechanism leads to an
increased net vectoral HCO
3 secretion
accompanied by a passive net H2O
movement into bile during the bombesin-stimulated
HCO
3-rich choleresis, while
Cl
shuttles in and out of
the cholangiocytes at the luminal membrane via this recycling pathway.
Detailed electrophysiological studies would be needed to better
elucidate this mechanism. At present, it is not clear whether
Cl
fluxes also occur at the
basolateral membrane during this process.
Although an intracellular acid load should follow the
bombesin-stimulated increase in HCO3
secretion into bile from increased
Cl
/HCO
3
exchange activity, bombesin had no measurable effect on the basal
pHi of IBDU, suggesting the
presence of a counterregulatory pH mechanism(s) in cholangiocytes.
Several acid-extruding mechanisms have been described in
cholangiocytes, including
Na+/H+
exchange, H+-ATPase, and
Na+-HCO
3
symport (1, 39, 44). However,
Na+/H+
exchange is unlikely to play any significant role, since bombesin did
not affect either basal pHi in KRB
in the presence of amiloride or in HEPES or the recovery rates after an
NH4Cl acid load in HEPES medium.
This conclusion is consistent with previous findings (1) that the
Na+/H+
exchanger is minimally involved in basal
pHi maintenance in physiological HCO
3-containing medium and is only
active at low pHi. Moreover,
Na+/H+
exchange also does not play a major role in secretin-stimulated biliary
HCO
3 secretion in pigs (19) and in the
isolated guinea pig liver (5).
The present study also presents the first convincing functional
evidence that Na+-independent
acid-extruding mechanisms, such as
H+-ATPase or the
K+/H+
exchanger, are unlikely to be counterregulatory mechanisms in rat
cholangiocytes. As shown in Fig. 6,
pHi did not recover after acidification when Na+ is excluded
from the medium and infusion of bombesin also could not reverse this
acidification until Na+ is
replaced, strongly suggesting the functional absence of
Na+-independent acid-extruding
mechanisms. Furthermore, immunostaining of rat bile ducts with specific
antibodies to H+-ATPase (Fig. 7)
also failed to detect this exchanger, providing additional evidence
that rat cholangiocytes lack
H+-ATPase. In addition, the
absence of pHi recovery in
HCO3-containing medium when
Na+ is withdrawn also suggests
that reversal of the
Cl
/HCO
3
exchanger by admitting HCO
3 in
exchange for Cl
or
nonspecific HCO
3 influx through
certain ion channels, such as the cystic fibrosis transmembrane
conductance regulator, does not occur (or is not enough) to compensate
for the acidification of pHi.
Thus, unlike cholangiocytes in pigs but analogous to those in humans
(40), rat cholangiocytes do not have
Na+-independent acid-extruding
mechanisms.
Having excluded
Na+/H+
exchange and Na+-independent acid
extrusion as counterregulatory mechanisms, our findings are instead most consistent with HCO3 entry at the
basolateral membrane via
Na+-HCO
3
symport after bombesin-stimulated HCO
3 secretion. A similar mechanism has been previously proposed for secretin-stimulated HCO
3 secretion in
cholangiocytes (1) and in rabbit and guinea pig gallbladder epithelium
(32, 48). Unlike the Na+/H+ exchanger, the
Na+-HCO
3 symporter is
active in maintaining basal pHi of
cholangiocytes in HCO
3-containing
medium (1, 39) and is much more active at basal than at lower or higher
pHi in the basolateral membrane of
rabbit renal proximal tubules (38). Moreover, the electrogenic
Na+-HCO
3
symporter also can be activated secondarily by changes in membrane
potential (15, 36, 49). Interestingly, measurements of the primary
activity of this acid extruder, as assessed by the recovery rates from
NH4Cl acid loading in KRB medium
in the presence of amiloride, suggest that
Na+-HCO
3
symport is not directly stimulated by bombesin. There are two plausible
explanations for this absence of stimulatory effects of bombesin on
Na+-HCO
3
symport activity assessed by this method. First, this
NH4Cl pH maneuver, which causes
intracellular acidification/alkalization by shifting
H+-HCO
3
balance inside the cell, can also alter membrane potentials as shown in
hepatocytes (21). Therefore, this maneuver cannot directly evaluate the
effects of membrane potential changes resulting from bombesin
stimulation on the
Na+-HCO
3
symport activity. Alternatively, the known dependence of certain
Cl
channels on
pHi (31, 45) can result in
Cl
channel inactivation at
low pHi used to test the symport
activity. Such an inhibition of
Cl
channel activity at low
pHi, in turn, would prevent the
membrane depolarization expected with bombesin stimulation that would
be necessary to increase electrogenic
Na+-HCO
3 symport
activity.
