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Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Ion and water transport across the
teleost Oncorhynchus mykiss gallbladder were studied in vivo by
comparing flow and composition of hepatic bile, collected by chronic
catheter, to volume and composition of terminally collected gallbladder
bile. Differences in composition were comparable with those of other
vertebrates, whereas bile flow (75 µl · kg
1 · h1)
was below values reported for endothermic vertebrates. The gallbladder concentrates bile acids five- to sevenfold and exhibits higher net
Cl than Na+ transport in vivo, in
contrast to the 1:1 transport ratio from gallbladders under
saline/saline conditions. Transepithelial potential (TEP) in the
presence of bile, at the apical surface, was 
13 mV (bile side
negative) but +1.5 mV in the presence of saline. Bile acid in the
apical saline reversed the TEP, presumably by a Donnan effect. We
propose that ion transport across the gallbladder in vivo involves
backflux of Na+ from blood to bile resulting in higher net
Cl than Na+ flux. This Na+
backflux is driven by a bile side negative TEP and low Na+
activity in bile due to the complexing effects of bile acids.
transepithelial potential; teleost freshwater fish; hepatic bile flow; ion and water reabsorption; bile acid
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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BILE IS A HEPATIC SECRETION that functions to promote digestion and absorption of lipids from the intestine via the action of bile acids or bile salts. Bile also acts as the medium for excretion of many endogenous and exogenous substances from the blood and liver that are not excreted through the kidneys. Excretory functions in fish have been described in detail (see Ref. 35 for review). The mechanisms of hepatic bile production in fish, however, have received much less attention.
Notable exceptions are certain components of the enterohepatic bile salt circulation. Bile acid composition (7) and mechanisms of hepatic uptake of bile salts (25) in the teleost rainbow trout as well as several elasmobranchs (3, 11, 21, 26, 31, 32) are well characterized. Furthermore, carrier-mediated intestinal bile salt absorption has been reported in teleost fish (17). These studies reveal some differences among elasmobranchs, teleosts, and higher vertebrates in the mechanisms of basolateral membrane bile salt uptake. Reports on hepatic bile flow and composition (i.e., the result of canalicular membrane transport mechanisms) in lower vertebrates are limited to one study of bile flow and composition in elasmobranchs (2) and two studies on hepatic bile flow rates in teleost fish (12, 30). Consequently, one aim of the present study was to refine a technique to continuously collect hepatic bile in vivo for the characterization of bile flow and composition in the teleost rainbow trout (Oncorhynchus mykiss).
The role of the gallbladder in storing and concentrating bile in fish
is not well described. By contrast, the gallbladder epithelia of
various amphibians and mammals have been studied in great detail; this
tissue has become a model for epithelia that transport salt and water
at high rates and in isosmotic proportions ("leaky epithelia").
The structural simplicity, i.e., a monolayered epithelium with an
exclusive or predominant cell type, only a few basic transport
processes, and, for some species, large cell size facilitating
electrophysiological studies, makes it an appealing model system (for
comprehensive reviews, see Refs. 27 and 28). Some of the early,
classical work describing the transport processes of this epithelium
was conducted on a teleost fish (8-10). Isolated gallbladder
epithelia of roach (Rutilus rutilus) exhibited a 1:1 Na+- to Cl-transport ratio (9). Later,
this was also reported for Japanese eel (Anguilla japonica)
(16) and seems to also apply to isolated gallbladder epithelia of
higher vertebrates in general (reviewed by Reuss in Refs. 27 and 28).
However, when comparing the composition of hepatic bile with
gallbladder bile in several teleosts and one elasmobranch, Diamond (8)
observed that the Na+ concentration of gallbladder bile was
higher than the corresponding concentration in the hepatic bile,
whereas the opposite was true for Cl. This
phenomenon also applies to amphibians and mammals (8). Indeed Hunn (18)
reported much higher Na+ than Cl
concentrations in the gallbladder bile of 25 different teleost fish
species. These findings suggest that the gallbladder epithelium in vivo
has a higher net Cl transport rate than
Na+ transport rate and not a 1:1 Na+- to
Cl-transport ratio as observed in vitro.
Surprisingly, considering that this discrepancy has been evident for almost four decades, no studies on ion transport of the gallbladder epithelium in vivo have been reported, to our knowledge. In the present study, we refined a technique for continuously collecting hepatic bile for several days in rainbow trout. By comparing the hepatic output of bile acids and major electrolytes, as well as bile volume, with the gallbladder bile volume and composition at the same times, ion and water transport of the gallbladder epithelium was analyzed in rainbow trout during progressive starvation. Starvation was employed as a tool to ensure that bile collected in the gallbladder.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Rainbow trout, Oncorhynchus mykiss, were obtained from Humber
Springs Trout Farm, Ontario. The fish were held in 400-l fiberglass tanks (up to 100 fish/tank), each supplied with a flow-through of
dechlorinated aerated Hamilton city tap water (in mM: 0.6 Na+; 0.7 Cl; 1.0 Ca2+; and
1.9 HCO3, pH 7.9-8.2) at a rate of at least 5 liters/min. The fish were held at 14.0 ± 1.0°C and were fed a maintenance ratio of dry trout pellets (Martin's Feed Mill, Ontario) at a rate of 1% of their body mass per day.
