Enterohepatic circulation of scymnol sulfate in an
elasmobranch, the little skate (Raja
erinacea)
Gert
Fricker1,
Ralph
Wössner2,
Jürgen
Drewe3,
Ruth
Fricker4, and
James L.
Boyer5
1 Institut für
Pharmazeutische Technologie und Biopharmazie, D-69120 Heidelberg,
Germany; 2 Novartis AG, 4002 Basel;
3 Medizinische Poliklinik und
Division für Gastroenterologie, Kantonsspital, 4031 Basel,
Switzerland; 4 Department of
Anesthesiology, Allgemeines Krankenhaus,University Hospital, A-1000
Vienna, Austria; 5 Liver Center,
Yale University School of Medicine, New Haven, Connecticut
06510
 |
ABSTRACT |
The sulfated
bile alcohol scymnol sulfate (ScyS),
3
,7
,12
,24
,26,27-hexahydroxy-5
-cholestane-26(27)-sulfate,
is the major bile salt in bile of an elasmobranch, the little skate. To
investigate hepatic transport of bile alcohols in skate liver,
[3H]ScyS and a
potential precursor, 3
,7
,12
-trihydroxy-5
-cholestane (chtriol), were used as model compounds. Their transport into isolated
hepatocytes was partially saturable, temperature sensitive, and
Na+ independent. The uptake of
ScyS was inhibited by cholyltaurine, and uptake of cholyltaurine was
inhibited by ScyS in a competitive manner. In contrast, uptake of
chtriol was not inhibited by cholyltaurine, suggesting separate
transport systems. ScyS and chtriol showed a choleretic effect in
isolated perfused livers. When ScyS was added to the perfusate of
isolated perfused livers, >25% was found in bile within 7 h. When
chtriol was added to the perfusate, 10% of the dose was secreted into
the bile mainly in the form of polar metabolites, whereas only
nonmetabolized chtriol remained in the livers. The slow bile flow of
40-50 µl/h and the high recovery in the liver suggest that
metabolism may be the rate-limiting step in the hepatic elimination of
chtriol. The major metabolites secreted into bile were identified by
mass spectrometry and chromatography as scymnol and ScyS. To study the
enterohepatic circulation,
[3H]ScyS or
[3H]chtriol was
administered into the duodenum of free-swimming skates, and bile was
collected through exteriorized indwelling cannulas over a 4-day period.
More than 90% of the radioactivity was recovered from bile, indicating
that there was a highly effective absorption in the intestinal
epithelium, as well as specific transport mechanisms for hepatic uptake
and biliary secretion of these compounds. This is the first direct
demonstration of an enterohepatic circulation for a bile alcohol
sulfate in fish liver.
cholyltaurine; bile alcohol transport; bile acid transport; liver
 |
INTRODUCTION |
ALL VERTEBRATE LIVERS transport a wide variety of
compounds from blood into bile. In most mammals, bile acids are the
major solutes in bile. They are synthesized by the liver, efficiently excreted into the bile (21, 25, 29), and reabsorbed in the lower part
of the small intestinal tract by active transport mechanisms (19, 22).
Several carrier systems have been identified in the sinusoidal plasma
membrane of rat hepatocytes, which transport bile acids from the portal
blood (8, 9, 14, 26, 33), thus maintaining an efficient enterohepatic
circulation. In some mammals, sulfated bile alcohols were
also identified as major bile salts, e.g., tetra- or pentahydroxy
alcohol sulfates in the bile of paenungulates (manatees, elephants) or
some perissodactyla (rhinoceros, tapirs; Refs. 10, 20), and it may be
assumed that they also undergo an enterohepatic circulation. There is very little information about the development of an enterohepatic circulation in lower vertebrates, but it seems to exist in reptiles and
amphibians (17, 34). No data are available about an enterohepatic circulation in fishes. Although bile flow in marine elasmobranch vertebrates, such as the little skate (Raja
erinacea), is ~100 times slower than in rodents
(3), hepatobiliary transport has been shown to be the major pathway for
the elimination of amphipathic organic anions, including cholyltaurine
(3, 5, 6, 27). But, in many species of fish, bile acids are only minor
components of bile, whereas sulfated bile alcohols are the major
constituents (16, 32). Therefore, we studied the mechanisms underlying the hepatocellular uptake and secretion of a bile alcohol and its
contribution to bile formation in isolated skate hepatocytes, isolated
perfused skate liver, and free-swimming fish. As substrates (Fig.
