From the Division of Drug Metabolism and Molecular
Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku
University, Sendai 980-8578, Japan and ¶ Laboratory of Metabolism,
NCI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, October 17, 2002, and in revised form, February 25, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Supplement of 1% lithocholic acid (LCA)
in the diet for 5-9 days resulted in elevated levels of the marker for
liver damage aspartate aminotransferase and alkaline phosphatase
activities in both farnesoid X receptor (FXR)-null and wild-type
female mice. The levels were clearly higher in wild-type mice than in
FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and
taurolithocholic acid, were clearly higher in wild-type mice than in
FXR-null mice after 1% LCA supplement. Marked increases in hepatic
sulfating activity for LCA (5.5-fold) and hydroxysteroid
sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null
mice. A 7.4-fold higher 3 Bile acids produced from cholesterol are associated with
various biological functions, including absorption of fatty acids and retinoids and excretion of lipids and cholesterol. Bile acids are
produced in liver and excreted into bile through canalicular bile acid
transporters such as Bsep1
and Mrp2 (1). The primary bile acids excreted are deconjugated and
deoxygenated by enterobacteria and are reabsorbed at intestine as
secondary bile acids such as LCA and deoxycholic acid. The liver has
sufficient capacity to transport bile acids into the bile duct mainly
after transformation into conjugates. Thus, limited amounts of bile
acids are detected in sera under normal conditions. Serum and liver
bile acid levels are, however, increased dramatically after hepatic
failure or bile duct obstruction. Accumulation of bile acids,
particularly secondary bile acids, is believed to be a risk factor for
liver toxicity.
Administration of LCA and its conjugates, which are hydrophobic
secondary bile acids, causes intrahepatic cholestasis (2). Xei et
al. (3) and Staudinger et al. (4) independently
reported that the pregnane X receptor (PXR) is involved in protection
against LCA-induced liver toxicity. Recently, Makishima et
al. (5) reported that the vitamin D receptor functions as a
receptor for LCA, as well as 1 LCA is mainly transformed to tauroconjugates and glycoconjugates in
liver and pumped out to the bile duct by the Bsep. Conjugated LCA is
also transformed to 3-sulfate and 3-glucuronides by sulfotransferase and UDP-glucuronosyltransferase, respectively (6, 7). LCA sulfate and
LCA glucuronide were less toxic than LCA (8). These conjugations make
the bile acid more hydrophilic and facilitate their elimination in the
feces and urine (9-12). Bile acid sulfation is catalyzed by the
hydroxysteroid sulfotransferase, St2a forms (13). In mice, two
cDNAs encoding St2a4 and St2a9 were isolated (14, 15).
The farnesoid X receptor (FXR) is activated by a number of bile acids
including chenodeoxycholic acid (CDCA), cholic acid, and LCA (16-18).
FXR mediates a number of bile acid-dependent regulatory processes (19-25) and plays an important role in lipid homeostasis (26). FXR-null mice, in which cholesterol-bile acid regulation is
disrupted, show an increase in serum total bile acid levels. Reduction
of bile acid pools and fecal bile acid excretion was also shown to
occur because of decreased expression of Bsep. Cholic acid feeding
resulted in severe liver damage in FXR-null mice, although no apparent
toxicity was detected in FXR-null mice fed control diet. Recently,
CYP3A forms were reported to be elevated in FXR-null mice (27).
Increased excretion of urinary bile acids was reported in FXR-null mice
fed diets supplemented with 1% cholic acid, compared with wild-type
mice (26). These data suggest a possible occurrence of alterations in
the metabolic pathway leading to bile acids in the FXR-null mice.
In our preliminary experiments, FXR-null male mice were more
susceptible to cholic acid- (26) and LCA-induced liver damage than wild-type male mice, whereas FXR-null female mice were less susceptible to LCA-induced damage than wild-type female mice. These
results suggested a specific mechanism for the protection against
LCA-induced liver damage in FXR-null female mice. Thus, in the present
study, the metabolic shift in bile acid metabolism was investigated
with FXR-null female mice in relation to the liver toxicity of LCA. A
clear increase in female-specific St2a form (5.8-fold) catalyzing LCA
sulfation was detected in livers of FXR-null female mice as compared
with wild-type mice. Furthermore, the hepatic level was inversely
correlated with serum ALP activity a marker of liver cholestasis,
indicating a critical role for St2a in protection against LCA-induced
liver damage.
Materials--
LCA, CDCA, taurolithocholic acid
(tauroLCA), taurochenodeoxycholic acid (tauroCDCA), and
1,12-dodecanedicarboxylic acid were purchased from
Sigma-Aldrich. [35S]PAPS (2000 mCi/mmol) was from
PerkinElmer Life Sciences. SDS-PAGE molecular weight markers (low
range) were from Bio-Rad. The HPLC column, Chemcosorb 5-ODS-H
(6.0 × 150 mm), was purchased from Chemco Scientific Co. (Tokyo,
Japan). L-column ODS (2.1 × 150 mm) was obtained from
Kagakuhinnkennsakyoukai (Tokyo, Japan). Enzymepak 3 Animal Treatment and Sample Collection--
FXR-null mice (26)
were back-crossed to strain C57BL/6 for at least five generations. Mice
were housed under standard 12-h light/12-h dark cycle. Prior to the
administration of special diets, mice were fed standard rodent chow
(CE-2; Clea) and water ad libitum for acclimation.
Experimental diets contained 0.5 or 1% (w/w) LCA mixed with the
control diet (CE-2). Age-matched groups of 8- to 12-week-old animals
were used for all experiments and were allowed to access to water
ad libitum. Bile, blood, and tissue samples were taken for
the biochemical assay after 9 or 5 days. Microsomal and cytosolic
protein concentrations were determined by the method of Lowry et
al. (28). Total RNA was prepared from livers using the
ULTRASPEC II RNA isolation system (Biotecx Lab., Houston, TX). The
total RNA content was determined by measuring the absorbance at 260 nm
using a spectrophotometer (Beckman DU 640).