Experimental design.
Bile was collected via chronic cannulation of the hepatic bile duct for
9 successive 12-h intervals (n 
9 fish in all
cases) and via terminal sampling from the gallbladder of noncannulated fish at 24, 48, 72, 96, 144, 168, and 240 h after last feeding (n = 10 at each sampling point). Gallbladder surface area was determined in 10 fish. In addition, transepithelial potential (TEP)
across the gallbladder (bile-to-blood fluid) was measured in vivo in
seven fish, and in vitro in 13 excised preparations. In the
latter, the influence of exchanging the mucosal bile for isotonic
saline and supplementing the saline with bile acids was also evaluated.
Surgical procedure.
Our surgical procedure is the product of extensive testing. The anatomy
of the gallbladder and bile duct system, and the common hepatic bile
duct cannulation technique are illustrated in Fig. 1. The ideal size of experimental animals
for this surgical procedure was ~225 g. Thirty-six hours of
starvation were found to provide the ideal conditions for the
cannulation of the common hepatic bile duct; the overall success rate
of this procedure was just under 50%. A total of 15 experimental
animals (average weight 232 g; range 179-278 g), out of 34 originally cannulated, contributed data to this experiment. The
remaining fish were not included in the experiment, because no bile was
obtained via the catheter in some or all of the 9 successive 12-h
intervals.
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Continuous collection of hepatic bile.
The fish were allowed to recover from surgery for 3-12 h in the
fish boxes. The catheter end was then placed in preweighed Eppendorf
tubes, which were kept on ice in styrofoam containers. At 12-h
intervals, the Eppendorf tubes and ice were replaced. The volumes of
bile collected were determined gravimetrically for all 9 12-h intervals
(i.e., 96 h). The samples were stored at 70°C. For the last
eight collection periods, the bile samples were analyzed for the
concentration of bile acid, Na+, Cl,
Ca2+, Mg2+, total CO2, and
osmolality as described in Analytical techniques. Bile from the
first collection period was not analyzed for composition, because it
was contaminated with the 140 mM saline initially present in the catheter.
Sampling of gallbladder bile.
Fish in a single large holding tank (400 liters) were fed to
satiation, and then groups of 10 fish were netted out of the holding
tank and anesthetized individually at 24, 48, 72, 96, 144, 168, and 240 h after feeding. A blood sample was drawn from the caudal artery with a
heparinized syringe, and plasma was obtained immediately by
centrifugation. The fish was then killed by a blow to the head,
weighed, and the gallbladder exposed by dissection. The gallbladder
bile was obtained by a 1-ml syringe fitted with a 26-gauge needle, and
volume was determined by weight using preweighed Eppendorf tubes.
Samples of plasma and gallbladder bile were frozen in liquid nitrogen
as soon as possible after sampling and stored at 70°C for
later analysis of bile acid. All bile samples and 10 randomly selected
plasma samples were analyzed for Na+,
Cl, Ca2+, Mg2+, total
CO2, and osmolality.
Determination of gallbladder surface area. Ten fish were netted out of the holding tank, anesthetized, killed by a blow to the head, and weighed. The entire gallbladder from each fish was obtained by dissection, opened with a longitudinal incision, and the surface area was determined using graph paper.
Determination of transepithelial potential in vivo and in
vitro.
For the determination of gallbladder TEP, "free flowing
bridges" (23) connected via AgCl electrodes to a
high-impedance voltmeter (Radiometer pHM84) were employed. The
substantial difference in Cl concentration between
gallbladder bile and plasma creates a significant junction potential
and thus inaccurate TEP measurements when AgCl electrodes are used in
combination with standard KCl-agar bridges (23). Free-flowing bridges
were constructed from barrels of disposable 1-ml plastic syringes that
were heated over a gas flame and pulled to form very thin and flexible
capillaries (~50 µm ID and 100 µm OD and ~50 cm long). The
modified syringe barrels were subsequently filled with 3 M KCl, and a
pressure of ~10 cmH2O was used to force the KCl solution
through the capillaries, resulting in a constant and similar
Cl concentration at the tip of the
free-flowing bridges. Because TEP recordings were performed
within 30 s, the low flow rate (<1 µl/min) negligibly
altered the Cl concentration in the bath or the
gallbladder (>500 µl). The AgCl electrodes were connected to
the KCl solution in the modified syringe for recording of
potential difference. Tests demonstrated that the junction
potential was reduced to <0.4 mV when the tip of the free-flowing
electrodes were bathed in asymmetrical solutions ranging from 1.5 to
150 mM NaCl. As an additional check, when one free-flowing electrode
was placed in saline and the other one in gallbladder bile connected to
the saline via a KCl-agar bridge, there was no junction potential
arising from differences in composition between saline and gallbladder bile.
