April 1995 AASLD A l 1 6 5

• CALCIUM-ACTIVATED CHLORIDE CHANNELS IN A HUMAN BILIARY CELL LINE: A POTENTIAL SITE FOR REGULATION OF DUCTULAR SECRETION T. Schlenker, R.M. Roman, J.G. Fitz. Division of Gastroenterology, Duke University Medical Center, Durham, NC

Anion channels represent a key point for regulation of secretion by biliary epithelial coils. Previous studies have focused on CFTR-associated channels that are localized to the apical membrane and ere opened by cAMP- dependent phosphorylation. Defective cAMP-dependent regulation leads to biliary drrhosis in some patients with the genetic disease cystic fibrosis. Since increases in intracollular calcium can activate a separate population of CI channels in other secretory cells, the purpose of these studies was to determine whether there is CaZ*-dependent regulation of Cfchannels in biliary epithelia. Single ion channel currents were measured using patch clamp techniques in Mz-ChA-1 cells from a human biliary adenocarcinoma that has previously been shown to posses phenotypic features of differentiated biliary cells and to express CFTR. In the coil-attached configuration, exposure to ionomycin to increase intracellular Ca =+ activated channels in 31/31 attempts; channel open probability increased >100 fold in individual cells and returned to basal activity following subsequent CaZ*-chelation (addition of EGTA to the bath). With CI'- containing solutions in the bath and pipette, two anion channel types with slope conductances for inward currents of 12.2+_3.3pS (n=11) and 6.6+2.5pS (n=5) could be identified. The larger channel was activated in 95.4% of studies and exhibited a mean open time of ~TinS; the smaller channel was activated in 59.1% of studies and exhibited a mean open time of ~30mS. In each case, channels carried inward current (efflux of CI) at the resting potential. Substitution of K + in the pipette solution with Na ÷ or NMDG + had no effect on channel properties, but lowering CI- (by replacement with the impermeant anion aspartate) caused a shift of the reversal potential in the direction anticipated for anion selective channels. These studies indicate that rises in intracellular Ca 2÷ concentration increase membrane Cf permeability through activation of at least two anion channel types. These channels represent a potential site for pharmacological therapy aiming to modulate bile flow and bypass the CI" secretory defect associated with cystic fibrosis.

HEPATOPULMONARY SYNDROME: A REVERSIBLE DISEASE BY ORTHOTOPIC LIVER TRANSPLANTATION.

* y * , + • VL Scott, M.D. , F Nelson, M.D. , AM DeWolf, M.D., I Paradis, M.D. , G Ziady, M.D. ®, Y Kang M,D.*, H Vargas, M.D. ¥, J Rakela, M.D. ¥, J Fung, M.D; Ph.D. f2, Department of Anesthesiology/CCM*, Pulmonary Medicine :~, Cardiology ~, Hepatology tr & Surgery ft. University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 INTRODUCTION: The hepatopulmonary syndrome (HPS) is a disease found in association with chronic liver disease with a reported incidence of 15-47%. 1,2 It is characterized by hypoxemia, clubbing, cyanosis and orthodeoxia. The hypoxemia is caused by impaired diffusion of oxygen due to intrapnimonary vascular dilations. Hypoxemia however is not the result of a true "shunt", since oxygenation improves when patients breath 100% oxygen. 3 Orthotopic liver transplantation (OLT) reverses this condition in mildly hypoxemic patients. 4 In this report we discuss the reversal of the HPS in patients with moderate to severe hypoxemia. METHODS: Between 1989-1994, 22 adult cirrhotic patients, age 38-68 yrs, were found to have HPS. All patients were evaluated by a contrast-enhanced echocardiography using micro-air bubbles either before or during surgery to confirm the presence of an intrapulmonary shunt and exclude intracardiac shunts. Preoperative pulmonary evaluation included arterial blood gas tension determination while breathing 21% and 100% oxygen, respirometry, DLCo, and shunt fraction estimation assuming a constant AVDO2. Technetium-99- macroaggregated-albumin scan was performed in some but not all patients preoperatively, as this study is only diagnostic for a right-to-left-shutu. A Fick shunt calculation (Qs/QT) was performed after induction of general anesthesia. All patients required home-oxygen by nasal canula prior to transplantation. RESULTS: The QS/QT of all patients ranged from 9-29%, with a room air PaO~ of 29-86 mmHg prior to OLT. Preoperatively, all patients demonstrated an improvement of arterial oxygen tension > 250 mmHg at Fi02 of 1.0 with the exception of one whose Pa t2 improved to only 150mmHg. Postoperatively, the length of stay in the intensive care unit varied from 4-120 days. Seventeen of the 22 patients demonstrated reversibility of the HPS with 5 pending follow-up evaluation. The timing to closure of the shunts varied from 3-9 months in all patients. The patient whose Pa t2 did not improve beyond 150 mmHg with an Fie2 of 1.0, died within 2 weeks of transplantation. Two other patients died at approximately 1 year after OLT due to opportunistic pulmonary infections, however after having documented reversal of the lIPS. DISCUSSION: The results confirm that the HPS with moderate to severe hypexemia is reversible after successful OLT. However, delayed closure of the shunt may occur in some patients, possibly secondary to the development of organ rejection, poor graft function, and infections. Patients who are unable to improve the Pa t2 to greater than 150 mmHg,should undergo further evaluation. In conclusion, hypoxemia secondary to the hepatopulmonary syndrome is reversible after liver transplantation. REFERENCES: (1) Mayo Clin Proc 1985; 60: 407-418. (2). Am J Cardiol 1992; 70:516-519. (3) Chest 1990: 97:1165-1170. (4)Sem Liver Dis 1993: 13: 414-422.

