Volume 122, Issue 4, Pages 963-973 (April 2002) Heat stress prevents impairment of bile acid transport in endotoxemic rats by a posttranscriptional mechanism  Ulrich Bolder, Andrea Schmidt, Lukas Landmann, Verena Kidder, Stefan Tange, Karl–Walter Jauch  Gastroenterology  Volume 122, Issue 4, Pages 963-973 (April 2002) DOI: 10.1053/gast.2002.32408 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of heat stress on biliary CT secretion in IPRLs perfused in single pass mode. Bile acid output (BAO) is shown compared with the infused dose. The time course shows the mean ± SEM of 4–6 IPRLs. (A) Experiments were performed 12 hours after injection of saline (●). Further perfusion studies were performed in HS animals (○). (B) In a further group, CT transport after intraperitoneal injection of LPS (■) was compared with transport observed when heat stress was applied 2 hours before intraperitoneal injection of LPS (□). Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Uptake of 10 μmol/L CT in isolated rat hepatocytes. Incubations were performed in (A and B) media containing Na+ or in (C and D) Na+-free choline Cl media. Hepatocytes were prepared from control rats (●), after HS (○), or from animals receiving LPS alone (■) or after HS/LPS (□). For transport assays, 2 mg of freshly isolated hepatocytes was resuspended in the following (in mmol/L): NaCl or choline Cl, 137; KCl, 5.2; MgSO4, 0.9; CaCl2, 0.12; and Tris/HEPES, 10 (pH 7.4). Transport was initiated by the addition of 1 mL media containing 10 μmol/L labeled CT (analysis of variance; P < 0.001; *P < 0.01 vs. LPS; **P < 0.001 vs. LPS). Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Initial rate of uptake of CT in freshly isolated hepatocytes. Kinetic parameters were calculated according to Michaelis and Menten. Hepatocytes were incubated in fresh media containing labeled CT (0, 5, 10, 20, 50, 100, and 200 μmol/L). Uptake was corrected by subtraction of values obtained in Na+-free choline-Cl media. Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunofluorescence of ntcp and bsep. Cryosections (0.5–1 μm) were probed simultaneously with a monoclonal antibody recognizing HSP70 (green) and antisera against ntcp (left column) or bsep (right column). The insets show a different illumination focusing on the depiction of the bile acid transporters. Controls (A and B) show the domain-specific expression in the basolateral (ntcp, A) and canalicular (bsep, B) membrane domains, respectively. Distribution of membrane proteins was not altered by any treatment protocol. Staining intensity was slightly decreased after HS treatment (C and D), a protocol that leads to the expression of HSP70 in nuclei and cytoplasm of perivenous hepatocytes. Administration of LPS (E and F) results in decreased staining intensity of ntcp and almost complete disappearance of bsep. This effect is not observed if LPS is preceded by HS treatment (G and H) (bar = 25 μm). Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunoblot analyses from hepatocyte homogenates. Time course of HSP70 and HSP25 in samples detected at various times after animals were subjected to heat stress. Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunoblot analyses of ntcp and bsep performed in basolateral or canalicular membranes. Membrane fractions were isolated from control animals, after heat stress (HS), after LPS injection (LPS), or after heat stress followed by LPS injection (HS/LPS). Purity of membranes was assessed according to methods described previously.51 Canalicular membranes assessed by Mg2+,K+–adenosine triphosphatase were concentrated 38- ± 4-fold compared with the homogenate. Basolateral membranes were concentrated 33- ± 5-fold measured by Na+,K+–adenosine triphosphatase. Contamination of canalicular or basolateral membrane fractions with mitochondria or microsomes was absent, assessed by succinate cytochrome c reductase and reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase, respectively. No differences were detected in the experimental groups. The pixel densities were calculated from 3 representative blots (analysis of variance, P ≤ 0.001; *P ≤ 0.01 vs. LPS, **P ≤ 0.001 vs. LPS, †P ≤ 0.05 vs. control). Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Time course of bsep and ntcp in membrane fractions isolated from control animals (●), after administration of LPS (■), or HS/LPS (□). Preparations were performed at 0, 4, 12, 24, 36, and 72 hours after the application of NaCl, LPS, or HS/LPS. HS alone was not tested. The Figure shows representative immunoblots. The densities of 3 blots were quantified and depicted in the lower panels. bsep: analysis of variance, P ≤ 0.001. *P ≤ 0.01 vs. control and HS/LPS, **P ≤ 0.01 vs. control and HS/LPS. ntcp: analysis of variance, P ≤ 0.001. **P ≤ 0.01 vs. control, *P ≤ 0.05 vs. LPS and control. Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Steady state mRNA levels of ntcp and bsep in livers prepared from control rats, after LPS injection (LPS), after heat stress (HS), or after heat stress and consecutive LPS injection (HS/LPS). A total of 20 μg RNA was probed for ntcp and bsep as described in Materials and Methods. Densities of 3 blots were normalized to 100% of glyceraldehyde-3-phosphate dehydrogenase, which was used as loading standard. ntcp, analysis of variance, P < 0.001. *P < 0.01 vs. control and HS, **P < 0.001 vs. controls. bsep: analysis of variance, P < 0.001. *P < 0.05 vs. control, **P < 0.001 vs. control; †P < 0.01 vs. HS and LPS. Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Time course of bsep and ntcp mRNA prepared from livers after intraperitoneal injection of NaCl (●), LPS (■), or combined HS/LPS (□) treatment. Preparations were performed at 0, 4, 12, 24, 36, and 72 hours after the respective treatments and compared by densitometry in the lower panels. The Figure shows representative Northern blot analyses (n = 3). Analysis of variance, P ≤ 0.001. **P ≤ 0.01 vs. control, *P ≤ 0.05 vs. control. Gastroenterology 2002 122, 963-973DOI: (10.1053/gast.2002.32408) Copyright © 2002 American Gastroenterological Association Terms and Conditions