Analytical techniques.
The Cl concentration of bile samples was determined
using the colorimetric assay of Zall et al. (37). Cations were analyzed using a Varian 1275 atomic absorption spectrophotometer (AAS) with
methods as documented by the manufacturer. The total
concentration of bile acids was determined using Sigma kit 450-A
modified for microtitre plate use. Osmolality of bile was measured
using a Wescor 5100C vapor pressure osmometer. The total
CO2 concentration of the bile was analyzed using a Corning
965 carbon dioxide analyzer.
70°C to establish that freezing did not influence Na+ activity.
Calculations, data presentation, and statistical evaluation.
Hepatic bile flow (expressed as
ml · kg1 · h1)
was calculated by relating the volume of bile collected over time to
the weight of the individual fish and the time elapsed. The summed bile
flow over time (Hepoutflow) was calculated as follows
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![Hep<SUB>outflow</SUB>(<IT>x</IT>) = <LIM><OP>∑</OP><LL><IT>T0</IT></LL><UL><IT>T1</IT></UL></LIM> {[V(<IT>x</IT>)]/W}](/content/vol278/issue6/fulltext/R1674/img002.gif)
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![GB(<IT>x</IT>) = V<SUB>GB</SUB>/[<IT>x</IT>]](/content/vol278/issue6/fulltext/R1674/img003.gif)
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hepatic bile flow and composition.
The mean bile flow rate in the common hepatic bile duct was constant at
~75
µl · kg1 · h1
during the entire 108 h of experimentation (Fig.
2A). There were significant
declines in the concentration of both bile acids (Fig. 2B) and
Na+ (Fig. 2D), which were complete by 60-72 h.
Over the same period, the Cl concentration rose
(Fig. 2C), although for Cl, the overall
effect of time was not significant. Osmolality of the hepatic bile
remained constant throughout (Fig. 2H). Concentrations of both
Mg2+ (Fig. 2F) and total CO2 (Fig.
2G) were low and constant, whereas Ca2+ levels
declined significantly by 60-72 h (Fig. 2E). Note that for
all biliary constituents except bile acids, concentrations in hepatic
bile were generally comparable with those in blood plasma (Table
1). Bile acid levels in plasma were below
the detection limit of the assay (20 µM) in contrast to 15-50 mM
in hepatic bile. This difference was associated with a small but
significant elevation in osmolality in hepatic bile (Fig. 2H)
relative to plasma (Table 1).
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Gallbladder bile volume and composition.
Total volume of bile in the gallbladder increased from 0.36 ± 0.14 ml/kg at 24 h after feeding to a maximum of 2.46 ± 0.14 ml/kg 120 h after feeding and stayed more or less constant
thereafter at ~2 ml/kg for up to 240 h (Fig.
3A).
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concentration of the
gallbladder bile did not change over time. However, in contrast
to Na+, the mean Cl concentration
in the gallbladder bile was much lower (18-31 mM) than the
corresponding concentration in the bile collected from the hepatic
common bile duct (~140 mM; Figs. 3C and 2C, respectively).
The mean Ca2+ concentration in the gallbladder bile
increased gradually with time after feeding (Fig. 3E). Thus by
the latter half of the experiment, the Ca2+ concentration
of the gallbladder bile was substantially higher than the corresponding
concentration of the bile collected from the hepatic common bile duct
(Fig. 2E). The mean Mg2+ concentration in the
gallbladder rose in a similar manner to values substantially higher
than the corresponding values from the hepatic bile (Figs. 2F
and 3F).
Total CO2 concentration in the gallbladder bile was
gradually reduced during 240 h of starvation (Fig. 3G) and was
much lower than the corresponding concentration in the hepatic bile
(Fig. 2G). Surprisingly, the osmolality of the gallbladder bile
remained much lower than might be expected from the concentrations of
the main constituents in the gallbladder bile (Fig. 3H).
Na+ and bile acid, the two major constituents in the
gallbladder bile together amounted to between 400 and 700 mM total
concentration throughout the entire 240 h of starvation. The
corresponding osmolality, however, remained constant and much lower at
around 305 mOsm (Fig. 3H), slightly lower than that in bile
collected from the common hepatic bile duct (~320 mOsm; Fig.
2H), but slightly higher than in blood plasma (Table 1). These
differences in osmolality are consistent with a reduction in
Na+ activity by conjugate formation between Na+
and bile acids (see DISCUSSION).
Fluid and ion reabsorption by the gallbladder epithelium.
