Volume 119, Issue 3, Pages 794-805 (September 2000) Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis  Esther Titos, Joan Clària, RamóN Bataller, Marta Bosch–Marcé, Pere Ginès, Wladimiro Jiménez, Vicente Arroyo, Francisca Rivera, Joan Rodés  Gastroenterology  Volume 119, Issue 3, Pages 794-805 (September 2000) DOI: 10.1053/gast.2000.17831 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (Upper panel) Representative Northern blot analysis of 5-LO mRNA expression in liver tissue from 2 control (CT1 and CT2) and 2 cirrhotic rats with ascites (CH1 and CH2). Poly(A)+ RNA was electrophoresed on denaturing 1% agarose gels, transferred onto nylon filters, and hybridized with 32P-labeled cDNA probes specific for human 5-LO and GAPDH. Total RNA isolated from rat lung tissue was used as a positive control. (Lower panel) Representative Western blot analysis of LTC4 synthase protein expression in liver tissue from control (CT) and cirrhotic (CH) rats. Proteins (100 μg) were electrophoresed on 18% SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes, and LTC4 synthase was detected with an anti-LTC4 synthase polyclonal antibody and a secondary horseradish peroxidase–linked donkey anti-rabbit antibody. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunoreactive cysteinyl-LT content in rat liver homogenates. Liver samples were obtained from untreated animals (week 0) and from rats treated with CCl4 for either 6 or 16 weeks. Cysteinyl-LT levels were measured by EIA after Sep-Pak extraction. Values represent mean ± SEM and were statistically different at weeks 6 and 16 compared with week 0. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Representative RT-PCR analysis of 5-LO mRNA expression in rat liver cells. RNA was obtained by the guanidinium isothiocyanate–cesium chloride method, and cDNA was produced by RT. PCR amplification was performed using specific oligonucleotide sequences of rat 5-LO and GAPDH. (A) 5-LO mRNA expression in liver tissue and parenchymal liver cells. Total liver samples from a control animal (lane 1) and from rats treated with CCl4 for 6 (lane 2) and 16 weeks (lane 3); purified hepatocytes from control (lane 4) and cirrhotic (lane 5) rats and CC-1 cells under resting conditions (lane 6) and after LPS (lane 7) and IL-6 (lane 8) stimulation. RNA isolated from RFL-6 cells (a rat fetal lung fibroblast cell line) was used as a positive control (lane 9). (B) 5-LO mRNA expression in Kupffer cells (lane 1) and HSCs (lane 2) from a control animal. RNA isolated from CRL-2192 cells (a rat alveolar macrophage cell line) was used as a positive control (lane 3). Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Representative RP-HPLC profile of products obtained from purified hepatocytes incubated with LTA4 (left panel). Materials from 3 different incubations were pooled, extracted with Sep-Pak cartridges, and injected into RP-HPLC. The column, a Tracer Supelcosil (5 μm, 4.5 × 250 mm), was eluted with an isocratic mobile-phase MeOH/H2O/acetic acid (65:35:0.01; vol/vol/vol; pH 5.7) at a flow rate of 1.2 mL/min, and the detector was set to record signals at 280 nm. Upper right panel: a representative profile of authentic materials. Lower right panel: on-line spectra of materials obtained from hepatocyte incubations. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Ability of rat liver cells to generate cysteinyl-LTs. Isolated rat hepatocytes (HEP), HSCs, Kupffer cells (KC), and SECs were exposed to vehicle (DPBS++; □) or LTA4 (20 μmol/L; ▨) and incubated with A23187 (5 μmol/L) for 20 minutes at 37°C. Inset: Effect of exogenous arachidonic acid (AA) and LTA4 on the release of cysteinyl-LTs by cultured CC-1 cells. Confluent CC-1 cells were exposed to vehicle (DPBS++), arachidonic acid (20 μmol/L), or LTA4 (20 μmol/L) and incubated with A23187 (5 μmol/L) for 20 minutes at 37°C. Cysteinyl-LT production was measured by EIA. Results represent the mean ± SEM of 3–10 experiments. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Concentration-dependent effects of LPS and IL-6 on the generation of cysteinyl-LTs by CC-1 cells. Confluent CC-1 cells plated in 24-well tissue culture plates were exposed to (A) LPS or (B) recombinant rat IL-6 for 18 hours, and incubated with LTA4 (15 μmol/L) and A23187 (5 μmol/L) in 0.2 mL DPBS++ for 20 minutes at 37°C. Cysteinyl-LT levels were measured by EIA. Results represent the mean ± SEM of 3 different experiments determined in duplicate. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Cysteinyl-LT formation from (A)endogenous and (B) exogenous sources of LTA4 by purified rat hepatocytes. Hepatocytes (5 × 106 cells/mL) were obtained from 6 control (CT) and 6 rats with cirrhosis and ascites (CH) and incubated with either vehicle (top) or LTA4 (20 μmol/L; bottom) in the presence of A23187 (5 μmol/L) for 20 minutes at 37°C. Cysteinyl-LT levels were quantified by EIA. The values represent mean ± SEM. Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of LTD4 on [Ca2+]i in individual Fura-2–loaded cultured HSCs. (A) Cells under basal conditions. (B) Two minutes after stimulation with 10−8 mol/L LTD4. (C) Calcium response to LTD4 in an individual cell. Arrow shows addition of the agonist (10−8 mol/L). Illustrations are representative of overlaid fluorescence images in which intensities are depicted in red and green for the 340-nm and 380-nm excitation wavelengths, respectively. (Original magnification 400×.) Gastroenterology 2000 119, 794-805DOI: (10.1053/gast.2000.17831) Copyright © 2000 American Gastroenterological Association Terms and Conditions