Adaptive regulation of the canalicular bile salt export pump in response to changes of bile salt pool size

A934 AASLD ABSTRACTS

GASTROENTEROLOGY Vol. 118, No.4

165 DlSCOORDINATE REGULATION OF THE ILEAL BILE ACID TRANSPORTER (ASBT) AND BILE ACID BINDING PROTEIN (ILBP) IN MOUSE ILEUM. Lin Ma, Ephraim Sehayek, Jan L. Breslow, Benjamin L. Shneider, Mount Sinai Sch of Medicine, New York, NY; Rockefeller Univ, New York, NY. Introduction: Active transport of conjugated bile acids by the ileum plays an important role in maintaining bile acid homeostasis. The following experiments were performed to determine the effects of bile acid load on ASBT and ILBP expression in the mouse. Methods: 15 mice were randomly divided into 3 group and fed with either normal chow, 0.5% cholic acid or 0.5% taurocholic acid for 3 weeks; Cholesterol 7a-hydroxylase knock-out mice were also produced. 5 heterozygous and 4 homozygous mice were confirmed by genetic analysis. The distal 1/3 and middle 1/3 of intestine from each single mouse was used to make homogenates and brush border membrane vesicles. ASBT and ILBP protein expression were detected by western blot. Quantification was performed using 125I-protein A secondary antibody and phosphorimager. Results: ASBT was down-regulated (p<0.05) while ILBP was up-regulated (p<0.05) by bile acid feeding. There was no significant difference between the effects of cholic acid and taurocholic acid feeding on ileal bile acid transporter system (see table). No ASBT or ILBP expression has been detected in the middle intestine of chow or bile acid fed mice. In Cholesterol 7a-hydroxylase knock-out mice, ASBT expression in distal intestine of the C7aH -1- mice increased compared with that in the +1- mice (155450 ::!:: 31134 U/mg vs. 76659 ::!:: 27434 U/mg, p<0.05). ASBT expression was also detected in middle intestine of the C7aH -1- mice but not in the +1- or wild type mice. ILBP expression decreased in the C7aH -1- mice compared with that in the +1- mice (10524 ::!:: 5387 U/mg vs. 5715 ::!:: 272lU/mg, p=0.09). Conclusions and Discussions: In mouse, ASBT and ILBP expression are not coordinately regulated. Bile acids have negative feedback effects on ASBT and positive feedback effects on ILBP. This leads to decreased uptake of bile salts and potential ILBP-mediated trapping of bile salts within enterocytes, akin to the effect of metallothionine. The exact opposite response is seen in the C7aH -1- mice when bile flow is decreased. In this model, novel expansion of the ASBT expression into the proximal intestine is also observed. Measurement Number of animals ASBT(U/~g)

ILBP (U/fl9)

Chow 4 53216 ± 11377 22718 + 2897

Chow+Cholic

Chow+Taurocholic

5

5

32582 ± 5002 67860 ± 17613

28406 ± 13489 58795 ± 14108

166 ADAPTIVE REGULATION OF THE CANALICULAR BILE SALT EXPORT PUMP IN RESPONSE TO CHANGES OF BILE SALT POOL SIZE. Marco A. Arrese, Margarita Pizarro, Nancy Solis, Luigi Accatino, Catholic Univ of Chile Sch of Medicine, Santiago, Chile.

Background: Adaptive up- and down-regulation of hepatic bile salt transport occurs in response to changes in bile salt (BS) pool size andlor bile salt flux in the liver (Arrese M. et ai, J. Hepatol 1997:26:694-702). BS are excreted into the canaliculi by an ATP-dependent carrier named BS export pump (Bsep). Aim. To assess whether changes in BS pool size are associated to changes in the expression of the Bsep. Methods: Male SpragueDawley rats were subjected to the following experimental conditions: I) BS Overload using a) exogenous BS feeding (1% wlw of cholic acid) during 4 days, or b) endogenous BS overload through a choledoco-jugular fistula for I and 2 days, and 2) depletion of the BS pool by external biliary drainage for 24 hrs. Bile flow, basal BS secretion and maximum taurocholate secretory rate (SRm) were determined. Bsep protein mass in isolated canalicular membranes was estimated by Western blotting. Protein mass of the basolateral sodium taurocholate cotransporting polypeptide (ntcp) was also assessed using a crude liver membrane fraction. Results: BS overload (groups 1a and 1b) was associated with significantly increased basal BS secretion (222%, 660% and 497%,respectively) and taurocholate SRm (172%, 199% and 148%,respectively). In these groups, a significant increase of membrane Bsep protein mass (533%, 317% and 174%, respectively) was apparent. In contrast, BS pool depletion, which determined a significant decrease of basal BS secretion (to 10%) and taurocholate SRm (to 65%), was associated with a significant decrease of Bsep protein mass (to 80%). Ntcp protein mass was not modified by BS feeding or BS depletion. Conclusion:these results support the role of Bsep in canalicular transport of BS and indicate that its expression is regulated by changes in BS pool size (Grants FONDECYT 1971\24 and 1990519). 167

