Volume 121, Issue 2, Pages 407-419 (August 2001) Tauroursodesoxycholate-induced choleresis involves p38MAPK activation and translocation of the bile salt export pump in rats  Anna Kordelia Kurz, Dirk Graf, Marcus Schmitt, Stephan Vom Dahl, Dieter Häussinger  Gastroenterology  Volume 121, Issue 2, Pages 407-419 (August 2001) DOI: 10.1053/gast.2001.26262 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.1 Effects of TUDC on the activities of p38MAPK, Erk-2, and JNK-1 in primary rat hepatocytes.Rat hepatocytes were maintained in primary culture for 24 hours and subsequently exposed to TUDC.MAP kinase activities were measured with immune complex assays using specific antibodies against p38MAPK, Erk-2, or JNK-1, respectively.MBP was used as substrate for the immunoprecipitated p38MAPK and Erk-2, GST-Jun for the JNK-1 immune complex.Densitometric estimation of MAP kinase activities is related to the corresponding controls from untreated cells and is indicated below each band.The immunoblots are representative for 3–8 experiments with similar results.(A) Time dependence.Rat hepatocytes were exposed to TUDC (500 μmol/L) for the indicated periods.(B) Concentration dependence.Rat hepatocytes were treated with the indicated concentrations of TUDC for 10 minutes. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.2 Effects of TUDC on the activities of p38MAPK, Erk-2, and JNK-1 in perfused rat liver.Livers were perfused in the presence of 100 μmol/L [3H]TC.After a preperfusion period of 50 minutes (t = 0 minute), TUDC (20 μmol/L) was added for another 30 minutes to the perfusion medium.Liver samples were taken after the periods indicated.MAP kinase activities were determined as described in legend to Figure 1 and analyzed by densitometry.MAP kinase activities are given relative to the activities found at t = 0 minute.Representative results, from a series of 3 independent perfusion experiments, are shown.For statistical analysis, see also Figure 3A.(A) After a 50-minute preperfusion period, TUDC (20 μmol/L) was added for 30 minutes.Infusion of (B) SB 202190 (100 nmol/L) or (C) PD098059 (250 nmol/L) was started 20 minutes before addition of TUDC.Inhibition of p38MAPK (SB202190) or Erk activation (PD098059) by TUDC had no effect on the TUDC-induced activation of Erk-2 and p38MAPK, respectively.(D) Control: when no further additions were made after 50 minutes of perfusion (t = 0 minute) with the TC-containing medium, no effect on MAP kinases was detectable. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.3 Time course of (A) TUDC– or (B) hypo-osmolarity–induced activation of p38MAPK, Erk-2, and JNK-1 in perfused rat liver.Livers were perfused in the presence of 100 μmol/L [3H]TC.MAP kinase activities were determined and analyzed densitometrically as described in the legends to Figures 1 and 2.Data are given as means ± SEM and are from 3 different perfusion experiments for each condition.(A) After a 50-minute preperfusion period (t = 0 minute), TUDC (20 μmol/L) was added for 30 minutes to the perfusion medium, and liver samples were taken at the time points indicated.(B) After a normo-osmotic (305 mosm/L) preperfusion period of 50 minutes (t = 0 minute), the perfusion medium was switched to hyposmolarity (225 mosm/L) and liver samples were taken for analysis at the time points indicated.When SEM values are omitted, they were within the symbol size. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.4 Effects of p38MAPK inhibition on (A) TUDC– or (B) hypo-osmolarity–induced TC excretion in perfused rat liver.Livers were preperfused in the presence of 100 μmol/L [3H]TC.Data are given as means ± SEM from 3–4 different experiments.(A) After a preperfusion period of 50 minutes, TUDC (20 μmol/L) was added for 30 minutes.Infusion of SB 202190 (□, 100 nmol/L), a specific inhibitor of p38MAPK, was started at 40 minutes of perfusion.In the control experiments, infusion of the biologically inactive MAP kinase control compound SB 202474 (■, 100 nmol/L) was started at 40 minutes and was present throughout the remainder of the experiment.