Serum AST and ALP Activities and Determination of Bile
Acid--
Serum AST activity was estimated by the POP-TOOS method
using a commercial kit, Transaminase CII-B-test Wako (Wako, Osaka, Japan). Serum ALP activity was estimated by the Bessey-Lowry method using Alkaliphospha B-test Wako (Wako, Osaka, Japan). Bile, liver, and
serum 3 LCA 6 LCA Sulfating Activity--
Sulfating activities were determined
by monitoring the radioactivities of the metabolites obtained with
[35S]PAPS as a sulfate donor after thin layer
chromatography. A typical incubation mixture consisted of 50 mM Tris-HCl buffer, pH 7.4, 1 mM
dithiothreitol, 20 mM MgCl2, 10 µM LCA, 5 µM [35S]PAPS, and 1 µg of cytosolic protein in a final volume of 10 µl. The reaction
was initiated by addition of [35S]PAPS and terminated by
addition of 5 µl of chilled acetonitrile after incubation at 37 °C
for 20 min. LCA dissolved in Me2SO was added to make
the final Me2SO concentration 1%. A portion (10 µl) of
the reaction mixtures was applied to a thin layer plate (thin layer
chromatography aluminum plate silica gel 60; Merck). Metabolites on the
chromatogram were developed with a solvent system of
chloroform/methanol/ammonia/water (60:35:0.5:7.5). The radioactive
spots were analyzed by a FLA-3000 image analyzer (Fuji Film, Tokyo,
Japan). The apparent kinetic parameters were developed from assays
carried out with several concentrations of each substrate (10 nM-1 µM LCA and tauroLCA, 1-25
µM CDCA and tauroCDCA). Each reaction was linear within
the concentrations examined.
Western Blot Analysis--
Cytosolic protein (3-10 µg/lane)
were separated by 12% SDS-PAGE and transferred to a nitrocellulose
sheet. The sheet was immunostained with a polyclonal antibody (1:2000
dilution) raised against the purified recombinant ST2A1 protein (32),
horseradish peroxidase-conjugated goat anti-rabbit IgG,
3,3'-diaminobenzidine, and hydrogen peroxide. The stained sheets were
scanned with an Epson GT-8700 scanner, and their intensities were
measured by use of the NIH image (version 1.59) software
(Bethesda, MD).
Northern Blot Analysis--
10 µg of total RNA was subjected
to electrophoresis in the 0.22 M formaldehyde-containing
1% agarose gel and transferred to GeneScreen Plus membrane
(PerkinElmer Life Sciences). Full-length St2a9 cDNA was
used as a probe. The cDNA was random-primer labeled with
[ Reverse Transcription-Polymerase Chain Reaction--
Messenger
RNA levels of differentially expressed genes were analyzed using
reverse transcription (RT)-PCR. Single strand cDNAs were
constructed using an oligo(dT) primer with the Ready-to-Go You-Prime
First-strand Beads kit (Amersham Biosciences). These cDNAs provided
templates for PCRs using specific primers at a denaturation temperature
of 94 °C for 30 s, at an annealing temperature of 58 °C for
30 s, and at an elongation temperature of 72 °C for 30 s
in the presence of dNTPs and Taq polymerase. The PCR cycle numbers were titrated for each primer pair to assure amplification in
linear range. The reaction was completed by a 7-min incubation at
72 °C. PCR products were analyzed in 2% agarose gel (w/v)
containing ethidium bromide for visualization. Either a set of primers
specific for the nucleotide sequence of each metabolizing enzyme and
transporter were followed: Bsep (bp 2094-2517 in AF133903) sense,
5'-ACAGCATTACAGCTCATTCAGAG-3' and antisense,
5'-TCCATGCTCAAAGCCAATGATCA-3'; Ntcp (bp 71-671 in U95131) sense,
5'-ACACTGCGCTCAGCGTCATTC-3' and antisense, 5'-GCCAGTAAGTGTGGTGTCATG-3';
Mrp2 (bp 386-1022 in NM 013806) sense, 5'-GGTTCCTGTCCATGTTCTGGATT-3'
and antisense, 5'-GCAGCTGAGGATTCAGAAACAAA-3'; Mrp3 (bp 42-342 in
AI391398) sense, 5'-CGCTCTCAGCTCACCATCAT-3' and antisense,
5'-GGTCATCCGTCTCCAAGTCA-3'; Mrp4 (bp 161-382 in W54702) sense,
5'-GGTTGGAATTGTGGGCAGAA-3' and antisense, 5'-TCGTCCGTGTGCTCATTGAA-3'; Oatp1 (bp 711-1036 in AF148218) sense, 5'-TGATACACGCTGGGTCGGTG-3' and
antisense, 5'-GCTGCTCCAGGTATTTGGGC-3'; Oatp2 (bp 22-779 in AB031814)
sense, 5'-GTTGCAACCCATGGGGTCAGATG-3' and antisense, 5'-GCTGGTCAGGATATTCACTCCTG-3'; Lst-1 (bp 1086-1410 in AB031959) sense,
5'-TGGTCAGACAGCATCGCAGG-3' and antisense, 5'-CCCACAGACAGGTTCCCATTG-3'; glyceraldehyde-3-phosphate dehydrogenase (bp 487-1018 in NM_008084) sense, 5'-TGCATCCTGCACCACCAACTG-3' and antisense,
5'-GTCCACCACCCTGTTGCTGTAG-3'; FXR (bp 1073-1656 in NM_009108) sense,
5'-CGGACATGCAGACCTGTTGGAAG-3' and antisense,
5'-CCAGTGGGGTTTCCTGAAGCC-3'; PXR (bp 243-710 in XM_148459) sense,
5'-GTTCCTGATTCTTCAAGGTGG-3' and antisense, 5'-TCTTCCTCTTGATCAAGGCC-3'; CYP7A1 (bp 119-701 in NM_007824) sense, 5'-CATACCTGGGCTGTGCTCTGA-3' and antisense, 5'-GCTTTATGTGCGGTCTTGAGC-3'; CYP3A11 (bp 1126-1531 in
NM_007818) sense, 5'-TGATGGAGATGGAATACCTGG-3' and antisense, 5'-GGTTGAAGAAGTCCTTGTCTGC-3'.