By relating the accumulated hepatic bile output of fluid
(Hepoutflow) and solutes
[Hepoutput(x)] to the corresponding
gallbladder pools [GBV and GB(x), respectively] at
any given time, the function of the gallbladder epithelium in vivo can
be assessed. This is based on the assumption that no bile is leaving
the gallbladder via the cystic bile duct during starvation. An almost
perfect match between the calculated hepatic bile output of bile acids and the corresponding gallbladder pool during the entire experimental period (Fig. 4B) shows that this
assumption was justified. For both fluid and all other solutes, it is
evident that the gallbladder epithelium of freshwater rainbow trout is
indeed an active epithelium involved in reabsorption of solutes and
fluid (Fig. 4, A and C-F). By relating the differences
in hepatic output and the corresponding gallbladder pools to the
gallbladder epithelium surface area and the time elapsed, the net
transport rates of solutes and fluid by the gallbladder epithelium in
vivo could be calculated.
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was the
electrolyte reabsorbed at the highest rate (0.87 µmol · cm2 · h1)
by the gallbladder epithelium and that this rate was constant regardless of time elapsed since feeding (Fig. 4C).
Na+ was reabsorbed by the gallbladder epithelium as well,
however, at a lower and more variable rate than Cl.
Na+ reabsorption was 0.32 µmol · cm2 · h1
averaged over the entire 108 h of starvation (Fig. 4D). Fluid reabsorption by the gallbladder epithelium covaried with
Na+ reabsorption, although the relative fluctuations were
smaller. The mean fluid reabsorption rate over the entire 108 h was
5.07 µl · cm2 · h1.
A substantial fraction of the Ca2+, Mg2+, and
total CO2 secreted by the liver into the bile were
reabsorbed by the gallbladder epithelium (Fig. 4, E-G),
but due to the low concentration, the absolute reabsorption rates
(expressed as
µmol · cm2 · h1)
were very low. As expected, the gallbladder epithelium did not reabsorb
bile acid (Fig. 4B). Reabsorption rates for osmolality were not
calculated because Na+-conjugate formation in the bile (see
Ion transport by the gallbladder epithelium) would result in
large overestimation of this reabsorption rate. The
average concentrations of Cl, Na+,
Ca2+, Mg2+, and CO2 in the fluid
reabsorbed by the gallbladder epithelium over the entire 108 h were 171 mM, 63.1 mM, 0.2 mM, 1.3 mM, and 3.6 mM, respectively.
Na+ activity versus
concentration.
Ion-selective electrode measurements revealed a much lower mean
activity of Na+ ions (130 mM) in randomly selected
gallbladder bile samples in which total Na+ concentration
averaged 356 mM (Fig. 5). Na+
activity was not affected by freezing. The composition of rainbow trout
plasma is given in Table 1. As for bile, the Na+ activity
was lower than the Na+ concentration (Fig. 5). However, the
relative difference between the total Na+ concentration and
the Na+ activity in plasma was much lower than in
gallbladder bile (Fig. 5).
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TEP in vivo and in vitro.
The potential across the gallbladder epithelium in vivo was
approximately 13 mV (bile side negative relative to blood; Fig. 6). The same potential was recorded in
isolated gallbladders containing bile, with saline as a reference. In
contrast, the potential was approximately +1.5 mV (i.e., bile positive)
in isolated gallbladders when the bile was replaced with saline, i.e.,
under symmetrical conditions (Fig. 6). In several isolated
gallbladders, the original bile side negative potential (approximately
13 mV) was completely restored when the saline was replaced with
the original bile.
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1.0 and
2.4 mV, bile side negative at 100 and 200 mM, respectively (Fig.
6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hepatic bile flow and composition.
The technique employed for collecting hepatic bile from the common
hepatic bile duct revealed a constant hepatic bile flow in resting
starved restrained rainbow trout for up to 108 h after cannulation.
Thus progressive starvation from 36 h after last feeding (starvation
time before surgery) throughout this study did not influence hepatic
bile flow. The mean bile flow rate of ~75
µl · kg
1 · h1
observed in the present study is consistent with values of 50-75 µl · kg1 · h1
previously reported for rainbow trout (12, 30). The latter flow rates
were obtained from both spinally transected and free-swimming rainbow
trout during experimental periods of a few hours, and different
surgical procedures were employed. The constant bile flow rate over 108 h observed in the present study thus seems realistic. Because it is at
the high end of the previously reported values, we were apparently
successful in preventing contact between the hepatic bile and the
gallbladder epithelium and thereby preventing reabsorption of fluid
from the hepatic bile. The observed bile flow rate in freshwater
rainbow trout is comparable with bile flow rates of two marine
elasmobranchs, the spiny dogfish, Squalus acanthias, with a
flow rate of 33-74
µl · kg1 · h1
and the skate, Raja erinacea, with a rate of 75-111
µl · kg1 · h1
(2). The only bile flow rates reported from elasmobranchs and teleosts
are considerably lower than the bile flow rates of higher vertebrates:
the rat with a bile flow rate of 3,900; rabbit, 4,920; dog, 336; cat,
780; guinea pig, 9,600; and finally humans with a bile flow rate of 216 µl · kg1 · h1
(20, and references therein). At least in part, this difference could
be related to temperature, because bile flow is generated by the active
transport of bile acids and other organic anions and cations. The
studies of bile flow in elasmobranchs and teleost fish were conducted
at temperatures at least 20°C lower than the mammalian studies.
Indeed, increased temperature has been reported to increase
hepatobiliary excretion of the bile acid, taurocholate, in rainbow
trout (6, 19).
concentration. With the
exception of the bile acid concentration, the concentrations of all
measured ions were similar to the corresponding ion concentrations in
the plasma. This is in close agreement with observations reported for
the above mentioned species of elasmobranchs and mammals. Despite the
similarity in ionic concentrations, the osmolality of the hepatic bile
was higher than plasma osmolality. This difference can be accounted for
by the high concentration of bile acid in the hepatic bile compared
with the very low concentration in plasma (<20 µM), which provides
part of the driving force for paracellular diffusion of water and
electrolytes, thereby contributing to the bile flow (1, 5, 20, 22, 33).
In higher vertebrates, two components of bile formation have been
documented extensively: 1) bile acid-dependent bile flow and
2) bile acid-independent bile flow (for reviews, see Refs. 1,
5, and 22). In brief, transcellular, carrier-mediated transport of bile
acids (bile acid-dependent bile flow) and of other organic anions as
well as HCO3 and organic cations (bile
acid-independent flow) creates a hyperosmotic environment in the bile
canaliculi. This hyperosmotic environment drives a passive paracellular
movement of water and electrolytes from the blood plasma into the bile.
Consequently, the electrolyte composition of hepatic bile has been
shown to parallel that of the blood plasma (in vivo studies) or
perfusate (in vitro studies).
The bile acid concentration in the hepatic bile of rainbow trout in the
present study (15-50 mM) is within the range reported for mammals
(20, 33), and a bile acid-dependent component to bile flow in rainbow
trout seems likely. The reduced bile acid concentration and thereby
reduced hepatic bile acid output observed as time progressed was,
however, not parallelled by a reduced bile flow. This could indicate
that even the lowest bile acid concentration observed (15 mM) was
sufficient for maintaining a constant bile flow, or that the bile
acid-independent component to bile flow could account for most, if not
all, of the bile formation. It is interesting that the osmolality of
the hepatic bile remained constant throughout the experimental period
despite the marked reduction in bile acid concentration. This could
indicate that the concentration of other components, such as
Cl (which was in fact increased), glutathione,
bilirubin, and/or organic cations (all involved in the bile
acid-independent bile flow) were increased in compensation
for the reduced bile acid concentration.
Gallbladder bile volume and composition. In the present study, progressive starvation of the experimental animals was used as a tool to collect bile in the gallbladder. The conservation of bile acid accumulation in the gallbladder (Fig. 4B) demonstrate that the approach was successful.
The maximal gallbladder bile volume in the rainbow trout is comparable with volumes reported for European eel, Anguilla anguilla (14), and a number of other marine and freshwater teleost fish species after 5-10 days of starvation (M. Grosell, C. M. Wood, and F. B. Jensen, unpublished data). The ionic composition of the gallbladder bile is very similar to compositions reported for other teleost fish species, reptiles, amphibians, and mammals (8, 18). For these organisms, gallbladder bile is characterized by Na+ concentrations exceeding those in hepatic bile and plasma and Cl
concentrations much lower than than those in hepatic bile and plasma.
The absolute concentrations of Na+ and
Cl in plasma, and thus in hepatic bile of marine
elasmobranchs, are quite different from those of higher vertebrates,
including teleost fish, due to their unique osmoregulatory stategy. The relative relationships, however, are the same as in the higher vertebrates, i.e., higher Na+ and lower
Cl concentrations of the gallbladder bile.
Ion transport by the gallbladder epithelium.
All these findings indicate that the gallbladder epithelium in vivo has
a higher net transport of Cl than of
Na+. Furthermore, these observations suggest that the
mechanism of this asymmetrical anion-to-cation transepithelial
transport ratio could be similar throughout a wide phylogenetic range
of vertebrates. This analysis is in contrast to all reported findings
of ion-transport properties of isolated gallbladder epithelia
determined under "symmetrical" conditions in vitro in the absence
of bile acid on the mucosal side (see introduction) and cannot be
explained by the current proposed transport models. Consequently, we
propose an alternative model for transepithelial ion transport based on the present analysis of in vivo ion transport by the gallbladder epithelium of freshwater rainbow trout (Fig.
7).
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is
transported in exchange for HCO3 and
Na+ in exchange for H+ into the epithelial cell
across the apical membrane. The catalytic action of carbonic anhydrase
in the gallbladder reforms CO2, which diffuses back into
the cell where it is hydrated and refuels the cycle of
Cl/HCO3 and
Na+/H+ exchange. Na+ is extruded
across the basolateral membrane by the
Na+/K+-ATPase in a 3:2 exchange for
K+, and K+ subsequently acts together with
Cl as a substrate for a basolateral
K+/Cl cotransporter mediating
Cl transport across the basolateral membrane. To
account for a 1:1 transepithelial
Na+/Cl-transport ratio in this model, in
which two K ions are available for K+/Cl
cotransport while three Na ions are extruded across the basolateral membrane, Reuss suggests diffusion of Na+ back across the
basolateral membrane to refuel the
Na+/K+-ATPase. This model proposed by Reuss
(27, 28) elegantly accounts for the transepithelial net
Na+/Cl-transport ratio observed in
vitro under symmetrical conditions. It comes short, however, in
explaining the evidently higher net Cl transport
than Na+ transport in most (if not all) vertebrates in
vivo in which bile rather than saline is present at the apical
surface of the gallbladder epithelium.