TRANSLOCATION OF ESTRADlOL-17·B-GLUCURONIDE IN RAT LIVER MICROSOMAL VESICLES BY A NOVEL MULTI· FUNCTIONAL TRANSPORTER. Eric Battaglia, John Gollan, UCSF, Metz, France; Univ of Adelaide, Adelaide, Australia. An array of compounds are glucuronidated in the liver by the action of the endoplasmic reticulum (ER)-bound UDP-glucuronosyltransferases. The process by which glucuronides, generated by this enzyme system within the ER lumen, are exported across the ER membrane to the cytosol (prior

to excretion via plasma membrane multidrug resistance associated transporters) is undefined. Hence, we examined the process by which glucuronides are translocated across purified rat liver microsomal vesicle membranes. Uptake and efflux of [3H]estradiol-17-~-D-glucuronide (3HE 217{3G) was determined using a rapid filtration technique. Time- and temperature-dependent uptake of intact 3H-E217~G was observed. The efflux of 3H-E217~G from rat liver microsomal vesicles (preloaded with this glucuronide), suggested that ER transport is a bidirectional process. Treatment of ER vesicles with the pore-forming reagent alamethicin resulted in loss of internalized 3H-E 217I3G, supporting the presence of a transport process rather than non-specific binding. The uptake process was saturable with a Km and Vrn " of 3.29 :!: 0.58 pM and 0.193 ::!:: 0.020 nmol.minl.mg' protein, respectively. Transport of 3H-E 217{3G under initial rate conditions was inhibited by anion-transport inhibitors (4,4diisothiocyanatostilbene-2,2-disulfonic acid, probenecid. Moreover, shrinkage of ER vesicles by modification of the osmolarity with nonpermeant raffinose lowered the steady-state level of glucuronide accumulation. Specificity of the transport process was investigated by studying the cis-inhibitory effect of various anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on 3HE 21713G uptake. Cis-inhibitory potency was as follow: phenolphtaleinglucuronide>estrone-3-sulfate> f3- estradiol-3-sulfate> f3- estradiol-3,17disulfate>etoposide. Conversely, p-nitrophenol-glucuronide, 4-methylumbelliferyl-glucuronide, Doxorubicin, UDP-a-D-glucuronic acid did not inhibit 3H-E 217{3G influx. These data are indicative of a protein carrier for glucuronides in the hepatic ER. This novel intracellular transporter may playa key role in the initial excretion process for glucuronic acid-conjugated compounds, and also may contribute together with the well characterized plasma membrane MDR-related proteins to the phenomenon of multidrug resistance.

168 REGULATION OF HEPATOCELLULAR TRANSPORTER EX· PRESSION DEPENDS ON BILE ACID HYDROPHOBICITY IN MICE. Peter Fickert, Rainer Zenz, Gernot Zollner, Christine Pojer, Andrea Fuchsbichler, Kurt Zatloukal, Helmut Denk, Michael Trauner, Dept of Medicine, Graz, Austria; Dept of Pathology, Graz, Austria. Background: Cholestasis is associated with retention of bile acids and profound alterations in hepatocellular transporter expression. The effects of ursodeoxycholic acid (UDCA), used in the treatment of cholestatic disorders, on hepatocellular transporter expression are unknown. Aim: To determine the effects of bile acid hydrophobicity on expression of Ntcp, mdrla, mdrlb, mdr2, and Bsep in the mouse liver. Methods: Swiss albino mice were either fed a UDCA or cholate (CA) supplemented diet (0.1%, 0.5%, I %) or control diet for 3 and 7 days, respectively (each group n=5). Transporter mRNA, protein expression, and tissue localization, were studied in mouse livers by quantitative RT-PCR, Western analysis, and immunofluorescence microscopy. Results: CA resulted in significant overexpression of mdrl a, mdrlb, mdr2 and Bsep, while Ntcp was downregulated. In contrast, UDCA had no effect on mdrla, mdrlb, mdr2 and Ntcp expression, while Bsep was upregulated. Summary & Conclusions: CA upregulates canalicular and downregulates basolateral transport systems. Differential regulation of hepatocellular transporters depends on bile acid hydrophobicity. The therapeutic effects of UDCA may in part be due to stimulation of Bsep and conservation of Ntcp expression.

169 CLONING OF THE FULL·LENGTH CDNA FOR HAMSTER LIVER NA·TAUROCHOLATE COTRANSPORTING POLYPEP· TIDE (NTCP) AND FUNCTIONAL ANALYSIS: IDENTIFICATION OF TWO DISTINCT TRANSCRIPTS. Natarajan Balasubramanian, I'Kyori Swaby, Frederick J. Suchy, Meenakshisundaram Ananthanarayanan, The Mount Sinai Med Ctr, New York, NY. It has been well established now that the hepatic basolateral uptake of conjugated bile acids occur via a sodium-taurocholate cotransporting polypeptide (Ntcp), a-50 kDa protein, localized exclusively to the sinusoidal membrane. While most of the studies on Ntcp regulation has been done using the rat model, there are significant differences between rats and humans in bile acid and sterol metabolism. Sterol and lipoprotein metabolism, bile salt composition, synthesis and pools are highly similar between hamsters and humans. As a tool to undersatand Ntcp regulation in humans, we have cloned the full-length cDNA for Ntcp from hamster liver and analyzed its function using transient transfection of COS-7 cells. Methods: A partial Ntcp fragment of 562 bp was initially isolated by RT-PCR of hamster liver mRNA using degenerate primers. Using the derived sequence, further 5'- and 3'- RACE reactions were done to isolate the full-length cDNA. Transient transfection of COS-7 cells by Lipofectin followed by eH)TC uptake was used to assay transport. Results: A fulllength cDNA of 1433 bp was isolated which contained an open reading frame for a protein of 362 aa(calculated MW 39,441 Da) and 88 and 258 bp of 5'- and 3'- untranslated sequences respectively. The DNA and aminoacid sequences were 86% and 80% identical to rat Ntcp. In vitro translation of the cDNA resulted in the synthesis of ~33 kDa polypeptide which increased to -45 kDa on incubation with canine pancreatic microsomes. Northern blot analysis of hamster liver RNA with the cDNA probe revealed the presence of two distinct transcripts of 1.5 and 1.7 kb, in