1. Volume 124, Issue 3, Pages 737-753 (March 2003)
Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and cAMP- dependent secretion in cholangiocytes 
Carlo Spirlì, Luca Fabris, Elena Duner, Romina Fiorotto, Giorgio Ballardini, Tania Roskams, Nicholas F. Larusso, Aurelio Sonzogni, Lajos Okolicsanyi, Mario Strazzabosco 
Gastroenterology 
Volume 124, Issue 3, Pages (March 2003)
DOI: /gast
Copyright © 2003 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Time course of NOx production on the (A) apical and (B) basolateral medium of NRC-1 cells at different time points after basolateral administration of single cytokines at the following concentrations: LPS (10 ng/mL; n = 7), IL-1 (20 U/mL; n = 7), TNF-α (500 U/mL; n = 7), and IFN-γ (100 U/mL; n = 6). Cytokines were administered from the basolateral side of the monolayer. Apical and basolateral medium was collected and NOx concentration was measured. Only IFN-γ increased NOx production significantly after 24 hours of incubation. Both IL-1 and IFN-γ increased NOx production significantly at 48 hours. (C, D) Time course of NOS2 and β-actin messenger RNA expression and NOS2/β-actin densitometric ratio, respectively. Both IL-1 and IFN-γ increased the gene expression of NOS2 in NRC-1 cells. NOS2 gene expression peaked at the 12th hour and then slowly decreased by the 48th hour. (Data are presented as mean ± SD; P < 0.05 vs. controls.) □, 12 h; ▨, 24 h; ■, 48 h.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Effects of different mixtures of cytokines: LPS 10 ng/mL + TNF-α 500 U/mL [n = 4]; LPS 10 ng/mL + IL-1 20 U/mL [n = 4]; IL-1 20 U/mL + TNF-α 500 U/mL [n = 7]; LPS 10 ng/mL + IL-1 20 U/mL + TNF-α 500 U/mL [n = 4]; LPS 10 ng/mL + IFN-γ 100 U/mL [n = 4]; IL-1 20 U/mL + IFN-γ 100 U/mL [n = 4]; LPS 10 ng/mL + IL-1 20 U/mL + IFN-γ 100 U/mL; TNF-α 500 U/mL + IFN-γ 100 U/mL [n = 4]; TNF-α 500 U/mL + IL-1 20 U/mL + IFN-γ 100 U/mL [n = 5]; LPS + TNF-α 500 U/mL + IFN-γ 100 U/mL [n = 4] on (A) apical and (B) basolateral NOx production; (C) NOS2 gene expression and (D) NOS2/β-actin densitometric ratio after 24 hours of incubation. Cytokine effects clearly were additive and were facilitated by IFN-γ; indeed, NOx production increased significantly only in the presence of IFN-γ. (Data are presented as mean ± SD; P < 0.05 vs. controls).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Effects of NO donors (DETA, PAPA, and SIN-1) on fluid secretion in IBDU. (A) Forskolin (10 μmol/L)-induced luminal area expansion (percent increase with respect to basal values). PAPA was administered at a dose of 1 mmol/L and cells were studied 30 minutes after; DETA was administered at a dose of 500 μmol/L for 24 hours; SIN-1 was given at a dose of 1 mmol/L for 30 minutes, and cells were studied 24 hours later. Among NO donors, DETAnonoate but not PAPAnonoate caused a dose-dependent inhibition of forskolin-stimulated secretory activities. Similarly, SIN-1 significantly blocked forskolin-dependent fluid secretion. ●, controls + F (n = 213); □,*, PAPA + F (n = 13); ▴, DETA F (n = 75); ○, controls (n = 14); *, SIN-1 + F (n = 60). (B) Dose-dependent inhibition of forskolin-dependent secretion in IBDU. Cells were incubated for 24 hours with DETA at indicated concentrations (*P < 0.05 vs. controls + forskolin 10 μmol/L; **P < vs. controls + forskolin 10 μmol/L). (C) Effects of RNOS scavengers (uric acid, trolox, DTT, and MnTBAP) on NO-induced inhibition of forskolin-dependent secretion in IBDU. Cells were incubated for 24 hours with DETA 500 μmol/L together with 100 μmol/L uric acid or 200 μmol/L MnTBAP or 80 μmol/L trolox. In the case of DTT, 1 mmol/L DTT was added after 24 hours of incubation with DETA 500 μmol/L. All scavengers are capable of removing DETA inhibition on fluid secretion. (▴, P < 0.05 vs. DETA 500 μmol/L + forskolin 10 μmol/L.)
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Effects of L-NIL (50 μmol/L) and MnTBAP (200 μmol/L) on cytokine-mediated inhibition of forskolin-stimulated fluid secretion on IBDU. Incubation of IBDU with the specific NOS2 inhibitor L-NIL and RNOS scavenger MnTBAP prevents cytokine-induced cholestasis. IBDU were incubated for 24 hours with cytokine mix (INF-γ 100 U/mL + TNF-α 500 U/mL) as described.8 ♦, cytokine mix + MnTBAP + F (n = 19); ▴, cytokine mix + L-NIL + F (n = 19); ○, controls + F (n = 213); □, cytokine mix + F (n = 17); ●, controls (n = 14).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Representative intracellular pH (pHi) tracings showing the effects of NO donors on Cl−/HCO3− exchanger. This is an electroneutral transporter that mediates HCO3− secretion in the bile in exchange with Cl−. Because at physiologic electrochemical gradients the anion exchanger acts as an acid loader, its activity was assessed from the rate of pHi recovery from an intracellular alkaline load induced by administration and withdrawal of the cell-permeant weak acid propionate. IBDU were first incubated with 30 mmol/L propionate for 10 minutes (not shown). When propionate was withdrawn, intracellular pHi increased. Cells recover from this alkali load by extruding HCO3 in exchange with Cl−. The slope of this recovery phase is a measure of Cl−/HCO3− exchanger activity. The tracing on the upper left panel shows that in control conditions Cl−/HCO3− exchanger activity was stimulated by agents increasing cellular cAMP levels (DBc-AMP 100 μmol/L + IBMX 100 μmol/L + forskolin 3 μmol/L). (B) The same experiment in cells treated with DETA 500 μmol/L for 24 hours. Basal Cl−/HCO3− exchanger activity was not significantly different from controls in DETA-pretreated groups, whereas anion exchanger isoform 2 activation by cAMP mix was abolished.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Representative experiments showing the effects of NO donor on cAMP-dependent Cl− efflux that was CFTR-mediated. Cl− efflux was monitored by measuring fluorescence intensity of MEQ, an indicator of Cl− concentration (an increase in relative fluorescence indicated a decrease in intracellular Cl− concentration). Experiments were performed in the presence of furosemide to eliminate confounding effect of basolateral Na+-K+-2 Cl− cotransport. (A) In control conditions, cAMP administration stimulated Cl− efflux, as shown by the increase in relative fluorescence; removal of external Cl− from the perfusion medium caused a further increase in fluorescence. (B) In DETA-pretreated cells, the cAMP-mediated Cl− efflux (which in cholangiocytes is mediated by CFTR) was abolished, but cells rapidly extrude Cl− when exposed to Cl−-free medium.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Effects of NO donor on intracellular cAMP levels. Controls or DETA (500 μmol/L)-pretreated cells were stimulated with forskolin (10 μmol/L) for 30 minutes, followed by ethanol extraction and measurement of cAMP by using a standard fluorimetric cAMP assay kit (Cayman Chemicals), following the manufacturer's instructions. DETA significantly inhibited forskolin-stimulated increase in cAMP, but not basal levels of cAMP; incubation with MnTBAP 200 μmol/L blocked the inhibitory effect. As already reported,8 incubation with cytokines (TNFα 500 U/mL + IFNγ 100 U/mL) inhibited forskolin-stimulated cAMP formation; these effects were inhibited by co-incubation with the NOS2 inhibitor L-NIL 50 μmol/L and by MnTBAP 200 μmol/L, indicating that cytokine effects are mediated by NOS2 induction and RNOS generation. Columns represent the mean ± SD of indicated replicas (*P < vs. controls + forskolin 10 μmol/L; **P < 0.01 vs. DETA + forskolin; ▴, P < 0.05 vs. cytokine mix + forskolin 10 μmol/L).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