We propose a transepithelial rather than a basolateral membrane
recycling of Na+ to account for the low Na+ net
flux compared with Cl net flux. In this model,
Na+ and Cl are extruded across the
basolateral membrane and Na+, in part, diffuses back across
the epithelium. This diffusion of Na+ back across the
epithelium reduces the net Na+ flux across the epithelium
and can thus account for a lower Na+ than
Cl transepithelial net flux. At first glance, the
diffusion of Na+ from the lower plasma Na+
concentration (152 mM) to the apparently higher Na+
concentration in the gallbladder bile (350 mM) seems unlikely. This
diffusion, however, is possible because of the low free Na+
activity in the gallbladder bile (130 mM in bile vs. 114 mM in plasma;
Fig. 5) and the TEP of 13 mV (bile side negative). The equilibrium potential required for maintaining this observed
Na+ activity gradient is approximately 7 mV (bile
side negative). Thus there are thermodynamically favorable conditions
for the proposed Na+ backflux. The low Na+
activity and reduction in osmolality in gallbladder bile observed in
the present study is in agreement with observations reported by Diamond
(8) and is due to the formation of bile acid-Na+
conjugates. Our observation that the addition of 100 mM hydrocholic acid (as Na salt) to saline only increased the Na+ activity
by 29 mM demonstrates this Na-conjugate formation in the presence of
bile acid acid.
The TEP of 13 mV (bile side negative) in the presence of bile at
the apical surface is unusually high for a leaky epithelium and cannot
be explained by the higher net Cl than
Na+ transport. The TEP is abolished when bile is replaced
with saline, indicating that the presence of one or more components of
gallbladder bile causes a diffusion or Donnan potential. Impermeant
anions have previously been suggested to be the reason for the lumen negative potential observed in isolated rat hepatocyte couplets (13).
If the substantial cation-to-anion gap in gallbladder bile is
considered, the presence of an anion other than Cl
in gallbladder bile seems highly likely, and bile acids are a probable
candidate. The concentration of impermeant anions needed to explain the
bile negative TEP of 13 mV can be estimated to be ~50 mM from
the following equation (1)
![<IT>e</IT><SUP>[(<IT>FR</IT>/<IT>T</IT>)(TEP)]</SUP> = (1 + A<SUP>−</SUP>/Cl<SUP>−</SUP>)<SUP>1/2</SUP>](/content/vol278/issue6/fulltext/R1674/img004.gif)
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is the impermeant anion, F is Faraday's
constant, R the gas constant, T is absolute
temperature, and the Cl concentration is set to 25 mM, the mean value at 240 h (see Fig. 3). Bile acids could account for
this 50 mM concentration of impermeant anions. The average
concentration of bile acid in the gallbladder bile was variable
(101-359 mM) but averaged ~220 mM at 240 h. This value is
substantially higher than the concentration of impermeant anions needed
to explain the TEP. However, Na+-bile acid conjugate
formation reduced the free Na+ activity by ~65% (Fig.
5). Similarly, the activity of the bile acid anion must be comparably
reduced by Na+-bile acid conjugate formation, which leaves
a predicted bile acid free anion concentration close to the 50 mM of
impermeant unknown anion needed to explain the TEP of 13 mV. In
isolated gallbladders, the addition of bile acid to the luminal saline under saline/saline conditions revealed that this impermeant anion can
indeed reverse the TEP from blood side negative to bile side negative.
Even addition of 200 mM hydrocholic acid (as Na salt), however, was not
as potent as gallbladder bile in terms of generating bile side negative
TEP. This could be due to the difference in ionic composition of the
saline and gallbladder bile and/or different characteristics of bile
acids, but could also indicate that other components in gallbladder
bile could act as impermeant anions.
On the basis of the observed ion fluxes, Cl
transport clearly exceeds the total recorded cation transport. Not all
cations were, however, included in the present study. K+
could have contributed to the total cation transport, but, given the
low K+ concentration in blood plasma and thus in hepatic
bile, it is unlikely that K+ alone could account for the
cation gap. Possibly, net acidic equivalent transport (not measured)
could explain the remaining cation gap maintaining macroscopical
electroneutrality. The observed cation gap was ~0.4
µmol · cm2 · h1,
and the gallbladder surface area was 11 cm2/kg, which would
be associated with a net acid flux of only 4.4 µEq · kg1 · h1
from bile to blood, which would be negligible relative to the net
acidic equivalent flux of the whole animal during starvation (36).