1), we used scymnol sulfate [ScyS;
3
,7
,12
,24
,26,27-hexahydroxy-5
-cholestane-26(27)-sulfate], 3
,7
,12
-trihydroxy-5
-cholestane (chtriol), which is an
uncharged precursor of many physiological bile salts, and
cholyltaurine.
 |
MATERIALS AND METHODS |
All studies were performed at the Mount Desert Island Biological
Laboratory in Salsbury Cove, ME.
Animals.
Male skates (R. erinacea) with a
mean body weight of 1.0 ± 0.3 kg were caught by local fishermen off
Southwest Harbour, ME. The animals were maintained in aerated tanks
supplied with continuously flowing seawater at 15°C and were used
within 1-3 days of capture. Before hepatectomy, they were
anesthetized with pentobarbital sodium (2.5 mg/kg), which was injected
via a caudal vein.
Chemicals.
Chtriol and
[3H]chtriol with a
specific radioactivity of 133 GBq/mmol were obtained from Prof. G. Kurz, University of Freiburg, Freiburg, Germany. ScyS was purified from
skate bile by preparative thin-layer chromatography and characterized
by mass spectrometry as descibed in detail previously (16). All other
chemicals were purchased from commercial sources in the highest purity
available.
Mass spectrometry.
With the use of the method of electron ionization, mass spectra were
made with a mass spectrometer MAT 312 (Finnegan, Bremen, Germany).
Samples were dissolved in acetone and injected at a concentration of 10 µg/µl. The ionization energy was 70 eV, and the temperature of the
ion source was 220°C. The spectra were analyzed with the Finnegan
data system MAT SS 200.
High-performance liquid chromatography analyses.
The high-performance liquid chromatography (HPLC) equipment consisted
of two high-pressure pumps LKB-2150 (Pharmacia, Freiburg, Germany), a
Rheodyne injector (Rheodyne, Cotati, CA), an ultraviolet detector rapid
spectral detector 2140 (Pharmacia), and a flow-through scintillation
counter Ramona 90 (Raytest, Straubenhardt, Germany). Bondapack
C18 columns (3.9 mm × 300 mm; Waters, Eschborn, Germany) were used for analytic separation. All
samples were diluted in the respective solvent and filtered through
polyvinylidene difluoride filters with a pore size of 22 µm before
injection.
Isolation of hepatocytes.
Liver parenchymal cells were isolated by a modified collagenase
perfusion technique, described in detail elsewhere (28). Briefly, the
liver was removed from the abdominal cavity and was perfused via the
portal vein at 15°C with
Ca2+/Mg2+-free
elasmobranch Ringer solution (in mM: 270 NaCl, 4 KCl, 3 MgCl2, 2.5 CaCl2, 0.5 Na2SO4,
1 KH2PO4,
8 NaHCO3, and 350 urea) before it
was perfused with collagenase (0.07-0.1% in elasmobranch Ringer
solution). Then, the liver was placed in 80 ml Ringer solution containing 100 units/ml deoxyribonuclease, and the cells were suspended
subsequent to removal of the connective tissue capsule with a forceps.
The cells were resuspended in elasmobranch Ringer solution to yield a
final concentration of ~5-6 × 106 cells/ml. Most of the
hepatocytes were obtained in clusters of three to six cells surrounding
a single bile canaliculus, thus maintaining morphological polarity even
in the isolated state. Only cells with a trypan blue exclusion >97%
were used in transport studies. All kinetic experiments were performed
within 2.5 h after cell isolation.
Transport studies with isolated cells.
Freshly isolated hepatocytes (2.5-5 × 106 cells) were incubated at
15°C in 2.5 ml of elasmobranch Ringer solution containing various
concentrations of radiolabeled and unlabeled chtriol, ScyS, or
cholyltaurine. For inhibition experiments, the cell medium was
supplemented with bile salts in concentrations as indicated in the
legend to Fig. 3. The incubation flasks were gently agitated to
minimize unstirred water layer effects and to keep the cells in
homogeneous suspension. At the indicated time points, 500-µl portions
of the suspension were removed and centrifuged in 1.5-ml polyethylene
tubes in a Beckman microfuge at 12,000 g for 10 s. The supernatant was
removed with a pasteur pipette, and the surface of the remaining pellet
was washed with ice-cold elsamobranch Ringer solution and carefully
blotted with filter tips to remove residual fluid on the surface.
Substrate trapped in extracellular fluid was determined in control
experiments by incubation of the cells with
[14C]inulin and
calculation of extracellular space in the pellet after centrifugation.
The tubes were cut with a razor blade, and the tips containing the
cells were transferred into scintillation vials filled with 250 µl of
4% sulfosalicylic acid. After 3-h incubation, the vials were
vigorously shaken to disrupt the dispersed cells, and 5 ml of
scintillation liquid (Optifluor; Packard Instruments, Downers Grove,
IL) were added. The radioactivity was determined in a Packard Tri-Carb
scintillation counter, by using the external standard ratio method to
correct for quenching. Initial uptake rates were calculated from the
cell-incorporated radioactivity, and curve analyses were performed with
the data analysis program Enzfitter (Elsevier Publishers, Amsterdam,
The Netherlands).
Isolated perfused skate liver.
For the preparation of isolated skate livers, we followed a
modification of previously described protocols (1, 24). Male skates
were anesthesized by injection of 1% pentobarbital sodium (500 µl/kg
body wt) into a tail vein. During surgery, the gills were superfused
with 15°C seawater. The peritoneum was opened by a midline
incision, and the bile duct was cannulated with a polyethylene tube.
The portal vein was cannulated with another polyethylene tube, and the
liver was perfused with
Ca2+/Mg2+-free
elasmobranch Ringer solution, containing 0.1% (wt/vol) heparin to
prevent clotting. Then, the liver was carefully removed from all
ligaments and transferred to a perfusion dish. The organ was first
perfused at a flow rate of 30 ml/min in a nonrecirculating system with
200 ml complete elasmobranch Ringer solution. Then, the system was
switched to a recirculation mode with 100 ml complete elasmobranch
Ringer solution. All test compounds added were dissolved in dimethyl
sulfoxide (final concentration 0.5% vol/vol) and injected within at
least 1 min at the portal vein. Bile was collected at distinct time
intervals in polyethylene tubes.
Sampling of radiolabeled ScyS.
Radiolabeled ScyS was not available. Therefore,
[3H]chtriol was
administered by intravenous injection into the tail vein to free-swimming, bile duct-ligated little skates. Bile was collected by
means of a balloon connected with a cannula, which had been inserted
into the common bile duct of the fishes during a short pentobarbital
sodium anesthesia. A plastic plug was placed in the gallbladder lumen
to prevent bile from accumulating. The balloon was changed once every
day over a 4-day period. More than 90% of the recovered radioactivity
was ScyS, which was purified by HPLC.
 |
RESULTS |
Isolation of ScyS.
ScyS was isolated from skate bile by preparative thin-layer
chromatography. One milliliter of skate bile yielded on an average 100 mg ScyS, corresponding to a concentration of 18.2 mM in gallbladder bile. With the consideration of a total gallbladder bile volume of
~1.5 ml in the fish, the total ScyS pool was estimated to be ~27
µmol/fish.
Uptake of bile salts into freshly isolated skate hepatocytes.
Uptake rates of chtriol, ScyS, and cholyltaurine (Fig. 1) into freshly
isolated skate liver cells were determined as a function of increasing
medium concentrations (0.1-10 µM chtriol and 5-100 µM
ScyS or cholyltaurine, respectively) at an incubation temperature of
15°C. At all given concentrations, the amount of bile alcohols taken up by the cells increased linearily over at least 1.5 min, allowing the calculation of uptake rates by linear regression. The
uptake of chtriol (Fig. 2, Table
1) and ScyS (Table 1) into isolated cells
incubated in standard elasmobranch Ringer solution (containing 279 mM
Na+) was not significantly
different from uptake into cells incubated in an elasmobranch Ringer
solution, where Na+ was replaced
by choline+. The observed
nonlinear relationship between flux rates of uptake and medium
concentration indicates the presence of
Na+-independent transport systems
in addition to passive diffusion. Calculation of the kinetic parameters
based on the assumption of a single transport system acting parallel to
passive diffusion resulted in a flux rate
(Jmax) of 134 ± 30 pmol · min
1 · mg
1,
a Michaelis constant
(KT) of 6.7 ± 3.1 µM, and a diffusion constant (KD) of 8.0 ± 1.6 µl · min
1 · mg
1
for chtriol and in a
Jmax of 575 ± 25 pmol · min
1 · mg
1,
a KT of 25 ± 12 µM, and a
KD of 0.6 ± 0.2 µl · min
1 · mg
1
for ScyS. When the transport rates were determined at 4°C, uptake rates were diminished by 30%.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Uptake rates (J) of
3 ,7 ,12 -trihydroxy-5 -cholestane (chtriol) into freshly
isolated skate hepatocytes as function of medium concentration
(A). Cells were incubated either in standard elasmobranch
Ringer solution ( ) or in a medium where
Na+ salts were replaced by
respective choline salts ( ). Line represents calculated saturable
component, and dots show diffusional fraction of total uptake.
|
|
Previous experiments have shown that bile acids are taken up by
isolated skate heptocytes by a
Na+-independent, saturable
carrier-mediated transport mechanism (5, 6, 27). To clarify whether
chtriol, ScyS, and cholyltaurine share common transport systems, the
uptake of ScyS and chtriol was determined in the absence and presence
of increasing concentrations of cholyltaurine. The uptake of ScyS was
competitively inhibited by cholyltaurine (Fig. 3A). Vice
versa, the uptake of cholyltaurine was competitively inhibited by ScyS
with an inhibitory constant of 20 ± 6 µM (Fig. 3B).
In contrast, no effect of 50-100 µM
bile acid on the uptake rates of chtriol was seen (Fig.
3C), suggesting that bile acids and
the uncharged alcohol do not share a common transport system for uptake
into skate liver cells. Correspondingly, the uptake of cholyltaurine
was noncompetitively inhibited by chtriol (Fig.
3D). This result confirms our recent
findings demonstrating noncompetitive inhibition of bile salt uptake by
uncharged bile alcohols (5).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
A: uptake of scymol sulfate (ScyS) in
absence ( ) and presence of 100 µM cholyltaurine ( ).
B: uptake of cholyltaurine in absence
( ) and presence of 50 µM ScyS ( ) and 100 µM ScyS ( ).
C: uptake of chtriol into freshly
isolated hepatocytes in absence ( ) and presence of 50 µM
cholyltaurine ( ) and 100 µM cholyltaurine ( ).
D: uptake of cholyltaurine in absence
( ) and presence of 25 µM chtriol ( ).
|
|
Isolated perfused skate liver.
Bile production from isolated perfused skate livers averages ~2
µl · h
1 · g
liver
1 or 40-50 µl/h
(24). Therefore, bile has to be collected over relatively long time
periods to obtain information about excretion or secretory maxima.
[3H]chtriol was
injected into the portal vein of isolated perfused skate livers, and
bile was collected for 7 h. Figure
4A
demonstrates the time course of secretion of radioactivity into bile
after a bolus injection of 0.1 µM
[3H]chtriol. The time
points are corrected for the hepatic dead space volume of 2.9 µl/g
liver according to Ref. 24 and for the dead space volume of the biliary
cannula. The secretion of radioactivity reached a maximum at 3 ± 0.5 h after bolus injection. After 7.5 h, ~10% of the added
radioactivity was recovered in the secreted bile, whereas 90% remained
in the liver. Ten minutes after injection of
[3H]chtriol, no
radioactivity could be detected in the perfusate, suggesting a very
rapid and complete uptake of
[3H]chtriol into the
liver. Binding to the tubing and glassware was neglegible. Over the
time of maximum excretion, a slight transitory increase in the rate of
bile flow could be observed (Fig.
4B).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
A: biliary excretion of radioactivity
by isolated perfused skate liver after injection of 0.1 µM chtriol
into portal vein. , Cumulative excretion in percent of dose; ,
excreted amount per time interval. B:
bile flow of isolated perfused skate liver after injection of 0.1 µM
chtriol into portal vein.
|
|
Because 90% of the added radioactivity was recovered from the liver,
the organ was minced by means of scissors and further homogenized with
a Potter-Elvejhem tissue grinder. Then, the homogenate was extracted
with methanol/chloroform. Analysis of the extract by reverse-phase HPLC
and comparison with reference chtriol (Fig. 5A)
revealed that no metabolites had accumulated and that only intact
chtriol was retained by the liver (Fig.