(B) Hypo-osmolarity (225 mosm/L) was installed after a normo-osmotic (305 mosm/L) preperfusion period of 60 minutes and the infusion of SB 202190 (○, 100 nmol/L) or its biologically inactive analogue SB 202474 (●, 100 nmol/L) was started 20 minutes before the institution of hypo-osmolarity.Hypo-osmolarity was achieved by omission of 40 mmol/L NaCl from the perfusion medium. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.5 Effect of TUDC (20 μmol/L) on the subcellular distribution of immunoreactive Bsep and Zo-1 in perfused rat liver.Rat livers were perfused with normo-osmolar (305 mosm/L), TC (100 μmol/L)–containing buffer, shock-frozen, cryosectioned, and immunostained for Bsep (red) and Zo-1 (green).Colocalization results in yellow signals.Under these control conditions, Bsep is largely localized between the linear Zo-1 staining/tight junction complex.(A) Immunoreactive Bsep (arrows) is also found inside the cells and beside the linear Zo-1 staining in putative subcanalicular vesicles.(B) Thirty minutes' addition of TUDC to the same liver results in the disappearance of intracellular immunoreactive Bsep.(C) When livers were perfused with a SB 202190 (100 nmol/L) containing Krebs–Henseleit buffer, Bsep (red) distribution did not differ from control conditions.(D) Addition of TUDC to the same SB 202190–treated liver does not affect the Bsep-bound fluorescence.For statistical analysis of the effects depicted in A–D, see Figure 6.Representative experiments from a series of 4 different perfusion experiments for each series are shown. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.6 (A) Densitometric analysis of fluorescence intensity profiles of Bsep staining.The ordinate shows the normalized intensity of Bsep-bound fluorescence, depending on the distance (μm) from the center of the canaliculus.(A) Under control conditions (●) normo-osmotic, TC-containing Krebs–Henseleit buffer), Bsep fluorescence profile shows 1 peak.Perfusion with TUDC (20 μmol/L) resulted in a significant (P < 0.05) increase of the Bsep-fluorescence peak height and a significant narrowing of its fluorescence profile (♦).(B) SB 202190 (100 nmol/L) perfusion (●) resulted in no significant increase and narrowing of the fluorescence profile compared with control conditions (SB 202190 alone or normosmotic control; compare with A).In presence of SB202190, addition of TUDC (20 μmol/L, Δ) did not significantly affect the Bsep-fluorescence profile.Means of 10 measurements in each of the 4 individual experiments for each condition are shown (means ± SEM).Statistical analysis was performed as described in Materials and Methods. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.7 Inhibition of hepatic autophagic proteolysis by TUDC.Livers were prelabeled in vivo by intraperitoneal injection of 150 μCi of [3H]leucine, and [3H]label release in the effluent was monitored as a measure of hepatic proteolysis in liver perfusion experiments (for further details see Materials and Methods).Under control conditions in the absence of SB 203580, the release of [3H]label from the liver was set to 100%.TUDC (20 μmol/L) led to inhibition of proteolysis by 14% ± 3.5% (n = 4; ●).In the presence of SB 203580 (250 nmol/L; ○), the antiproteolytic effect of TUDC was completely abolished (n = 4).SB 203580 by itself had no effect on proteolysis35 and was infused 30 minutes before addition of TUDC.Data are given as means ± SEM and are from 4 different perfusion experiments for each condition. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.8 Hypothetical scheme on the TUDC– and hypo-osmotic–induced signaling toward biliary TC excretion in perfused rat liver.The scheme is based on signal-transduction and inhibitor studies with respect to the TUDC– and hypo-osmolarity–induced stimulation of bile acid excretion in primary rat hepatocytes and perfused rat liver.For details see references 8, 10, 11, 22, and 37 and Discussion. Gastroenterology 2001 121, 407-419DOI: (10.1053/gast.2001.26262) Copyright © 2001 American Gastroenterological Association Terms and Conditions