The water reabsorption of the gallbladder in vivo in the present study
was 4.5-6.0
µl · cm2 · h1,
which is approximately half of the values reported by Hirano and Bern
(16) and Diamond (10) for isolated gallbladders of a number of teleost
fish species under symmetrical saline/saline conditions in
vitro. The osmolality of the rainbow trout gallbladder bile is similar
but slightly higher than the corresponding plasma osmolality (a
difference of 7-10 mOsm). If assumed that the interstitial fluid
and plasma osmolalities are similar, it thus appears that water is
transported against an osmotic gradient. Water transport against an
osmotic gradient was also observed by Diamond (10) in isolated
gallbladders of teleost fish. Diamond tested the effect of osmolality
difference on the water transport rate and found water transport
against an osmotic gradient of up to 30 mOsm. At an osmotic difference
of 10 mOsm as in the rainbow trout in vivo, Diamond (10) found water
transport rates of 7-8
µl · cm2 · h1,
in close agreement with the in vivo water transport rates determined in
this study.
Perspectives
The present investigation suggests that the currently accepted model for ion transport by the gallbladder may not apply to in vivo conditions. Further studies on the ion-transport properties of the gallbladder epithelium are needed to verify our proposed model. These studies would have to employ isolated gallbladder epithelia but should now include the potential role of bile acids and other gallbladder bile components in modulating transepithelial net ion fluxes and TEP. The influence of temperature and bile acid concentration on hepatic bile flow in lower vertebrates remains unclear. Studies employing intravenous infusion of bile acids combined with continuous hepatic bile collection at different temperatures are needed to evaluate the presence of a bile acid-dependent bile flow and temperature-dependent bile flow in lower vertebrates.| |
ACKNOWLEDGEMENTS |
|---|
We greatly appreciate the excellent technical assistance of Erin Fitzgerald.
| |
FOOTNOTES |
|---|
This work was supported by a Danish National Research Council grant (9700849) to M. Grosell and National Sciences and Engineering Research Council research grants to C. M. Wood and M. J. O'Donnell.
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: M. Grosell, McMaster Univ., Dept. of Biology, 1280 Main St. West, Hamilton, ON, Canada L8S 4K1 (E-mail: grosellm{at}mcmaster.ca).
Received 2 June 1999; accepted in final form 11 January 2000.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Boyer, JL,
Graf J,
and
Meier PJ.
Hepatic transport systems regulating pHi, cell volume and bile secretion.
Annu Rev Physiol
54:
415-438,
1992[ISI][Medline].
2.
Boyer, JL,
Schwarz J,
and
Smith N.
Biliary secretion in elasmobranchs. I. Bile collection and composition.
Am J Physiol
230:
970-973,
1976.
3.
Boyer, JL,
Schwarz J,
and
Smith N.
Biliary secretion in elasmobranchs. II. Hepatic uptake and biliary excretion of organic anions.
Am J Physiol
230:
974-981,
1976.
4.
Brown, AC.
Biophysics of transport across membranes.
In: Physiology and Biophysics (19th ed.), edited by Ruch TC,
and Patton HD.. Philadelphia and London: W. B. Saunders, 1965, 820-842.
5.
Curtis, BJ,
and
Wood CM.
Renal and urinary bladder responses of trout to isosmotic NaCl and NaHCO3 loading.
J Exp Biol
173:
181-203,
1992[Abstract].
6.
Curtis, LR,
Kemp CJ,
and
Svec AV.
Biliary excretion of [14C]taurocholate by rainbow trout (Salmo gairdneri) is stimulated at warmer acclimation temperature.
Comp Biochem Physiol
84C:
87-90,
1986.
7.
Denton, JE,
Yousef MK,
Yousef IM,
and
Kuksis A.
Bile acid composition of rainbow trout, Salmo gairdneri.
Lipids
9:
945-951,
1974[ISI][Medline].
8.
Diamond, JM.
The reabsorptive function of the gallbladder.
J Physiol (Lond)
161:
442-473,
1962.
9.
Diamond, JM.
The mechanism of solute transport by the gallbladder.
J Physiol (Lond)
161:
474-502,
1962b.
10.
Diamond, JM.
The mechanism of water transport by the gallbladder.
J Physiol (Lond)
161:
503-527,
1962c.
11.
Fricker, G,
Hugentobler G,
Meier PJ,
Kurz G,
and
Boyer JL.
Identification of a single sinusoidal bile salt uptake system in skate liver.
Am J Physiol Gastrointest Liver Physiol
253:
G816-G822,
1987
12.
Gingerich, WH,
Weber LJ,
and
Larson RE.
Hepatic accumulation, metabolism and biliary excretion of sulfobromophthalein by rainbow trout (Salmo gairdneri).
Comp Biochem Physiol
58C:
113-120,
1977.
13.
Graf, J,
Henderson RM,
Krumpholz B,
and
Boyer JL.
Cell membrane and transepithelial. Voltages and resistance in isolated rat hepatocyte couplets.
