73 IMMUNOPEROXIDASE LOCALIZATION OF BILE SALTS IN R A T LIVER CELLS

Y. Lamri, A. Roda*, M. Dumont , G. Feldmann, S. Erlinger. INSERM U-24, H6pltal Beaujon, Clichy, Laboratot re de Biologic Cel lulaire , Facultfi de Mfidecine Xavier-Bichat , Paris, France , and *Is t i tu to di Scienze Chimiche , Faco l ta di Fa rmac ta , Univers i ta Deglt Studi di Bologna, Italy.

The m e c h a n i s m s of in t racel lu lar t ranspor t of bile acids f rom the sinusoidal pole to the canal icular pole of the hepa tocy te are poorly understood. Bile acid binding proteins have been identif ied, but there is physiological and autoradiographic evidence for a ves icular pathway. The purpose of this s tudy was to de t e rmine the local izat ion of na tura l bile acids in the liver using ant ibodies aga ins t bile acids labelled with peroxidase. Rabbit ant ibodies aga ins t cholic acid conjuga tes (CCA) and aga ins t ursodeoxycholic acid (UDCA) were obtained and an indirect immunoperoxidase technique was used on rat liver sect ions fixed e i ther with pa ra formaldehyde (PF) and saponin (S), a me mbra ne -pe rme a b i l i z i ng agent which allows pene t ra t ion of ant ibodies into the cell, or with PF alone. Sect ions were examined by light and e lec t ron microscopy. When sec t ions fixed with PF and S were incubated with the ant ibody agains t (2.CA, by light microscopy, a granular cy top lasmic s ta in ing was observed in all hepa tocy tes . By e lec t ron microscopy, s t rong e lec t ron dense deposi ts were observed most ly on vesicles of the Golgi appara tus (GA) and, some t imes , in the smooth endoplasmic re t icu lum (SER). Af te r t aurochola te infusion, the in tens i ty of the react ion increased. When the liver was fixed with PF alone a lmos t no react ion was visible on light microscopy but on e lec t ron microscopy the labeling was localized on the hepa tocy te plasma membrane , mainly on the bile canal icular domain and to a lesser e x t e n t on the sinusoidal domain. With the ant ibody aga ins t UDCA, no s ta ining was observed in 3 of 4 livers, and a sl ight s ta ining in one. However, a f t e r infusion of UDCA, a s ta in ing of GA and SER vesicles was observed when the liver was fixed with PF and S. With PF alone, the reac t ion was in tense on the canal icular membrane . These resul ts support the view that , a f t e r uptake by the sinusoidal p lasma membrane , vesicles from the GA and the SER are involved in the in t racel lu lar t ranspor t of bile acids before canal icular secret ion.

74 EFFECTS OF THROMBOXANE (TX) A2 ON FUROSEMIDE-INDUCED SODIUM AND WATER DIURESIS IN PATIENTS WITH CIRRHOSIS AND ASCITES.

G. La Vi l la, M. Pinzani, G. Laff i , E. Meacci, A. Birardi, F. Cominelli & P. Gentil ini. Ist i tuto di Clinica Medica I I , University of Florence, Italy.

Cirrhotic patients (C) with ascites have increased renal TXA2 synthesis, as shown by increased urinary (U-) excretion of TXB2. Since renal TXA2 has been suggested to act as an anti-natriuretic factor and/or positive modulator of antidiuretic hormone (ADH) within the kidney, we assessed the possible role of renal TXA2 on furosemide (F)-induced sodium and water diuresis in C with ascites and avid sodium retention. We studied 6 C inpatients, a l l of whom had been of f diuretics for at least 72 hours and drugs containing cyclo-oxygenase inhibitors for at least 4 weeks. On day l F (40 mg) was given i.v. and blood/urine samples were collected every 20' for l hour and after 4 hours. On day 2, the above protocol was repeated after pretreatment with the selective TX-synthase inhibitor OKY 046 (Ono Pharmaceutical, Japan, 200 mg b. i .d. ) . 6 age-matched healthy controls underwent the same schedule. U-TXB2 was measured by RIA after HPLC extraction. OKY 046 administration reduced basal and post-F (60') U-TXB2 respectively by 53.5 and 71.7% in C and by 51.7 and 60.8% in controls. TX-synthase inhibit ion enhanced post-F free water clearance (ml/h) in C (280.13 + 79 vs. 130.5 ± 63, p L O.OOl) and in controls (715.43 ± 376.01 vs. 531.26 + 377.18, p O.OOl). Sodium excretion (mEq/h) was also sl ight ly increased on day 2 in both C-(78.1 + 19.7 vs. 54.6 + 22.5) and controls (135.41 + 29.17 vs. 122.52 + 28.9). We conclude that: l) renal TXA2 does play a role in F-induced water diuresis in cirrhotics as well as in healthy subjects; 2) TXA2 could act as a positive modulator of (ADH) within the kidney.

S39