Influence of Bile Acid Feeding on Liver Biochemical
Parameters--
3 LCA Metabolism in Vitro--
LCA undergoes the 6 Enzyme of LCA Sulfation--
Among cytosolic
sulfotransferases, hydroxysteroid sulfotransferase termed ST2A (SULT2A)
is known to catalyze sulfation of bile acids. Using anti-rat ST2A1
antibodies, cytosolic levels of mouse St2a were measured (Fig.
2A). A band of about 31 kDa detected in the liver cytosol of female mice was 5.8-fold higher with
FXR-null mice than wild-type mice (Fig. 2B). Liver content of St2a was increased by the treatment of both wild-type and null mice
with 0.5% LCA. A clear correlation (r2 = 0.888) was
observed between individual cytosolic sulfation of LCA and St2a content
as assessed by Western blotting (Fig. 3).
In addition, an inverse correlation (r2 = 0.645) was
observed between ALP activity and St2a content in liver of wild-type
mice (Fig. 4). Upon 1% LCA treatment, no
clear relationship was obtained between ALP activity and St2a content, probably because of the extensive damage to the liver.
Detection of Bile Acid--
Profiles of bile acids
were determined by HPLC with liver of mice fed LCA. Several bile acids
with free 3-hydroxy group were detected in liver of female mice fed
LCA. In the LCA supplement groups, tauroLCA was detected as a major
peak in liver of wild-type and FXR-null mice. The peak was 12.8-fold
higher with wild-type than with FXR-null mice (Fig.
5A). In addition, free LCA was
detected as a minor but clear peak in the treated wild-type mice.
Furthermore, tauroCDCA was detected as a major peak in liver of
wild-type and FXR-null mice. The content was 3.2-fold higher with
wild-type than with FXR-null mice (data not shown). A clear correlation was observed between ALP activity and hepatic tauroLCA content (Fig.
5B). No LCA and tauroconjugate was detected in livers of untreated female mice (less than 5 nmol/g liver), although taurocholic acid and taurodeoxycholic acid were observed. Bile and liver obtained from female mice fed 1% LCA diet for 5 days were used to determine 3 Measurement of Kinetic Parameters for Sulfation of LCA, CDCA, and
Their Tauro Conjugates--
Kinetic parameters for sulfation of major
bile acid compounds, LCA, tauroLCA, CDCA, and tauroCDCA, were
determined in liver of untreated FXR-null female mice. LCA and tauroLCA
had more lower Km values and higher
Vmax values than did CDCA and tauroCDCA (Table
III). The Km value of
LCA was 36.4-fold lower than that of CDCA. Tauro conjugates were found
to have a higher Km value than the free
compounds.
Influence of PXR on the Expression of St2a--
Relative levels of
St2a expression were measured by Northern blotting. Consistent with
St2a protein levels, St2a mRNA level was elevated in FXR-null mice
(Fig. 6). Similar increase was also detected in liver of PXR-null mice. The level of St2a mRNA was further elevated in PXR-FXR double-null mice.
Changes in Hepatic Expression of Bile Acid-related
Genes--
Various transporters are known to participate in hepatic
influx and efflux of bile acids. Hepatic levels of their specific mRNAs were compared between wild-type and FXR-null mice using RT-PCR. Bsep mRNA levels were lower in FXR-null mice than the wild-type controls (Fig. 7A).
No clear change was observed in mice treated with LCA. Ntcp
transporting bile acids to hepatocytes did not differ between control
groups, but the mRNA level was reduced after LCA treatment. The
reduction was more profound with wild-type than FXR-null animals. A
similar phenomenon was observed with the anion transporter Oatp1. The
levels in both wild-type and FXR-null mice were abolished after LCA
treatment, although higher levels of the mRNA were observed with
control FXR-null mice than control wild-type mice. Lst-1 also showed a
profile similar to that of Oatp1. No obvious change was observed with Mrp2, Mrp3, and Mrp4 mRNAs. On the other hand, CYP3A11 and
CYP7A1 mRNA levels were higher in FXR-null mice than wild-type mice
(Fig. 7B). Furthermore, marked decrease in CYP7A1 mRNA
level was observed in the wild-type mice fed LCA. In contrast, no
significant change in CYP3A11 mRNA level was found in wild-type
mice after LCA feeding.
In the present study, female FXR-null mice were found to be
resistant to the toxic effects of LCA as compared with wild-type mice
despite the decreased expression of Bsep. In LCA-fed mice, liver
tauroLCA concentration correlated with a marker for liver damage, ALP
activity. Furthermore, female FXR-null mice had higher LCA sulfating
activity than wild-type mice. Hepatic level of St2a catalyzing LCA
sulfation inversely correlated with the ALP activity, indicating that
St2a plays a role in the protection against LCA-induced liver damage.
Recent studies (3, 4) suggested a protective role of activation of PXR
against severe liver damage induced by LCA. Cyp3a11 mRNA detected
by RT-PCR and Cyp3a proteins by Western blotting (data not shown) were
higher in the liver of FXR-null mice than wild-type mice in this study
consistent with what was found in an earlier report (27) showing that
Cyp3a11 levels were higher in FXR-null mice. Although the catalytic
activities of mouse Cyp3a forms remain unknown, human CYP3A4 is
reported to catalyze LCA 6 It was reported that in human, intravenously injected radiolabeled LCA
were excreted rapidly and predominantly in bile, and 60% of excreted
radioactivity were derived from sulfated forms (34). An increase in LCA
sulfate that is poorly reabsorbed from intestine results in rapid fecal
excretion of LCA (35). In bile of female FXR-null mice, sulfated bile
acid concentration was 7.4-fold higher compared with that of wild-type
mice, whereas 3 In contrast to 1% LCA feeding, no significant increase in AST and ALP
activities was found in wild-type mice fed a diet supplemented with
0.25% LCA for 5 days (data not shown). Under these conditions, a rise
in liver 3 Liver St2a mRNA level is strikingly elevated in FXR-null, PXR-null,
and FXR-PXR double-null mice. The expression of FXR and PXR is thus
likely to be directly or indirectly involved in the repression of basal
St2a expression. Liver total bile acid concentration was 2.8-fold
higher for FXR-null mice than for wild-type mice. Furthermore, hepatic
St2a level in FXR-null and wild-type mice increased after LCA (0.5%)
feeding. These results suggest the possibility that the intracellular
accumulation of hepatic bile acids results in the hepatic St2a
induction through nuclear receptors such as PXR and vitamin D receptor.
Makishima et al. (5) have shown recently that vitamin D
receptor also functions as a receptor for the secondary bile acid LCA.
Runge-Morris et al. (42) have proposed, using an in
vitro reporter assay, that the expression of rat hydroxysteroid
sulfotransferase is stimulated by a dual mechanism in which both PXR
and the glucocorticoid receptor are involved. A reporter construct
carrying an FXR binding element from the rat hydroxysteroid
sulfotransferase promoter is stimulated in Caco-2 cells upon CDCA
treatment (43). Recently, the element was shown to mediate
PXR-dependent transcriptional activation of the rat
hydroxysteroid sulfotransferase (44). Our in vivo results
with mice lacking PXR and/or FXR showing elevated mRNA encoding
St2a were contradictory with these reports. Although we can not explain
the conflicting results, our in vivo results suggest a
complicated mechanism for regulating St2a expression.
The level of Bsep mRNA in control FXR-null mice was less than that
of wild-type mice. Increased expression of Bsep mRNA was not
observed in LCA-fed mice. This result is consistent with the recent
report that LCA decreases expression of Bsep through FXR antagonist
activity (45). Mrp2, Mrp3, and Mrp4, which mediate export of anionic
chemicals, showed no clear changes at their mRNA levels. Relative
mRNAs of transporters working for entry of anionic chemicals in
liver such as Ntcp, Oatp1, and Lst-1 were all decreased under
conditions of LCA supplement, which may suggest an adaptive response to
high levels of LCA. The results of RT-PCR indicate that liver
transporters function to lower liver bile acid levels. Bile acid
sulfates are known to undergo excretion by canalicular and basolateral
transporters such as Mrp2, Mrp3, and Mrp4 (37-40). No clear changes in
levels of transporters working for LCA disposition were observed in the
present experiments. Thus, sulfating activity of LCA, not transporting
activity of LCA sulfate, might be a rate-limiting step for excreting
LCA. In fact, 3 There are reports showing high excretion of sulfated bile acids in
liver disorders (46, 47). Urinary excretion of sulfated bile acid was
shown to be high in bile-duct ligation in experimental animals and in
liver failure in humans. These data also suggest the possibility that
liver St2a induction is a general adaptive response to extranormal
levels of bile acids, particularly of LCA in liver of mammals. The
present result also implies hepatic levels of St2a sulfotransferase to
be a diagnostic marker for LCA-associated liver disorder.
-sulfated bile acid concentration was
observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels
of alkaline phosphatase. In contrast, microsomal LCA
6
-hydroxylation was not increased and was in fact lower in
FXR-null mice compared in wild-type mice. Clear decreases in mRNA
encoding sodium taurocholate cotransporting polypeptide, organic anion
transporting polypeptide 1, and liver-specific organic anion
transporter-1 function in bile acid import were detected in LCA-fed
mice. These transporter levels are higher in FXR-null mice than
wild-type mice after 1% LCA supplement. No obvious changes were
detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate
hydroxysteroid sulfotransferase-mediated LCA sulfation as a major
pathway for protection against LCA-induced liver damage. Furthermore,
Northern blot analysis using FXR-null, pregnane X receptor-null, and
FXR-pregnane X receptor double-null mice suggests a repressive role of
these nuclear receptors on basal St2a expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25-dihydroxyvitamin D3 (5). These
facts suggest the involvement of several nuclear receptors in
LCA-induced toxicity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HSD
column was purchased from Jasco (Tokyo, Japan). 5
-Cholanic
acid-3
, 6
-diol and 5
-cholanic acid-3
, 6
-diol were
purchased from Steraloids, Inc. (Newport, RI). The PXR-null mice were
kindly provided by Dr. Steven A. Kliewer at the University of Texas
Southwestern Medical Center under a Material Transfer Agreement with
GlaxoSmithKline (4).
-hydroxy bile acid concentrations were estimated by an
enzyme-colorimetric method using Total bile acid-test Wako (Wako,
Osaka, Japan). 3
-Sulfated bile acid concentrations were determined
by an enzyme-colorimetric method using UBASTEC-AUTO (Daiichi Pure
Chemical, Tokyo, Japan) (29, 30). All absorptions were measured using
spectrophotometer (Beckman DU 640). The liver 3
-hydroxy bile acid
contents were measured by HPLC. A portion (100 µl) of liver
homogenate was mixed with 1 ml of ethanol containing 2 nmol of
androstandiol and treated at 85 °C for 1 min and then centrifuged at
1,000 × g for 5 min. After the supernatant was isolated, the precipitate was extracted twice with 1 ml of ethanol. Combined extracts were dried and re-dissolved in 200 µl of methanol. HPLC analyses were performed with a Jasco intelligent model PU-980 pump
(Jasco, Tokyo, Japan), Waters M-45 pump (Waters, Milford, MA)
and FP-920S fluorescence detector (Jasco). The bile acid extracts were
separated at 35 °C with L-column ODS (2.1 × 150 mm)
(Kagakuhinnkennsakyoukai, Tokyo, Japan). The eluates were mixed with an
NAD+ solution prior to introduction to 3
-hydroxysteroid
dehydrogenase immobilized on Enzymepak 3
-HSD column. NADH produced
was measured by the fluorescence using an excitation wavelength of 365 nm and an emission wavelength of 470 nm. The separation was started at 0.5 ml/min with a 60-min linear gradient of solution A (10 mM phosphate buffer, pH 7.2/acetonitrile (60:40))/solution
B (30 mM phosphate buffer, pH 7.2/acetonitrile (80:20))
mixture (25:75) to solution A/solution B mixture (55:45) and then
continued with solution A/solution B mixture (55:45) for 25 min. The
eluates were passed through a 3
-HSD column after mixing with
solution C (10 mM phosphate buffer, pH 7.2, 1 mM EDTA, 0.05% 2-mercaptoethanol, and 0.3 mM
NAD+) (1:1).