IMMOBILIZED ARTIFICIAL MEMBRANE (IAM) CHROMATOGRAPHY FOR ANALYSIS OF BILIARY VESICLE-ASSOCIATED PROTEINS

R. Secknus. G. Yamashita, S. Ginanni Corradini, A. Chernosky, K. Krivacic, R.T. Holzbach. G.I. Research Unit, Cleveland Clinic Fdn., Cleveland, OH. In a recent study, the hydrophilic/hydrophobic balance of bile proteins was proposed to account for their effects on cholesterol crystallization in man (Ahrnsd,H .A. et aI.,JLFI 35:211,1994). Vesicle-associated proteins are a rich source of such proteins. However, it has previously not been possible to separate proteins passively trapped inside vesicles from those that interact specifically with vesicular lipids. Aim: To devise a method capable of separating specifically membrane-associated biliary proteins from non- membrane-associated proteins. Methods; Human gallbladder bile from stone- free patients was delipidated using either organic solvent delipidation or sodium taurodeoxycholate washes. Size exclusion chromatography (SEC) of bile was performed on a HPLC system (exclusion limit 170kD). Chromatography of delipidated biliary proteins solubilized in TBS, TBS/STDC, or TBS/Triton X-100 was performed on lAId-PC cartridge columns (Pidgeon,C. et al.,AnaI.Biochem.194:163,1991). Eluents were 0.1% (low affinity fraction) and 2% (high affinity fraction) solutions of Triton X-100 in TBS. SDS-PAGE and Western blotting of albumin, Apolipoprotein A-I, cccantitrypsin, and transferdn were performed. Also, biliary haptoglobin was prepared for IAM-PC study by immunoaffinity chromatography. Results; Biliary vesicles eluted in the void volume of native bile on SEC, and they were associated with a wide array of proteins as shown by SDS-PAGE. Among bile proteins soluble in TBS alone, albumin showed the highest IAM-binding affinity. Of proteins solubilized in the presence of a detergent, Apolipoprotein A-I was confined to the lower affinity fraction and selectively mare concentrated compared to native bile and total vasicle-associatod proteins. Other proteins binding with low affinity to the IAM-column were transferrin, ¢x~-antitrypsin, and immunopurified haptoglobin. Conclusions: Vesicle-associated proteins are capable of directly interacting with vesicular lipids, e.g., IAM. Such lipid interactions, however, do not predict effects on cholesterol crystallization (e.g., albumin). Comparative affinity of a protein to the IAM column does not correlate with either inhibition or promotion of cholesterol crystallization (e.g., Apolipoprotein A-I, haptoglobin).

THE MANAGEMENT OF ESOPlIAGO-GASTRIC VAR1CES' BLEEDING A.Sedici, G.Passalacqua, D. De Angelis, P.Filanri, L.Marzio SS Filippo and Nicola General Hospital, Avezzano (ITALY)

INTRODUCTION Esophago-gastric varices bleeding still represent a very important clinical problem in cirrhotic patients due to high incidence of mortality (33% mortality cause). MATERIALS AND METHODS After diagnostic endoscopic exam performed in our service in urgent condition (within 12 hours after bleeding), we have observed 480 cases of upper digestive hemorrhages in the time span going from 1/I/90 to 31/10/94. Out of them 90 (18,7%) were due to esophago- gastric varices ruptures (60 males and 30 females, with mean age of 67.5). 90% of the patients had portal hypertension caused by cirrhosis post-epatitis or alcoolic, 10% had distrectual or pre-hepatie hypertension. 63% belonged to Child "B" class, 27% to Child "C" class. About 90 cases we observed, 74 underwent endoscopic selerotherapy of esophageal varices, 49 in deetive and 25 in urgent conditions, 5 were treated surgically, 3 with distal spleno-~nal diversion and 2 with porto-eaval diversion. From 1/1/93 to 31/10/94 19 cases were treated with TIPS; patients' selection was done on the basis of clinieal- instrumental considerations. The effectiveness of treatments were valued on the basis of: varices' eradication, variees' recurrence or rebleeding, survival. RESULTS About 49 cases treated with sclerotherapy in elective conditions, 13 (26.5%) had a recurrence of bleeding, 6 of wbom during following 6-12 mounths, 3 cases with rebleeding died for acute hepato-renal syndrome. 5 cases (10.2%) had a premature rebleeding (within 30 days from the beginning of treatment). About 25 cases treated in urgent conditions, 6 had a premature rebleeding. Totally 6 patients died. 5 cases, treated surgieally with portceaval diversion, showed during endoscopy varieeal eradication in 15 days. In 3 eases, treated with splenorenal diversion, 2 had a reduction of varieeal grade (two Paquet classes) and one had not modifications probably caused by anastomosis trombosis. During follow up (6-36 mounths) all patients treated surgically, had not bleeding reeerrences or died. All t9 patients, treated with TIPS, had endoscopic variceal reductions (2-3 Paquet classes) in 7 days. CONCLUSION Endoscopic sclerosis, an easy and economic method, represent nowdays the first choice treatment of variees bleeding. The T.I.P.S. and other therapeutic devices are recently become a valid alternative to surgical porto-systemic diversion.