5B). Reverse-phase HPLC of bile
samples indicated that only polar metabolites of chtriol were secreted
into the bile, since unmetabolized chtriol was not found in the samples
(Fig. 5C). The chromatogram
exhibited one major metabolite, with a retardation factor
(Rf) value corresponding to that
of ScyS. It was purified by semipreparative
C18 HPLC followed by
absorption chromatography with a cleanup C18
cartridge. Analysis by negative-ion fast atom bombardment
mass spectrometry showed one peak at a mass of 468 Da
corresponding to the mass of scymnol (Fig.
6). Only a minor peak was found at a mass
of 547 Da corresponding to the mass of ScyS. Treatment of ScyS in
the same solvents as the metabolite indicated that under the
conditions used, the sulfate residue is solvolized, resulting
in the recovery of nonsulfated scymnol. These findings suggest that
chtriol is a physiological precursor of scymnol and ScyS, respectively,
in skate liver.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
A: reverse-phase HPLC chromatogram of
chtriol. B: reverse-phase HPLC
chromatogram of bile alcohol extracted from isolated perfused skate
liver. C: reverse-phase HPLC
chromatogram of bile alcohol metabolites excreted into bile of isolated
perfused skate liver.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Fast atom bombardment mass spectrum of major bile alcohol metabolite
secreted into bile of isolated perfused skate liver. Relative mass of
468 Da is indicated for scymnol.
|
|
To study its hepatobiliary secretion,
[3H]ScyS was also
added to the perfusate of isolated perfused skate livers. The
radioactivity was rapidly extracted from the circulating perfusate.
Approximately 5 min after addition, radioactivity could be detected in
bile (after correction for dead space), suggesting rapid and complete uptake into the liver. After 7.5 h of perfusion, >25% of the added radioactivity was recovered from bile (Fig.
7A).
Only [3H]ScyS was
found by HPLC, indicating that no further metabolism had occurred
during the hepatic transit. The bile flow increased from a basal flow
of 0.9 ± 0.2 to 1.5 ± 0.3 µl · h
1 · g
liver
1 at the appearance of
ScyS and remained at that level over 4 h. At the secretory maximum, the
increase in bile flow (0.6 µl · h
1 · g
liver
1 over basal flow)
corresponded to 4.4 pmol · h
1 · g
ScyS secreted
1. When the
perfusion medium was supplemented with 50 µM ScyS, a significant
choleretic effect was observed. Bile flow rates increased more than
twofold within 15-30 min after addition of the sulfated bile
alcohol into the perfusate (Fig.
7B).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
A: biliary excretion of radioactivity
by isolated perfused skate liver after injection of 5 µM ScyS into
portal vein. , Cumulative excretion in percent of dose; ,
excreted amount per time interval. B:
bile flow of isolated perfused skate liver after injection of 50 µM
chtriol into portal vein.
|
|
Enterohepatic circulation in the free-swimming skate.
Both [3H]chtriol and
[3H]ScyS were
administered into the lumen of the gastrointestinal (GI) tract of
free-swimming skates. For that purpose, animals were anesthetized, and
the bile duct was cannulated. The cannula was exteriorized and
connected to a small balloon fixed at the skin of the free-swimming
fish, and bile was collected for up to 4 days. The test compound,
diluted with 1 ml skate bile, was injected into the jejunum of the
animal, ~1 cm below the papilla duodeni. At the end of the study,
samples were collected from intestine, liver, and bile and analyzed for radioactivity. Table 2 summarizes the
recovery rates. The data clearly demonstrate that the added compounds
were nearly completely absorbed from the GI tract, taken up by the
liver, and excreted into bile. Analysis of the biliary samples
confirmed the complete transformation of chtriol to ScyS. The enteral
administration of ScyS and the very high biliary recovery demonstrate
for the first time an enterohepatic circulation of a sulfated bile
alcohol in fishes.
View this table:
[in this window]
[in a new window]
|
Table 2.
Recovery of radioactivity after administration of chtriol and ScyS into
the upper gastrointestinal tract of free-swimming skate
|
|
 |
DISCUSSION |
The present study was performed to characterize the transport of bile
alcohols by skate liver.
The most important finding was that the sulfated bile alcohol ScyS
undergoes an efficient enterohepatic circulation in the little skate.
Existence of an enterohepatic circulation has previously been shown for
mammals, birds, and reptiles, all animals that possess C-27 or C-24
bile acids or taurine/glycine conjugates (11). Similar data in lower
vertebrates like fishes have been lacking. The bile salts in
cyclostomes and elasmobranches appear to be largely sulfated bile
alcohols rather than bile acids (12), and ScyS has been shown to be the
major bile salt in bile of several species of sharks and rays (16, 32).