- and 6
-Hydroxylase Activities--
LCA 6
- and
6
-hydroxylations were determined by HPLC. A typical incubation
mixture consisted of 0.1 M phosphate buffer, pH 7.4, 4.8 mM MgCl2, 0.32 mM
NADP+, 2.4 mM glucose 6-phosphate, 0.26 units/ml glucose-6-phosphate dehydrogenase, 0.25 mM LCA,
and 250 µg of microsomal protein in a final volume of 500 µl. The
mixture was incubated for 20 min at 37 °C. The incubation was
terminated by addition of 1 ml of ethyl acetate. After incubation,
1,12-dodecanedicarboxylic acid was added to the incubation mixture as
an internal standard. The mixture was centrifuged at 700 × g for 5 min. An organic phase was evaporated to dryness
under nitrogen. The residue was dissolved in 0.1 ml of acetonitrile
containing 10 mM
3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one and 0.1 ml of 56 mM 18-crown-6 in acetonitrile saturated with K2CO3, and the resulting solution was allowed
to stand at 40 °C for 30 min. Following the addition of 50 µl of
2% acetic acid, the solution was subjected to HPLC analyses (31). HPLC
analyses were performed with Jasco intelligent model PU-980 pump and
FP-920S fluorescence detector. The derivatives were separated with a
5-ODS-H column (6.0 × 150 mm) and detected by using an excitation
wavelength of 355 nm and an emission wavelength of 450 nm. The column
was eluted with acetonitrile containing 3% THF/1% acetic acid (52:48) for 15 min and then with a 35-min linear gradient of acetonitrile containing 3% THF/1% acetic acid (52:48) to acetonitrile containing 3% THF. The final elution with acetonitrile containing 3% THF was
continued for 10 min. LCA 6
- and 6
-hydroxylations were also determined using a 3
-HSD column. The data were consistent with the
data obtained with the post-labeling method described above.
-32P]CTP (Amersham Biosciences). After
prehybridization overnight with 15 mM sodium chloride
solution containing 1.5 mM sodium citrate and
0.5% SDS, the membrane was hybridized with 32P-labeled
probe (1 × 106 cpm/ml) in 0.5 M sodium
phosphate buffer, pH 7.2, containing 7% SDS, 1% bovine serum albumin,
and 1 mM EDTA overnight. After washing with 20 mM sodium phosphate buffer, pH 7.2, containing 1% SDS and
1 mM EDTA, the membrane was exposed to an imaging plate for
20 min. The radioactive spots were analyzed by a BAS1000 image analyzer
(Fuji Film, Tokyo, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Hydroxy bile acid levels determined
colorimetrically are shown in Table I.
Serum 3
-hydroxy bile acid levels were low in both wild-type and
FXR-null female mice fed control diets, although 2-fold difference was
observed between two groups. A liver 3
-hydroxy bile acid
concentrations exhibited a 2.8-fold difference in the two groups, and
the level was increased by an 1% LCA diet for 9 days. The increase in
serum was more evident in wild-type mice (82-fold) than in FXR-null
mice (5.3-fold), although no significant difference in food consumption
was observed between FXR-null and wild-type mice. A 7.7-fold higher
serum 3
-hydroxy bile acid level was observed in wild-type mice
compared with FXR-null mice after LCA treatment. Liver 3
-hydroxy
bile acid levels were also increased in mice treated with 1% LCA and
were higher in wild-type mice than in FXR-null mice. Typical diagnostic
marker activities for liver damage, serum AST activities, were slightly higher in control FXR-null mice than in wild-type mice, and ALP activities were similar between the two strains (Table I). Higher levels were detected after LCA feeding in both strains. Consistent with
the bile acid levels, AST and ALP activities were higher in wild-type
than in FXR-null mice after feeding LCA. In male mice fed 1% LCA diet
for 9 days, AST activities, ALP activities, and liver 3
-hydroxy bile
acid concentrations of FXR-null mice were 290 ± 280 IU/liter,
131 ± 87 IU/liter, and 715 ± 444 nmol/g liver,
respectively. These of wild-type mice were 35 ± 18 IU/liter, 13 ± 2 IU/liter, and 211 ± 44 nmol/g liver,
respectively.
Changes in 3-hydroxy bile acid levels and liver damage diagnostic
markers
-hydroxy bile acid levels and serum AST and ALP
activities were determined as described under "Experimental
Procedures." Control or 1% lithocholic acid-supplemented diets were
given for 9 days to wild-type and FXR-null female mice. Basal levels of
both liver bile acid and serum AST were significantly different between
wild-type and FXR-null mice (p < 0.01). In LCA-treated
animals, all parameter levels were significantly different between
wild-type and FXR-null mice (p < 0.05). LCA, AST, and
ALP indicated lithocholic acid, aspartate aminotransferase and alkaline
phosphatase, respectively. 3
-OH bile acid indicated 3
-hydroxy
bile acid.
- and
6
-hydroxylations followed by sulfation prior to excretion. Thus,
microsomal LCA 6
- and 6
-hydroxylation activity was determined
(Fig. 1). The rate of liver LCA
6
-hydroxylation was roughly 2-fold higher in wild-type than in
FXR-null mice, despite the 1.5-fold higher activity for testosterone
6
-hydroxylation in FXR-null mice (data not shown). The rate of liver
6
-hydroxylation was 2-fold higher in FXR-null mice than in wild-type
mice but was low compared with the 6
-hydroxylation. Cytosolic
3-O-sulfation of LCA was low in wild-type animals but was
5.5-fold higher in FXR-null mice. The sulfating activity tended to be
slightly increased after feeding 0.5% LCA.
View larger version (14K):
[in a new window]
Fig. 1.
LCA 6 and
6
-hydroxylations and sulfation in liver.
A, microsomal 6
- and 6
-hydroxylations of LCA.
