294A AASLD ABSTRACTS HEPATOLOGY October 1995

749 CATALASE INHIBITS COLLAGEN SYNTHESIS AND ACTIVATION OF RAT LIPOCYTES IN PRIMARY CULTURE. A M S o w e i d R O'Neill . R D

B r w . Division of Gastroenterology and Hepatology, Saint Louis University Health Sciences Center, St. Louis, MO.

750 BILE ACID UPTAKE I N HUMAN HEPATOBLASTOMA, CELLS IS MEDIATED BY THE ORGANIC ANION TRANSPORTING POLYPEPTIDE. GA Kullak-Ublick. U Beuers. and G Paum_oartner. Dept. of Medicine II, Klinikum Grosshadern, Munich, Germany.

Activation ofbepatic lipoeytes (stellate, Ire cells) is the key pathogenetic event in the initiation of hepatic fibrosis. Lipocytes cultured on plastic undergo spontaneous activation. This process is characterized by proliferation, acquisition of a myofibroblastic phenotype including the expression of a-smooth muscle actin (¢-SMA), and production of type I collagen. Reactive oxygen intermediates (ROI) may be involved in the activation process, as we have previously demonstrated (Gastroenterology 108:A 1175, 1995 ). Catalase (CAT) is an antioxidant enzyme that scavenges hydrogen peroxide, an important ROI. Aim: To determine the effect of CAT on parameters of activation of rat lipocytes. Methods: Lipocytes were isolated from normal rat livers and were plated on uncoated plastic in serum-containing media. CAT was added to the media to achieve a concentration of 100 units/ml. At day seven, proliferation was determined by automated cell counting, and expression of a-SMA was assessed by Western blot analysis. Collagen production was determined by ~H-proline incorporation. Levels of mRNA for proeollagen I (¢ 1) were measured using ribonuclease protection assay (RPA) and quantified by densitometry. Results: Exposure of lipocytes to CAT resulted in a decrease of 55% in collagen production compared to controls (p<0.05). RPA results demonstrated a comparable decrease (48%) in procollagen I (~i) mRNA in the CAT group. CAT exposure suppressed a-SMA expression by more than 90%. Proliferation was inhibited by approximately 15% (10<0.05) in cells treated with CAT compared to controls. There was no evidence of CAT-related cytotoxicity as determined by trypan blue exclusion test. Coneluslons: Exposure of lipocytes to the hydrogen peroxide scavenger CAT significantly decreased collagen production, apparently via an effect on collagen gene transcription: This result was accompanied by a dramatic suppression of a- SMA expression and a modest decrease in proliferation. These results support the hypothesis that ROI may be involved in lipocyte activation. (Supported by NIH DK-03907 and Glaxo Basic Research Award)

Bile acids are taken up into the liver via Na+-dependent bile acid specific (NTCP) and Na+-independent multispecific (OATP) basolateral transport proteins. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of bile acids is markedly reduced. However, the degree to which Na+-dependent uptake is impaired in relation to Na+-independent uptake is unknown. Therefore, we have studied the transport of bile acids in the human hepatoblastoma cell line HepG2. Uptake of [3H]taurocholic acid was found to be saturable and entirely Na ÷- independent, with an apparent K m of 64 p.M and a Vma x of 24.7 pmol/min/mg protein. This K m value was identical with the K m of taurocholate uptake in cRNA-injected Xenopus laevis oocytes expressing the OATP of human liver. Injection of HepG2 mRNA into oocytes significantly stimulated taurocholate uptake in a Na+-free choline chloride medium, indicating the expression of a Na +- independent bile acid transport protein. OATP, but not the Na +- dependent bile acid transporter NTCP, was detectable by RT-PCR analysis of HepG2 RNA. Transfection of HepG2 cells with an OATP- specific phosphorothioate-antisensa oligonucleotide resulted in 77% inhibition of temperature-sensitive taurocholate uptake, suggesting that OATP is the major bile acid uptake system expressed in the HepG2 cell line. These data indicate that (1) bile acid uptake into human hepatoblastoma cells is mediated by the organic anion transporting polypeptide OATP, and (2) HepG2 cells have a complete absence of the Na+-taurocholate cotransporting polypeptide NTCP. Since the multispecific transport system OATP is preserved, strategies for the treatment of hepatoceilular carcinoma, employing cytostatic drugs coupled to bile acids or to other organic anions, appear feasible.

