Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and cAMP-dependent secretion in cholangiocytes.
OBJECTIVE
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.
Spirlì
Carlo
C
Department of Medical and Surgical Sciences, University of Padova, Italy.
Fabris
Luca
L
Duner
Elena
E
Fiorotto
Romina
R
Ballardini
Giorgio
G
Roskams
Tania
T
Larusso
Nicholas F
NF
Sonzogni
Aurelio
A
Okolicsanyi
Lajos
L
Strazzabosco
Mario
M
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism*; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D004357:Drug Synergism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008107:Liver Diseases / Q000201:enzymology; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D009566:Nitrates; D020030:Nitric Oxide Donors; D009573:Nitrites; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D000242:Cyclic AMP; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000262:Adenylyl Cyclases