B, cytosolic sulfation of LCA. Details for microsomal 6
-
and 6
-hydroxylations of LCA and cytosolic sulfation of LCA are
described under "Experimental Procedures." Liver samples obtained
from mice treated with control, 0.5, or 1% LCA diets were used for
both assays. Data are shown as the mean ± S.D. from four to six
different mice. Significant differences (p < 0.01)
were observed between wild-type and FXR-null mice on both the 6
- and
6
-hydroxylations and sulfation.
View larger version (29K):
[in a new window]
Fig. 2.
Relative levels of hepatic St2a and the
influence of LCA supplement. A, Western blot analysis
of St2a in mouse liver cytosol. Cytosolic proteins (10 µg) were
subjected to SDS-PAGE on 12% gel and electrically transferred to a
nitrocellulose membranes for the immunostaining with anti-rat ST2A1
antibody. B, St2a levels were quantitated as described under
"Experimental Procedures." Control or 1% LCA-supplemented diets
were given to wild-type and FXR-null female mice for 9 days. Data are
shown as the mean ± S.D. (n = 4-6). In
LCA-treated groups, significant differences (p < 0.01)
were observed between wild-type and FXR-null mice.
View larger version (18K):
[in a new window]
Fig. 3.
Correlation between LCA sulfating activity
and St2a content. Cytosolic LCA sulfating activity and St2a levels
were determined as described for Fig. 2.
View larger version (17K):
[in a new window]
Fig. 4.
Correlation between serum ALP activity and
St2a content. Experimental details for the determination of ALP
activity and St2a content were described under "Experimental
Procedures." Liver samples obtained from wild-type mice treated with
control and 0.5% LCA diets were used.
-sulfated bile acid level. Sulfated bile acid levels are shown in
Table II. A 7.4-fold higher sulfated bile
acid level was observed in bile of FXR-null mice fed an LCA diet
compared with that of the wild-type mice, although no significant
difference in the level of 3
-hydroxy bile acids was found.
Hepatic 3
-hydroxy bile acid level was 2.1-fold higher in wild-type
mice than in FXR-null mice, whereas hepatic sulfated bile acid was not
detectable (<0.06 µmol/g liver) in both mice.
View larger version (18K):
[in a new window]
Fig. 5.
Hepatic bile acid contents. The contents
of bile acids were determined with HPLC. Experimental details for the
assay of bile acid are described under "Experimental Procedures."
Liver samples obtained from mice treated with 1% LCA diet were used
for the assay. A, hepatic bile acid concentrations were
significantly different between wild-type and FXR-null mice
(p < 0.01). B, the correlation between
hepatic tauroLCA content and ALP activity.
Changes in bile acid 3-sulfate levels in liver and bile
-Hydroxy bile acid and 3
-sulfated bile acid concentration were
determined as described under "Experimental Procedures." One
percent lithocholic acid-supplemented diets were given for 5 days to
wild-type and FXR-null mice. 3
-OH and 3
-sulfate indicated
3
-hydroxy bile acid and 3
-sulfated bile acid, respectively.
Apparent kinetic parameters for sulfation of LCA, CDCA, and their tauro
conjugates by mouse cytosols
View larger version (36K):
[in a new window]
Fig. 6.
Northern blot analysis of mouse St2a.
Wild-type (lanes 1 and 2), PXR-null (lanes
3 and 4), FXR-null (lanes 5 and
6), and PXR-FXR double-null (lanes 7 and
8) mice were fed a control diet. Total hepatic RNA was
isolated, and 10 µg were separated on 1.1% agarose gel, transferred
to a nylon membrane, and hybridized with the indicated
32P-labeled cDNA probes.
View larger version (22K):
[in a new window]
Fig. 7.
Liver expression of bile acid-related
genes. Hepatic mRNA levels of bile acid-related genes were
measured by RT-PCR. Hepatic mRNAs were prepared from wild-type and
FXR-null female mice fed a control diet or the diet supplemented with
1% LCA for 9 days. Specific primers described under "Experimental
Procedures" were used to determine levels of expression of each
mRNA encoding hepatic bile acid transporters (A) and
nuclear receptors and metabolizing enzymes (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and 6
-hydroxylations in
vitro (3, 33). These data suggested the possible involvement of
mouse Cyp3a in microsomal 6
- and 6
-hydroxylations of LCA as a
detoxification pathway of LCA-induced toxicity. However, microsomal LCA
6
-hydroxylation activity was higher for wild-type mice than for
FXR-null mice (Fig. 1). Microsomal LCA 6
-hydroxylation activity was
very low in both mouse livers. These data, as well as in
vivo profiles, indicate that CYP3A metabolism is not rate-limiting
in LCA detoxification. In contrast, cytosolic sulfation of LCA and the
amounts of St2a were markedly elevated in FXR-null mice. Indeed, an
inverse relationship was observed between liver content of St2a and ALP
activity in mice treated with LCA. These data suggest a protective role
for St2a in catalyzing LCA sulfation rather than CYP3A catalyzing LCA
hydroxylation in the LCA-induced liver damage, at least in female mice.
-sulfated bile acid in livers of both mice was not
detectable (<0.06 µmol/g liver) (Table II). These data also support
the idea that hydroxysteroid sulfotransferase-mediated LCA sulfate is
rapidly excreted from liver resulting in the decrease of LCA level in liver of female FXR-null mice. Because of the efficient LCA-sulfating activity in female mice, as well as humans, female mice rather than
male mice lacking the activity might represent a feasible model for
human LCA-induced toxicity. Despite lacking bile acid sulfating
activity, male wild-type mice are resistant to LCA-induced toxicity,
indicating the presence of the protective mechanisms that are different
from bile acid sulfation.
-hydroxy bile acid levels is likely to be prevented through enhanced excretions of tauro conjugate of bile acids by Bsep
and of sulfated bile acids by Mrp2, Mrp3, and Mrp4 (36-40). Further
the increase in LCA intake might overwhelm the LCA excretion capacity
resulting in an accumulation of hepatic bile acids in wild-type mice
having lower LCA sulfating activity. Bsep expression level was not
increased in mice fed with a high LCA-containing diet. Furthermore,
Bsep-mediated bile acid transports were inhibited by LCA sulfate (41).
