cAMP inhibits inducible nitric oxide synthase expression and NF-kappaB-binding activity in cultured rat hepatocytes.
BACKGROUND
The inducible nitric oxide synthase (iNOS) is strongly expressed following inflammatory stimuli. Adenosine 3',5'-cyclic monophosphate (cAMP) increases iNOS expression and activity in a number of cell types but decreases cytokine-stimulated iNOS expression in hepatocytes. The mechanisms for this effect are unknown.


METHODS
Rat hepatocytes were stimulated with cytokines to induce iNOS and cultured with cAMP agonists dibutyryl-cAMP (dbcAMP), 8-bromo-cAMP, and forskolin (FSK). Nitric oxide synthesis was assessed by supernatant nitrite levels and iNOS expression was measured by Northern and Western blot analyses. Nuclear factor kappaB binding was assessed by electromobility shift assay.


RESULTS
Cyclic AMP dose dependently decreased NO synthesis in response to a combination of proinflammatory cytokines or interleukin-1beta (IL-1beta) alone. The adenylate cyclase inhibitor SQ 22,536 increased cytokine- or IL-1beta-stimulated NO synthesis. dbcAMP decreased iNOS mRNA expression and iNOS protein expression. Both dbcAMP and glucagon decreased iNOS promoter activity in rat hepatocytes transfected with the murine iNOS promoter and decreased DNA binding of the transcription factor NF-kappaB.


CONCLUSIONS
These data suggest that cAMP is important in hepatocyte iNOS expression and agents that alter cAMP levels may profoundly alter the response of hepatocytes to inflammatory stimuli through effects onthe iNOS promoter region and NF-kappaB.
Harbrecht
B G
BG
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA. harbrechtbg@msx.upmc.edu
Taylor
B S
BS
Xu
Z
Z
Ramalakshmi
S
S
Ganster
R W
RW
Geller
D A
DA
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005934:Glucagon / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D007375:Interleukin-1 / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000502:physiology; D018805:Sepsis / Q000378:metabolism / Q000503:physiopathology; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; C017759:9-(tetrahydro-2-furyl)-adenine; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D005934:Glucagon; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D000262:Adenylyl Cyclases; D000225:Adenine