9. Fig. 8
AC activity in cholangiocyte (NRC-1 cell line) membranes was inhibited by NO donors. Membranes were preincubated with the indicated concentrations of SNP and DETA for 30 minutes on ice. Membranes were then assayed for forskolin-stimulated AC activity expressed as pmol/mg/min. cAMP concentrations were determined by a radioimmunoassay procedure. Both NO donors inhibit AC activity in a dose-dependent manner. Columns represent the mean ± SD of indicated replicas. (**P < vs. controls + forskolin 10 μmol/L). ■, no stimulation; ●, forskolin 10 μmol/L.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

10. Fig. 9
The scatter plot shows the distribution of histologic score for NOS2 staining intensity in hepatocytes, interlobular/septal ducts (LBD), and cholangioles (SBD). NOS2 expression was analyzed by using a semiquantitative scale ranging from 0 to 4. Zero was equal to no staining, 1 denoted the positivity in some structures, 2 indicated a diffuse mild staining, 3 indicated a moderate diffuse positivity, and 4 was for strong diffuse staining. Only stainings performed in paraffin-embedded biopsy samples were used for semiquantitative analysis. Statistical significance vs. controls, calculated by using the Kruskal-Wallis test, are reported between brackets. *P < 0.05; **P < 0.01; ***P <
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

11. Fig. 10
Immunolocalization of NOS2 in (A, B) hepatitis C virus–related chronic liver disease, (C, D) PBC, (E, F) PSC, and of (G, H) nitrotyrosine in PSC (paraffin sections, 3-amino-9-ethylcarbazole method). In hepatitis C virus–related chronic liver disease, (A) a variable NOS2 expression was present in hepatocytes throughout the nodule (original magnification ×10), and (B) in some marginal reactive ductules, but not in major ducts (original magnification ×40). (C) In PBC, a mild NOS2 immunoreactivity was found in hepatocytes and marginal ductular structures (original magnification ×10), (D) whereas it was consistently negative in major ducts (original magnification ×20). (E) In contrast, in PSC a strong NOS2 labeling was observed extensively not only in reactive ductules (original magnification ×20), but also in (F) major ducts, some of which showed an onion-like bulb periductular fibrosis (original magnification ×20). (G, H) Nitrotyrosine expression was also easily detectable, as seen in PSC where it extensively decorated periportal hepatocytes and bile ducts of different sizes (G, original magnification ×10, H, original magnification ×40).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions