Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and cAMP-dependent secretion in cholangiocytes

BACKGROUND; AIMS: The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis. METHODS: To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (3) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases. RESULTS: Our results show that: (1) tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl(-) and HCO(3)(-) transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (3) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC). CONCLUSIONS: Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO(3)(-) and Cl(-) secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies.