Al182 AASLD ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• INHIBITION OF BINDING TO THE CYTOSOL Y' BILE ACID BINDERS (BAB) INCREASES SIN U SOID A L EFFLUX AND DECREASES EXCRETION OF BILE ACIDS IN THE INTACT RAT LIVER. Id. T A K I ~ Y. SUGIYAMA, J.C. FERNANDEZ-CHECA, J. KUHLENKAMP, M~ OOKHTENS, N. KAPLOWITZ. Dept. of Medicine, Teikyo Univ. School of Medicine, Tokyo, Dept. of Pharmaceutical Sciences, Univ. of Tokyo, Tokyo, Hospital Clinic i Provincial, Univ. de Barcelona, Barcelona, Division of GI/Liver, Dept. of Medicine, USC School of Medicine, Los Angeles, CA

We previously identified that Y' BAB (3ct-hydroxysteroid dehydro- genases; 3ct-HSD) interact with bile acids in intact rat hepatocytes. Using [313-3H, C24-14C]bile acids, indomethacin (IND), a competitive inhibitor of 3ct-HSD, inhibited 3H-loss from the C3-position of bile acids and inhibited hepatic bile acid extraction and excretion (JCI 80:861,1987). To study the kinetics of these inhibitory effects and distinguish effects at various steps, the transport of a tracer dose of [3H]glycocholate (C-gly) injected as a bolus in the absence and presence of IND (50 ~M in the porfusate) was studied over 40 min in the single-pass perfused rat liver. IND markedly increased the sinusoidal efflux into perfusate of C-gly which had been taken up by the liver following single pass extraction (N)-a:15% with vs 9+5% without IND) and decreased biliary C-gly excretion (48~ 14% with vs 96~ 14% without IND). To more precisely define the effects of IND on C-gly transport, perfusate and bile samples were collected at frequent intervals during 10 rain after [3H]C-gly and [14C]inulin bolus injection. Moment analysis of the radioactivity in the perfusate during the first 30 see indicated 2.5 fold increased C-gly availability in the porfusate with IND (0.0564- 0.017 with vs 0.0224-0.014 without IND), compatible with inhibition by IND of Na+-dependent C-gly uptake. The model-dependent analyses of the release of C-gly into the perfusate and bile suggested an increase in the fraction of C~gly in the intrahepatic pool available for sinusoidal efflux and a decrease in the pool available for biliary excretion. These observations indicate that the effect of IND to delay biliary C-gly excretion is relaWxl to either intraceUular or canalicular C-gly transport. However, IND up to 2 mM did not inhibit electrogenic or ATP-dependont C-gly uptake (50 p,M) by canalicular membrane vesicles. In conclusion, we evaluated the kinetics of inhibition by IND of bile acid handling in the intact liver which allowed us to assess the effects on intracellular transport and reflux of extracted bile acids. Our findings support an important role for binding of bile acids to cytosol Y' t3AB in overall hepatic transport and suggest that specific interference with cytosol binding can interfere with the excretion of bile acids and may result in a novel mechanism for cholestasis.

OLIGOCLONAL T CELL CLONOTYPES ARE RANDOMLY ACCUMULATED IN THE LIVER OF NORMAL MICE. A.Tanaka 12, N.Hashimoto 2, K.Kurokawa 2, K.Nishioka 1, and K.Yamamoto 1. 1institute of Medical Science, St. Marianna University, School of Medicine, Kanagawa, and 2First Department of Internal Medicine, University of Tokyo, Tokyo, Japan.

In the annual meeting of AASLD in 1994, we reported that antigen-specific T cell clonotypes were oligoclonally accumulated in the liver of patients with chronic hepatitis C(CH-C) and autoimmune hepatitis(AIH), using an RT/PCR-SSCP system, which can dis- criminate the difference of junctional region of T cell receptor(TCR) l~ chain to detect clonal accumulation of T cells. We could not examine, however, if oligoclonal T cell clonotypes were accumulated in the normal liver, in which T cells were scarcely infiltrated. Nevertheless, in mice, it has been reported that some T cell populations reside in the normal liver. Therefore, we investigated if there were oligoclonality in T cells residing in the liver of normal mice. Materials and Methods Eight C57B/6 mice, four were 4w/old and four were 22w/old, were investigated. After perfusing PBS into the portal vein to eliminate blood contained in the liver, we removed the liver and separated into two portions to compare T cell clonality of two different sites. We then isolated mono-nuclear ceils by Ficoll-lsopaque density gradient from each portion of the liver, extracted total RNA, and performed an RT-PCR/SSCP analysis. Results In all mice, we found oligoclonal accumulation of T cell clonotypes in the liver, although there was a preference in V!3 chains used. Between 4w/old mice and 22w/old mice, we could not find any significant difference in the number of detected bands, i.e. in the number of accumulated T cell clonotypes. By comparing two samples obtained from each separated portion of the liver, we failed to identify the same T cell clonotypes that commonly existed between these two different sites of the normal liver of mice, in contrast to the liver of patients with CH-C or AIH. Conclusion These results suggested that, in normal liver of mice, oligoclonal T cell clonotypes were randomly accumulated, and that there would be no common antigenic stimulation.

