Volume 120, Issue 5, Pages 1227-1240 (April 2001) Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver  Takanori Kyokane, Shinji Norimizu, Hisashi Taniai, Tokio Yamaguchi, Shinji Takeoka, Eishun Tsuchida, Makoto Naito, Yuji Nimura, Yuzuru Ishimura, Makoto Suematsu  Gastroenterology  Volume 120, Issue 5, Pages 1227-1240 (April 2001) DOI: 10.1053/gast.2001.23249 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Overproduction of NO and CO through iNOS and HO-1 in livers undergoing LPS exposure. (A) Western blotting analyses showing effects of LPS on expression of iNOS and HO-1. m, molecular markers; LDD(+), Kupffer cell–depleting procedure by LDD. (B) Measurements of NO2− and CO in the venous perfusate and bilirubin (BR)-IXα in bile collected from LPS-treated livers. ▨, Data collected from livers perfused with 5 mmol/L AG, an inhibitor of iNOS. Note that the AG treatment completely attenuated LPS-induced overproduction of NO and inversely increased generation of CO and BR-IXα (†P < 0.05). #P < 0.05 compared with the 6-hour LPS-treated group. Data indicate mean ± SD of measurements from 5–7 livers. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of HbO2, metHb, and HbV-O2 on the vascular resistance and tissue cGMP contents in the (left) LPS-untreated and (right) -treated livers. Shaded area indicates a 15-minute interval for perfusion with the Hb derivatives. The cGMP contents were measured in snap-frozen tissue samples collected at 15 minutes (arrows in the upper panels), the end of perfusion with the reagents. *P < 0.05 compared with the baseline in the LPS-untreated control. †P < 0.05 compared with the data from livers untreated with Hb derivatives in the same panel. #P < 0.05 compared with the data in the Hb-perfused LPS livers. Data indicate mean ± SD of measurements from 5–7 livers. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of HbO2, metHb, and HbV-O2 on the baseline bile output and biliary flux of bilirubin (BR)-IXα in the (left) LPS-untreated and (right) -treated livers. Shaded area indicates a 15-minute interval for perfusion with the Hb derivatives. Insets in the lower panels illustrate net increases in the biliary BR-IXα flux during the 15-minute period for perfusion of the Hb derivatives (ΔBF(15)BR-IXα). *P < 0.05 compared with the baseline in the LPS-untreated control. †P < 0.05 compared with the data from livers untreated with Hb derivatives in the same panel. #P < 0.05 compared with the data in the Hb-perfused LPS livers. Data indicate mean ± SD of measurements from 5–7 livers. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of depletion of Kupffer cells on biliary flux of bilirubin (BR)-IXα in the LPS-pretreated liver. LDD(−)/(+) indicate the livers treated without and with LDD, the Kupffer cell–depleting reagent, respectively. Upper panels indicate representative microfluorographs showing phagocytosis of Nile red–coated latex particles in the 6-hour LPS-treated rats. Bar = 30 μm. The BR-IXα values were measured at 30 minutes after the onset of perfusion with the Hb derivatives, indicating mean ± SD of 6 and 4 experiments in the LDD(−) and LDD(+) groups, respectively. Note that the Kupffer cell depletion significantly attenuated the HbV-O2–induced elevation of BR-IXα (*P < 0.05), but not that induced by HbO2 or by metHb. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of suppression of vasorelaxing gases by AG and ZnPP on the (A) vascular resistance and (B) bile output in the LPS-pretreated livers. Shaded area indicates a 10-minute period for perfusion of 300 nmol/L ZnPP and/or 4 μmol/L CO, or that of SNAP. Inset in the upper panel illustrates differences in tissue contents of cGMP among data points indicated by a–d (*P < 0.05 compared with a; †P < 0.05 compared with b). *P < 0.05 compared with the baseline value at 0 minutes. †P < 0.05 compared with the data measured at 30 minutes. #P < 0.05 compared with the data in the group treated with ZnPP. Data indicate mean ± SD of measurements from 5–7 livers. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Representative pictures showing patterns of sodium fluorescein labeling in perfused livers undergoing 6-hour LPS treatment. (A) The LPS-untreated control liver. (B) The liver exposed to 6-hour LPS treatment. (C and D) The LPS-treated liver perfused with 1.5 g/dL of HbO2 and metHb, respectively. Note that the HbO2 treatment induces a marked reduction of the fluorescence labeling in periportal regions. P, periportal regions; C, central venules. Bar = 100 μm. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Vasorelaxing effects of cytochrome P450 inhibitors, CTZ and metyrapone (MP), on the AG-elicited vasoconstriction in the LPS liver. (Left) Dose-dependent vasorelaxation by supplementation of CO. Note that 8 μmol/L CO induced a maximum vasorelaxation. (Right) Vasorelaxing effects of MP and CTZ. As seen, CO supplementation at 4 μmol/L does not induce additive relaxing effects in the presence of 10 μmol/L CTZ. *P < 0.05 and #P < 0.05 compared with the nontreating baseline (none). Data indicate mean ± SD of measurements from 5 livers. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of administration of HbO2 and metHb on basal bile output and biliary bilirubin (BR)-IXα flux in LPS-treated rats in vivo. Alterations in bile output were expressed as percentage changes in the output measured at 20 minutes vs. that measured before the administration of HbO2 or metHb (%ΔBile output). ΔBR-IXα flux denotes net increases in BR-IXα excreted into bile during the initial 20 minutes after the administration of HbO2 or metHb was started. Data collected from the (A) LPS-untreated control rats and (B) those undergoing the 6-hour LPS exposure, respectively. *P < 0.05 compared with the value of the vehicle-treated LPS rats. †P < 0.05 compared with the values in the HbO2-treated LPS rats. Data indicate mean ± SD of measurements from 5–7 experiments. Gastroenterology 2001 120, 1227-1240DOI: (10.1053/gast.2001.23249) Copyright © 2001 American Gastroenterological Association Terms and Conditions