The excretion pathway of LCA sulfate thus might become relatively
important in the protection system of LCA-induced damage observed with
mice fed high LCA diet. Because the apparent Km
value of LCA sulfating activity in female mouse liver cytosol was very
low (0.14 µM), hepatic LCA should be efficiently
sulfated. These results support the contention that LCA sulfation is a
critical determinant for regulation of hepatic LCA concentrations.
-sulfated bile acid level in bile correlated with
hepatic St2a level and LCA sulfating activity.
![]() |
ACKNOWLEDGEMENTS |
---|
The technical assistance of Hijiri Otsuka is greatly appreciated. We thank Dr. Steven A. Kliewer (University of Texas Southwestern Medical Center) for providing PXR-null mice.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Human Science Foundation.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. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel.: 81-22-217-6829; Fax: 81-22-217-6826; E-mail: miyata@mail.pharm.tohoku.ac.jp.
Present address: Dept. of Pharmacology, Dalhousie University,
Halifax, Nova Scotia B3H 4H7, Canada.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M210634200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Bsep, bile salt export pump; FXR, farnesoid X receptor; LCA, lithocholic acid; CDCA, chenodeoxycholic acid; St, sulfotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ODS, octadeyl-silica; HSD, hydroxysteroid dehydrogenase; THF, tetrahydrofuran; Mrp, multidrug resistance protein; Ntcp, sodium taurocholate cotransporting polypeptide; Oatp, organic anion transporting polypeptide; Lst-1, liver-specific organic anion transporter-1; PXR, pregnane X receptor; tauroLCA, taurolithocholic acid; tauroCDCA, taurochenodeoxycholic acid; PAPS, adenosine 3'-phosphate,5'phosphosulfate; HPLC, high pressure liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kullak-Ublick, G. A., Stieger, B., Hagenbuch, B., and Mieer, P. J. (2000) Semin. Liver Dis. 20, 273-292[CrossRef][Medline] [Order article via Infotrieve] |
2. | Yousef, I. M., Bouchard, G., Tuchweber, B., and Plaa, G. L. (1997) Drug Metab. Rev. 29, 167-181[Medline] [Order article via Infotrieve] |
3. |
Xei, W.,
Radominska-Pandya, A.,
Shi, Y.,
Simon, C. M.,
Nelson, M. C.,
Ong, E. S.,
Waxman, D. J.,
and Evans, R. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3375-3380 |
4. |
Staudinger, J. L.,
Goodwin, B.,
Jones, S. A.,
Hawkins-Brown, D.,
MacKenzie, K. I.,
LaTour, A.,
Liu, Y.,
Klaassen, C. D.,
Brown, K. K.,
Reinhard, J.,
Willson, T. M.,
Koller, B. H.,
and Kliewer, S. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3369-3374 |
5. |
Makishima, M.,
Lu, T. T.,
Xei, W.,
Whitfield, G. K.,
Domoto, H.,
Evans, R. M.,
Haussler, M. R.,
and Mangelsdorf, D. J.
(2002)
Science
296,
1313-1316 |
6. | Stiehl, A. (1974) Eur. J. Clin. Invest. 4, 59-63[Medline] [Order article via Infotrieve] |
7. | Dionne, S., Tuchweber, B., Plaa, G. L., and Yousef, I. M. (1994) Biochem. Pharmacol. 48, 1187-1197[Medline] [Order article via Infotrieve] |
8. | Takikawa, H., Tomita, J., Takemura, T., and Yamanaka, M. (1991) Biochim. Biophy. Acta 1091, 173-178[CrossRef][Medline] [Order article via Infotrieve] |
9. | Stiehl, A., Earnest, D. L., and Admirant, W. H. (1975) Gastroenterology 68, 534-544[Medline] [Order article via Infotrieve] |
10. | Carey, M. C., Wu, S. F., and Watkins, J. B. (1979) Biochim. Biophys. Acta 575, 16-26[Medline] [Order article via Infotrieve] |
11. | Oelberg, D. G., Chari, M. V., Little, J. M., Adcock, E. W., and Lester, R. (1984) J. Clin. Invest. 73, 1507-1514[Medline] [Order article via Infotrieve] |
12. | Kuipers, F., Heslinga, H., Havinga, R., and Vonk, R. J. (1986) Am. J. Physiol. 251, G189-G194[Medline] [Order article via Infotrieve] |
13. | Nagata, K., and Yamazoe, Y. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 159-176[CrossRef][Medline] [Order article via Infotrieve] |
14. | Kong, A.-N. T., Tao, D. L., Ma, M. H., and Yang, L. D. (1993) Pharm. Res. 10, 627-630[Medline] [Order article via Infotrieve] |
15. | Kong, A. N. T., and Fei, P. W. (1994) Chem. Biol. Interact. 92, 161-168[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Makishima, M.,
Okamoto, A. Y.,
Repa, J. J.,
Tu, H.,
Learned, R. M.,
Luk, A.,
Hull, M. V.,
Lustig, K. D.,
Mangelsdorf, D. J.,
and Shan, B.
(1999)
Science
284,
1362-1365 |
17. |
Parks, D. J.,
Blanchard, S. G.,
Bledsoe, R. K.,
Chandra, G.,
Consler, T. G.,
Kliewer, S. A.,
Stimmel, J. B.,
Willson, T. M.,
Zavacki, A. M.,
Moore, D. D.,
and Lehmann, J. M.
(1999)
Science
284,
1365-1368 |
18. | Wang, H., Chen, J., Hollister, K., Sowers, L. C., and Forman, B. M. (1999) Mol. Cell 3, 543-553[Medline] [Order article via Infotrieve] |
19. | Lu, T. T., Makishima, M., Repa, J. J., Schoonjans, K., Kerr, T. A., Auwerx, J., and Mangelsdorf, D. J. (2000) Mol. Cell 6, 507-515[Medline] [Order article via Infotrieve] |
20. | Goodwin, B., Jones, S. A., Price, R. R., Watson, M. A., McKee, D. D., Moore, L. B., Galardi, C., Wilson, J. G., Lewis, M. C., Roth, M. E., Maloney, P. R., Wilson, T. M., and Kliewer, S. A. (2000) Mol. Cell 6, 517-526[Medline] [Order article via Infotrieve] |
21. |
Urizar, N. L.,
Dowhan, D. H.,
and Moore, D. D.
(2000)
J. Biol. Chem.
275,
39313-39317 |
22. |
Repa, J. J.,
Turley, S. D.,
Lobaccaro, J.-M. A.,
Medina, J.,
Li, L.,
Lustig, K.,
Shan, B.,
Heyman, R. A.,
Dietschy, J. M.,
and Mangelsdorf, D. J.
(2000)
Science
289,
1524-1529 |
23. |
Chiang, J. Y. L.,
Kimmel, R.,
Weinberger, C.,
and Stroup, D.
(2000)
J. Biol. Chem.
275,
10918-10924 |
24. |
Ananthanarayanan, M.,
Balasubramanian, N.,
Makishima, M.,
Mangelsdorf, D. J.,
and Suchy, F. J.
(2001)
J. Biol. Chem.
276,
28857-28865 |
25. | Kast, H. R., Nguyen, C. M., Sinal, C. J., Jones, S. A., Laffitte, B. A., Reue, K., Gonzalez, F. J., Willson, T. M., and Edwards, P. A. (2001) Mol. Endocrinal. 15, 1720-1728 |
26. | Sinal, C. J., Tohkin, M, Miyata, M., Ward, J. M., Lambert, G., and Gonzalez, F. J. (2000) Cell 102, 731-744[Medline] [Order article via Infotrieve] |
27. |
Schuetz, E. G.,
Strom, S.,
Yasuda, K.,
Lecureur, V.,
Assem, M.,
Brimer, C.,
Lamba, J.,
Kim, R. B.,
Ramachandran, V.,
Komoroski, B. J.,
Venkataramanan, R.,
Cai, H.,
Sinal, C. J.,
Gonzalez, F. J.,
and Schuetz, J. D.
(2001)
J. Biol. Chem.
276,
39411-39418 |
28. | Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1954) J. Biol. Chem. 193, 265-275 |
29. | Tazuke, Y., Matsuda, K., Adachi, K., and Tsukada, Y. (1994) Biosci. Biotech. Biochem. 58, 889-894[Medline] [Order article via Infotrieve] |
30. | Kato, T., Yoneda, M., Nakamura, K., and Makino, I. (1996) Digest. Dis. Sci. 41, 1564-1570[Medline] [Order article via Infotrieve] |
31. | Yamada, J., Sakuma, M., and Suga, T. (1991) Anal. Biochem. 199, 132-136[Medline] [Order article via Infotrieve] |
32. | Shimada, M., Yoshinari, K., Tanabe, E., Shimakawa, E., Kobashi, M., Nagata, K., and Yamazoe, Y. (2001) Brain Res. 920, 222-225[CrossRef][Medline] [Order article via Infotrieve] |
33. | Araya, Z., and Wikvall, K. (1999) Biochim. Biophys. Acta 1438, 47-54[Medline] [Order article via Infotrieve] |
34. | Cowen, A. E., Korman, M. G., Hofmann, A. F., and Cass, O. W. (1975) Gastroenterology 69, 59-66[Medline] [Order article via Infotrieve] |
35. | Hofmann, A. F. (1994) in The Liver: Biology and Pathobiology (Arias, I. M. , Boyer, J. L. , Fausto, N. , Jakoby, W. B. , Schachter, D. A. , and Shafritz, D. A., eds) , pp. 677-718, Raven Press, New York |
36. |
Gerloff, T.,
Stieger, B.,
Hagenbuch, B.,
Madon, J.,
Landmann, L.,
Roth, J.,
Hofmann, A. F.,
and Meier, P. J.
(1998)
J. Biol. Chem.
273,
10046-10050 |
37. | Konig, J., Nies, A. T., Cui, Y., Leier, I., and Keppler, D. (1999) Biochim. Biophys. Acta 1461, 377-394[Medline] [Order article via Infotrieve] |
38. | Suzuki, H., and Sugiyama, Y. (1998) Semin. Liver Dis. 18, 359-376[Medline] [Order article via Infotrieve] |
39. |
Hirohashi, T.,
Suzuki, H.,
and Sugiyama, Y.
(1999)
J. Biol. Chem.
274,
15181-15185 |
40. |
Hirohashi, T.,
Suzuki, H.,
Takikawa, H.,
and Sugiyama, Y.
(2000)
J. Biol. Chem.
275,
2905-2910 |
41. | Akita, H., Suzuki, H., Ito, K., Kinoshita, S., Sato, N., Takikawa, H., and Sugiyama, Y. (2001) Biochim. Biophys. Acta 1511, 7-16[Medline] [Order article via Infotrieve] |
42. |
Runge-Morris, M.,
Wu, W.,
and Kocarek, T. A.
(1999)
Mol. Pharmcol.
56,
1198-1206 |
43. |
Song, C. S.,
Echchgadda, I.,
Baek, B.-S.,
Ahn, S. C.,
Oh, T.,
Roy, A. K.,
and Chatterjee, B.
(2001)
J. Biol. Chem.
276,
42549-42556 |
44. |
Sonoda, J.,
Xie, W.,
Rosenfeld, J. M.,
Barwick, J. L.,
Guzelian, P. S.,
and Evans, R. M.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
13801-13806 |
45. |
Yu, J.,
Lo, J.-L.,
Huang, L.,
Zhao, A.,
Metzger, E.,
Adams, A.,
Meinke, T.,
Wright, S. D.,
and Cui, J.
(2002)
J. Biol. Chem.
277,
31441-31447 |
46. | Takikawa, H., Beppu, T., and Seyama, Y. (1984) Gastroenterol. Jpn. 19, 104-109[Medline] [Order article via Infotrieve] |
47. |
Makino, I.,
Shinozaki, K.,
Nakagawa, S.,
and Mashimo, K.
(1974)
J. Lipid Res.
15,
132-138 |