Membrane Biol
95:
241-254,
1987.
14.
Grosell, MH,
Hansen HJM,
and
Rosenkilde P.
Cu uptake, metabolism and elimination in fed and starved European eels (Anguilla anguilla) during adaptation to water-borne Cu exposure.
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol
120:
295-305,
1998[ISI][Medline].
15.
Grosell, MH,
Hogstrand C,
and
Wood CM.
Cu uptake and turnover in both Cu acclimated and non-acclimated rainbow trout (Oncorhynchus mykiss).
Aquat Toxicol (Amst)
38:
257-276,
1997.
16.
Hirano, T,
and
Bern HA.
The teleost gall bladder as an osmoregulatory organ.
Endocrinol Jpn
19:
41-46,
1972[Medline].
17.
Hokanen, RE,
and
Patton JS.
Bile salt absorption in killifish intestine.
Am J Physiol Gastrointest Liver Physiol
253:
G730-G736,
1987
18.
Hunn, JB.
Inorganic composition of gallbladder bile from freshwater fishes.
Copeia
3:
602-605,
1975.
19.
Kemp, CJ,
and
Curtis LR.
Thermally modulated biliary excretion of [14C]taurocholate in rainbow trout (Salmo gairdneri) and the Na+,K+-ATPase.
Can J Fish Aquat Sci
44:
846-851,
1987.
20.
Klaassen, CD,
and
Watkins JB.
Mechanisms of bile formation, hepatic uptake, and biliary excretion.
Pharmacol Rev
36:
1-67,
1984[ISI][Medline].
21.
Miller, DS,
Fricker G,
Schramm U,
Henson JH,
Hager DN,
Nundy S,
Ballartori N,
and
Boyer JL.
Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters.
Am J Physiol Gastrointest Liver Physiol
270:
G887-G896,
1996
22.
Nathanson, MH,
and
Boyer JL.
Mechanisms and regulation of bile secretion.
Hepatology
14:
551-566,
1991[ISI][Medline].
23.
Neher, E.
Correction for liquid junction potentials in patch-clamp experiments.
Methods Enzymol
207:
123-131,
1992[ISI][Medline].
24.
O'Donnell, MJ,
Dow JAT,
Huesmann GR,
Tublitz NJ,
and
Maddrell SHP
Separate control of anion and cation transport in Malpighian tubules of Drosophila melanogaster.
J Exp Biol
199:
1163-1175,
1996[Abstract].
25.
Råberg, CMI,
Ziegler K,
Isomaa B,
Lipsky MM,
and
Eriksson JE.
Uptake of taurocholic acid and cholic acid in isolated hepatocytes from rainbow trout.
Am J Physiol Gastrointest Liver Physiol
267:
G380-G386,
1994
26.
Reed, JS,
Smith ND,
and
Boyer JL.
Determinants of biliary secretion in isolated perfused skate liver.
Am J Physiol Gastrointest Liver Physiol
242:
G319-G325,
1982
27.
Reuss, L.
Ion transport across gallbladder epithelium.
Physiol Rev
69:
503-545,
1989
28.
Reuss, L.
Salt and water transport by the gallbladder epithelium.
In: Handbook of Physiology. The Gastrointestinal System. Bethesda, MD: Am. Physiol. Soc, 1991, sect. IV, p. 303-322.
29.
Robinson, RA,
and
Strokes RH.
Electrolyte solutions (2nd ed.). London: Butterworths, 1970.
30.
Schmidt, DC,
and
Weber LJ.
Metabolism and biliary excretion of sulfobromophthaein by rainbow trout (Salmo gairdneri).
J Fish Res Board Can
30:
1301-1308,
1973.
31.
Smith, DJ,
Grossbard M,
Gordon ER,
and
Boyer JL.
Isolation and characterization of a polarized isolated hepatocyte preparation in the skate, Raja erinacea.
J Exp Zool
241:
291-296,
1987a[ISI][Medline].
32.
Smith, DJ,
Grossbard M,
Gordon ER,
and
Boyer JL.
Taurocholate uptake by isolated skate hepatocytes: effect of albumin.
Am J Physiol Gastrointest Liver Physiol
252:
G479-G484,
1987
33.
Strange, RC.
Hepatic bile flow.
Physiol Rev
64:
1055-1102,
1984
34.
Wolf, K.
Physiological salines for freshwater teleosts.
Prog Fish Cult
25:
135-140,
1963.
35.
Wood, CM.
Excretion.
In: Physiology and Ecology of the Pacific Salmon, edited by Groot C,
Margolis L,
and Clarke WC.. Vancouver: UBC Press, 1995.
36.
Wood, CM.
Acid-base and ionic exchanges at the gills and kidney after exhaustive exercise in the rainbow trout.
J Exp Biol
136:
461-481,
1988
37.
Zall, DM,
Fisher D,
and
Garner MD.
Photometric determination of chlorides in water.
Anal Chem
28:
1665-1678,
1956.
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