To circumvent these problems, we assessed indirectly the role of
membrane depolarization as a secondary activator of the electrogenic Na+-HCO3
symport activity. As shown in Fig. 5, this study was performed near
basal pHi so that changes in
pHi should not significantly
affect the activities of various ion transporters and channels with
possible pHi dependence. Such
secondary activation of the electrogenic
Na+-HCO
3
symport is evident by an increase in basal pHi from
HCO
3 influx (Fig. 5 and Table 4) after depolarization of cholangiocytes by valinomycin and high
K+. Although less likely, this
increase in pHi could also be from an activation of K+-dependent
transporters such as
K+/H+
exchanger due to shifts in the K+
gradient. However, as discussed above, no significant
Na+-independent acid-extruding
mechanisms are present in cholangiocytes. Thus it is unlikely that this
increase in pHi is secondary to transporters such as K+/H+ exchanger.
In addition, as we have shown previously in isolated cholangiocytes
(1), the depolarization of cholangiocytes in IBDU by valinomycin and
high K+ also increased
Cl/HCO
3
exchange activity (Fig. 5 and Table 4), providing evidence for coupling
of the electrogenic
Na+-HCO
3
symport with
Cl
/HCO
3
exchange. Thus, when bombesin stimulates biliary
HCO
3 secretion, the electrogenic
Na+-HCO
3
symporter is secondarily activated, not only from the increased
HCO
3 concentration gradient due to an
increase in HCO
3 efflux as luminal Cl
/HCO
3
exchange is stimulated, but also from the membrane depolarization that
presumably occurs from Cl
channel activation. This secondary activation of the
Na+-HCO
3 symporter then,
in turn, may function to maintain the
pHi of the cholangiocytes.
Although the underlying ion transport mechanisms mediating bombesin-stimulated biliary secretion are quite similar to secretin, some important differences appear to exist. Unlike secretin, preliminary studies (7) suggest that the bombesin response is not dependent on the function of microtubules or on cAMP as a secondary messenger. In addition, previous Ca2+ measurements (29) indicate that bombesin does not change intracelluar Ca2+ in isolated cholangiocytes or IBDU, suggesting that bombesin-stimulated biliary secretion also does not involve the Ca2+ messenger system. Although preliminary, these findings suggest a unique signal transduction pathway for this bombesin response in cholangiocytes.
In summary (Fig. 8), the present study demonstrates for
the first time that bombesin stimulates luminal
HCO3 secretion by increasing
Cl
/HCO
3
exchange, presumably coupled to basolateral HCO
3 entry via electrogenic
Na+-HCO
3
symport mediated by changes in the
HCO
3 gradient and/or in the
membrane potential. In addition, we also present the
first functional evidence that, unlike pig cholangiocytes but analogous
to those in human BDE, rat cholangiocytes have no Na+-independent acid-extruding
mechanisms.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. F. Boron and J. Geibel for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-25636 and DK-34989 to J. L. Boyer and to the cell isolation, culture, organ perfusion, and morphology cores of the Yale Liver Center, respectively. W. K. Cho was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-07356 and an American Gastroenterology Association Advanced Research Award.
Part of this work was presented at the American Gastroenterology Association meeting in San Diego, CA, May 1995, and AASLD meeting in Chicago, IL, November 1995, and published in abstract form (Gastroenterology 108: A1049, 1995; Hepatology 22: 315A, 1995).
Address for reprint requests: W. K. Cho, Division of GI/Hepatology, Indiana Univ. School of Medicine, 975 West Walnut St., IB 424, Indianapolis, IN 46202.
Received 30 December 1997; accepted in final form 22 July 1998.
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