751 EXPRESSION IN XENOPUS LAEVIS OOCYTES OF A NOVEL ORGANIC ANION TRANSPORTER FROM RAT LIVER. M Runneaar. J Yi. S Kona and N Kaolowitz, Center for Liver Diseases,University of Southern California, Los Angeles CA

The liver extracts a large variety of organic anions from the portal circulation by carrier mediated transport. In the rat two sinusoidal transporters have been cloned: the sodium- taurocholate (TC) transporting polypeptide (Ntcp, 1.6 kb) and an organic anion-transporting polypeptide (oatp 2.8 kb). However, inhibition and kinetic studies indicate that additional organic anion transporters must be present. Microcystin (Mcyst) is one of a family of cyanobactedal cyclic pepUdes that are exclusively hepatotoxic because of carrier mediated transport at the sinusoidal pole. Uptake is Na + independent and fully inhibited by I~M concentrations of BSP Rifamycin (RIO, cyclosporine, trypan blue and trypan red, and partially by TC and cho ate. Toxicity of these peptides results from the specific, sustained inhibition of ser/thr protein phosphatases in the hepatocyte. As a first step in cloning the hepatic transporter for these and related xenobiotics and metabolites, we used a radiolabeled derivative of Mcyst to study uptake in the X. laevis oocytes. Injection of oocytes with rat liver poly(A)+RNA (36 ng RNA/oocyte) resulted in the functional expression of Mcyst uptake (10 nM 1251-Mcyst-YM) within 4-5 days. Uptake (10-15 individual measurements with at least two separate oocyte preparations) was 114-+17 for RNA injected oocytes and 44-+7 fmol Mcyst/8 oocytes/hr for water injected controls, and was linear over 3 hr: By sucrose gradient size fractionation of total rat liver mRNA a single fraction of size greater than 4 kb was found to express Mcyst uptake (injection 2 ng RNA/oocyte, uptake 248-+80 vs. 49-+10 fmol Mcyst/8 oocytes/hr for water injected oocytes). Mcyst uptake was fully inhibited by the addition of 50 I~M Rif (24_3÷ vs. 30_6÷ frnol Mcyst/8 oocytes/hr for water controls). Conclusions: Distinct size o f RNA fraction and Rif inhibition (which is nor exhibited by Ntcp or oatp) indicate that Mcyst is transported by a novel organic anion transporter from rat liver different from Ntcp and oatp.

752 VIP IS A POTENT STIMULUS OF BICARBONATE AND FLUID SECRETION IN BILE DUCTS. WK Cho. A Mennone. SA Rvdber~. and JL O B_Qy.~. Liver Center, Yale University School of Medicine, Ne~v Haven, CT.

VIP is known to stimulate bile flow in various animal models, but its underlying mechanism and site of action are not clearly defined. In order to study the actions and mechanisms of VIP, novel functional polarized intrahepatic bile duct units(IBDU) with an enclosed lumen were isolated from rat liver as previously described(PNAS in press). Infusion of 1 - 1000 nM VIP increased the luminal area of IBDU in a dose-dependent fashion indicating stimulation of ductular secretion. With 100 nM VIP, the luminal area increased by 91.6 + 24.5%(n=7) in 30 min, in contrast to -4.8 + 4.5%(n=7) with buffer control. Simultaneous infusions of somatostatin(100-500 nM) or substance P(100-500 nM) with VIP(100 nM) did not inhibit the secretory response. In addition, omission of bicarbonate or chloride from the perfusion medium reduced the secretory response by 52.8% and 60.8% respectively suggesting partial dependence on bicarbonate and chloride. Basal phi measured by BCECF microfluorospectrophotometry using a 490/440 ratio method was not affected by acute exposure to 100 nM VIP. VIP increased maximal rates of alkalinization after C1- removal (0.16 + 0.07 pHU/min) and of pHi recovery after C1- readmission(0.27 + 0.06 pHU/min) compared with the control values(0.11 + 0.04 and 0.19 + 0.02 pHU/min, respectively(n--4)), consistent with stimulation of C1- /HCO3- exchange activity. In normal isolated perfused rat liver without bile acid supplementation, VIP(100 nM) significantly increased pH, [HCO3"] and HCO3- output in bile without changing bile flow. However, in 2 week bile duct ligated rats, bile flow and bicarbonate output increased over 6 and 13 fold respectively. Conclusion: VIP can stimulate bile secretion by a direct action on bile duct epithelial cells. This secretory response is more potent than secretin, and is dependent on bicarbonate and chloride, thus presumably mediated by C1-/HCO3- exchange. This secretory response is not inhibited by somatostatin or substance P.