STUDY TO THE EXISTENCE OF THE HB VIRUS WITH THE MUTATION AT PRE-CORE REGION IN THE PATIENTS WITH HB VIRUS INFECTION

N.Tanaka , M. M0riyama, H.0hkub0, H. Ishizuka, Y.Arakawa 3rd nep. of Int . Hed.,Nihon University,Tokyo,JAPAN

[Purpose] The ex is tence of HDV with the mutation at p re - core region is important in the prognosis of chronic hcpa- t i t s type B. Therefore we s tud ied to the ex is tence of the

HBV with the mutation a t pre-core region(pre-core mutants) in the p a t i e n t s with HB v i rus in fec t ion . [Materials and Methods] The sub jec t s of t h i s study were 76 p a t i e n t s with HB v i rus in fec t ion . There Were 52 men and 24 women, mean age 39.0 years . 4 persons were asymptomtic HBV ca r r i e r , and 69 persons were chro-nic h e p a t i t i s type B, 3 persons were c i r r h o s i s . The ex i s t ence of pre-core mu- t a n t s were examinated with mutat ion s i t e spec i f i c assay (M SSA)method by the d iagnos t ic de r i s ion of 0htsuka pharmacy.

which used nestedPCR. [Resul ts] 38 p a t i n t s (50~) had pre-core mutants. In t h i s p a t i e n t s ,17 p a t i e n t s were HBe Antigen pos i t ive ,and 21 p a t i e n t s were HBe Antibody pos i t i ve . In the p a t i n e t s of HBe Ab pos i t i ve , 12 p a t i e n t s had l i ve r dysfunct ion. In the other s ide , the p a t i e n t s of pre-core mutants negat ive and wild type negat ive were mainteined HBe Ab and normal l i ve r f unc t i n in the long term. [Conclusion] This r e s u l t s were suggested tha t the p a t i e n t s with pre-coe mutants may have the p o t e n t i a l i t y of l i ve r

dysfunct ion. Therfore we conclude the examination of the pre-core mutants by MSSA method i s very useful information in the c l i n i c a l assessment of HB v i rus in fec t ion .

• OVEREXPRESSION OF NITRIC OXIDE SYNTHASE GENE AND PROTEIN IN PORTAL HYPERTENSIVE GASTRIC MUCOSA. K Tanoue, IJ Sarfeh, A Tamawski, KJ Wahlstrom, FL Irwin Jr, JJ Lee, TH Ngnyen, and K Sugimachi. DVA Ned Cent, Long Beach CA, Univ of Calif, Irvine and Kyushu Univ, Fukuoka, Japan.

Nitric oxide (NO), a potent endothelial-derived vasodilator, is a key regulator of vascular tone under physiological conditions. In the portal hypertensive (PHT) state, NO is implicated as a mediator of the hyperdynamic circulation and may precipitate PHT gastropathy. However, the expression of NO synthase (NOS) mRNA and localization of its protein in the PHT gastric mucosa remains unexplored, forming the basis of this study. METHODS: Portal hypertension was produced by staged portal vein ligation. In normal control rats and PHT rats at 3, 7, and 14 postoperative days (POD), the stomach was removed and fixed in 4% paraformaldehyde or frozen in liquid nitrogen. STUDIES: 1) Portal pressure; 2) Reverse transefiption-polymerase chain reaction (RT/I?CR) with specific primers for NOS. 3) Immunohistoehemieal staining for constitutive and inducible NOS (eNOS and iNOS) with specific antibodies and quantitation of fluorescence signal with video image analysis system. RESULTS: Portal pressure at 3 POD was significantly higher than that in controls (33+4 and 15:~3 cmH20, respectively, p<0.01), and persisted elevated at 7 and at 14 POD (28+2 and 274-2 cm H20, respectively). With RT/PCR, NOS mRNA expression at 7 and 14 POD was significantly increased vs controls by 58 % and 71%, respectively (p<0.01). Expression of eNOS and iNOS was localized to endothalia of microvessels and submucosal veins, the epithelium, the museularis macosac, museularis propria and nerves. Fluorescence signal of eNOS in endothelia ofPHT submucosal veins was 674-5, 70~2 units at 7 and 14 POD vs 58~-6 units in controls (p<0:01). Als0, eNOS expression in gastric mucosa of PHT rats was significantly increased at 3, 7, 14 POD (83*7; 984-8, 96-&-6 units) vs controls (664-1 lunits, p<0.05). Fluorescence intensity for iNOS in endothelia of submucosal veins at 3, 7,14 POD were also significantly increased vs normal controls, while in gastric mueosa only at 14 days. CONCLUSIONS: 1) Portal hypertension activates NOS genes in gastric muensa and submuensa with resulting overexpression of both eNOS and iNOS proteins. 2) The excess NO generated by gastric NOS overexpression may play an important role in gastropathy of portal hypertension.