Our previous studies have demonstrated that bile acids are substrates
for carrier-mediated transport in skate liver (3, 5, 6, 27). In
contrast to bile acid transport in mammals, they appear to be taken up into hepatocytes by a single
Na+-independent transport system
(5, 6, 27). However, only small amounts of cholic acid have been found
in the bile of skates and sharks (32), and it is likely that ScyS
rather than bile acids is the end product of cholesterol metabolism in
elasmobranches. The present studies provide evidence that chtriol is a
physiological precursor of
3
,7
,12
,24
,26,27-hexahydroxy-5
-cholestane-26(27)-sulfate. Because it is metabolized to C-24 amidates in rodents, a transformation into a sulfated bile salt in skate liver was not unexpected. As indicated by the hepatic excretion of more polar metabolites, the
hepatic permeation of chtriol occurs predominantly via a transcellular pathway. Both chtriol and ScyS are taken up by skate liver cells. Uptake of chtriol appears to occur by a saturable process, which is
separate from the previously identified organic anion transport system
recognizing bile acids. In contrast, ScyS and cholyltaurine seem to
share the same uptake system. Because bile acids are only a minor
component in elasmobranch bile, it may be assumed that bile acids, when
given as exogenous substrates, use an organic anion transport system
with a broader substrate specificity. Considering the structural
similarity between cholyltaurine and ScyS, it is likely that this
transport system may also represent the cellular uptake system for
ScyS. Previously, a membrane polypeptide with an apparent molecular
mass of 54,000 Da has been identified by photoaffinity labeling to be
involved in Na+-independent
cholyltaurine uptake into skate hepatocytes (6). This transport system
remains to be characterized at the molecular level but seems not to
share homology to the
Na+-independent organic anion
transporter cloned from rat and human liver (2, 13-15).
The biliary recovery of enterally administered ScyS in the
free-swimming fish was >90%. At present, we do not know whether part
of the sulfated alcohol is also eliminated via the urine of the fish.
This may be the case, because a similar observation has been made in
other species. Sulfated bile acids (3
- and 7
-sulfates) have been
detected in both animal and human (18, 23), and it is known that such
sulfated bile acids can be eliminated via the liver, but with a reduced
biliary clearance (4, 7). Sulfation enhances the urinary excretion of
such sulfated bile acids (30, 31).
In summary, ScyS, a sulfated bile alcohol, is taken up into skate
hepatocytes by a Na+-independent,
saturable transport system. The uncharged chtriol is a precursor of
ScyS, which is secreted by the hepatocytes and efficiently reabsorbed
from the skate intestine, providing the first evidence for an
enterohepatic circulation in a marine species.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Kurz, University of Freiburg, Freiburg, Germany, for
providing chtriol and David Hager, Doug Jutte, Alister Donald, and
David Seward for excellent technical assistance.
 |
FOOTNOTES |
This study was supported by the SmithKline Beecham Foundation Germany,
the Lucille P. Markey Charitable Trust, North Atlantic Treaty
Organization Collaborative Research Grant CRG-960281 (to G. Fricker),
Mundipharma Switzerland, Mount Desert Island Biological Laboratory's
Center for Membrane Toxicity Studies (National Institute of
Environmental Health Sciences Grant P30-ES-03828), and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34989
and DK-25636 (to J. L. Boyer).
Address for reprint requests: G. Fricker, Institut für
Pharmazeutische Technologie und Biopharmazie, Im Neuenheimer Feld
366, D-69120 Heidelberg, Germany.
Received 11 February 1997; accepted in final form 16 July 1997.
 |
REFERENCES |
1.
Ballatori, N.,
and
J. L. Boyer.
Slow heptobiliary elimination of methyl mercury in the marine elasmobranches, Raja erinacea and Squalus acanthias.
Toxicol. Appl. Pharmacol.
85:
407-415,
1994.
2.
Boyer, J. L.,
B. Hagenbuch,
M. Ananthanarayanan,
F. Suchy,
B. Stieger,
and
P. J. Meier.
Phylogenic and ontogenic expression of hepatocellular bile acid transport.
Proc. Natl. Acad. Sci. USA
90:
435-438,
1993[Abstract].
3.
Boyer, J. L.,
J. Schwarz,
and
N. Smith.
Biliary secretion in elasmobranches. I. Hepatic uptake and biliary excretion of organic anions.
Am. J. Physiol.
230:
974-981,
1976[Medline].
4.
Cleland, P. D.,
T. C. Bartholomew,
J. A. Summerfield,
and
B. H. Billing.
Hepatic transport of sulfated and nonsulfated bile acids in the rat after relief of bile duct obstruction.
Hepatology
4:
477-485,
1984[Medline].
5.
Fricker, G.,
V. Dubost,
K. Finsterwald,
and
J. L. Boyer.
Characteristics of bile salt uptake into skate hepatocytes.
Biochem. J.
299:
665-670,
1994[Medline].
6.
Fricker, G.,
G. Hugentobler,
P. J. Meier,
G. Kurz,
and
J. L. Boyer.
Identification of a single sinusoidal bile salt uptake system in skate liver.
Am. J. Physiol.
253 (Gastrointest. Liver Physiol. 16):
G816-G822,
1987[Abstract/Free Full Text].
7.
Gärtner, U.,
T. Goeser,
A. Stiehl,
R. Raedsch,
and
A. W. Wolkoff.
Transport of chenodeoxycholic acid and its 3-
- and 7-
sulfates by isolated perfused rat liver.
Hepatology
12:
738-742,
1990[Medline].
8.
Hagenbuch, B.,
H. Lübbert,
B. Stieger,
and
P. J. Meier.
Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes.
J. Biol. Chem.
265:
5357-5360,
1990[Abstract/Free Full Text].
9.
Hagenbuch, B.,
B. Stieger,
M. Foguet,
H. Lübbert,
and
P. J. Meier.
Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.
Proc. Natl. Acad. Sci. USA
88:
10629-10633,
1991[Abstract].
10.
Hagey, L. R. Bile Acid Biodiversity in
Vertebrates: Chemistry and Evolutionary Implications (PhD thesis).
San Diego: Univ. of California, 1992.
11.
Haslewood, G. A. D.
Evolution and bile salts.
In: Handbook of Physiology. Alimentary Canal. Washington, DC: Am. Physiol. Soc., 1968, sect. 6, vol. V, chapt. 111, p. 2375-2390.
12.
Haslewood, G. A. D.
The biological importance of bile salts.
In: Frontiers of Biology, edited by A. Neuberger,
and E. L. Tatum. New York: North-Holland, 1978, vol. 47.
13.
Jaquemin, E.,
B. Hagenbuch,
B. Stieger,
A. W. Wolkoff,
and
P. J. Meier.
Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes.
J. Clin. Invest.
88:
2146-2149,
1991[Medline].
14.
Jacquemin, E.,
B. Hagenbuch,
B. Stieger,
A. W. Wolkoff,
and
P. J. Meier.
Expression cloning of a rat liver Na+-independent organic anion transporter.
Proc. Natl. Acad. Sci. USA
91:
133-137,
1994[Abstract].
15.
Jaquemin, E.,
B. Hagenbuch,
A. W. Wolkoff,
P. J. Meier,
and
J. L. Boyer.
Expression of sodium-independent organic anion uptake systems of skate liver in Xenopus laevis oocytes.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G18-G23,
1995[Abstract/Free Full Text].
16.
Karlaganis, G.,
S. E. Bradley,
J. L. Boyer,
A. K. Batta,
B. Salen,
B. Egestad,
and
J. Sjövall.
A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea).
J. Lipid Res.
30:
317-322,
1989[Abstract].
17.
Kihara, K.,
M. Yasuhara,
T. Kuramoto,
and
T. Hoshita.
New bile alcohols 5
and 5
dermophols from amphibians.
Tetrahedron Lett.
8:
690-697,
1977.
18.
Kirkpartick, R. B.,
L. Lack,
and
P. G. Killenberg.
Identification of the 3-sulfate isomer as the major product of enzymatic sulfation of chenodeoxycholat conjugates.
J. Biol. Chem.
255:
10157-10159,
1980[Abstract/Free Full Text].
19.
Kramer, W.,
G. Burckhardt,
F. A. Wilson,
and
G. Kurz.
Bile salt-binding polypeptides in brush-border membrane vesicles from rat small intestine revealed by photoaffinity labeling.
J. Biol. Chem.
258:
3623-3627,
1983[Abstract/Free Full Text].
20.
Kuroki, S.,
C. D. Schteingart,
L. R. Hagey,
B. I. Cohen,
E. H. Mosbach,
S. S. Rossi,
A. F. Hofmann,
N. Matoba,
M. Une,
T. Hoshita,
and
D. K. Odell.
Bile salts of the West Indian manatee, Trichechus manatus latirostris: novel bile alcohol sulfates and absence of bile salts.
J. Lipid Res.
29:
509-522,
1988[Abstract].
21.
Müller, M.,
T. Ishikawa,
U. Berger,
C. Klünemann,
L. Lucka,
A. Schreyer,
C. Kannich,
W. Reutter,
G. Kurz,
and
D. Keppler.
ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.
J. Biol. Chem.
266:
18920-18926,
1991[Abstract/Free Full Text].
22.
Mullins, J. G. L.,
R. B. Beechey,
G. W. Gould,
F. C. Campbell,
and
S. P. Shirazi-Beechey.
Characterization of the ileal Na+/bile salt co-transporter in brush border membrane vesicles and functional expression in Xenopus laevis oocytes.
Biochem. J.
285:
785-790,
1992[Medline].
23.
Raedsch, R.,
B. H. Lauterburg,
and
A. F. Hofmann.
Altered bile acid metabolism in primary biliary cirrhosis.
Dig. Dis. Sci.
26:
394-401,
1981[Medline].
24.
Reed, J. S.,
N. D. Smith,
and
J. L. Boyer.
Hemodynamic effects on oxygen consumption and bile flow in isolated skate liver.
Am. J. Physiol.
242 (Gastrointest. Liver Physiol. 5):
G313-G318,
1982[Abstract/Free Full Text].
25.
Ruetz, S.,
G. Fricker,
G. Hugentobler,
K. Winterhalter,
G. Kurz,
and
P. J. Meier.
Isolation and characterization of the putative canalicular bile salt transport system of rat liver.
J. Biol. Chem.
262:
11324-11330,
1987[Abstract/Free Full Text].
26.
Schramm, U.,
G. Fricker,
H.-P. Buscher,
W. Gerok,
and
G. Kurz.
Fluorescent derivatives of bile salts. III. Uptake of 7
-NBD-NCT into isolated hepatocytes by the transport systems for cholyltaurine.
J. Lipid Res.
34:
741-757,
1993[Abstract].
27.
Smith, D. J.,
M. Grossbard,
E. R. Gordon,
and
J. L. Boyer.
Taurocholate uptake by isolated skate hepatocytes: effect of albumin.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G479-G484,
1987[Abstract/Free Full Text].
28.
Smith, D. J.,
M. Grossbard,
E. R. Gordon,
and
J. L. Boyer.
Isolation and characterization of a polarized isolated hepatocyte preparation in the skate (Raja erinacea).
J. Exp. Zool.
241:
291-296,
1987[Medline].
29.
Stieger, B.,
B. O'Neill,
and
P. J. Meier.
ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles.
Biochem. J.
284:
67-74,
1992[Medline].
30.
Stiehl, A.,
E. Ast,
P. Cszygan,
W. Fröhling,
R. Raedsch,
and
B. Kommerell.
Pool size, synthesis, and turnover of sulfated and nonsulfated cholic acid and chenodeoxycholic acid in patients with cirrhosis of the liver.
Gastroenterology
74:
572-577,
1978[Medline].
31.
Stiehl, A.,
R. Raedsch,
G. Rudolph,
U. Gundert-Remy,
and
M. Senn.
Biliary and urinary excretion of sulfated glucuronidated and tetrahydroxylated bile acids in cirrhotic patients.
Hepatology
5:
492-495,
1985[Medline].
32.
Tammar, A. R.
Bile salts in fishes.
Chem. Zool.
8:
595-661,
1974.
33.
Von Dippe, P.,
M. Amoui,
R. H. Steelwagen,
and
D. Levy.
The functional expression of sodium-dependent bile acid transport in Madin-Darby canine kidney cells transfected with the cDNA for microsomal epoxide hydroxylase.
J. Biol. Chem.
271:
18176-18180,
1996[Abstract/Free Full Text].
34.
Yousef, I. M.,
W. G. Bradley,
and
M. K. Yousef.
Bile acid composition of some lizards from southwestern United States.
Proc. Soc. Exp. Biol. Med.
154:
22-26,
1977.
AJP Gastroint Liver Physiol 273(5):G1023-G1030
0193-1857/97 $5.00
Copyright © 1997 the American Physiological Society