Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1.
OBJECTIVE
The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-kappaB in macrophages. ANP was also shown to protect from ischemia-reperfusion injury of the rat liver. This study aimed to investigate the effects of this immunomodulatory hormone and its second messenger cGMP on the activation of the two redox-sensitive transcription factors AP-1 and NF-kappaB and the expression of corresponding pro-inflammatory target genes during ischemia and reperfusion of the liver. The identification of the mechanisms underlying the protection by ANP should reveal new aspects concerning the pathomechanisms of ischemia/reperfusion injury.


METHODS
Rat livers were perfused with and without ANP or 8-Br-cGMP preceding 24 h of cold storage in University of Wisconsin solution. During reperfusion NF-kappaB and AP-1 DNA binding activities were determined in freeze-clamped liver samples by electrophoretic mobility shift assay. Protein levels of p50, p65, and of IkappaB were determined by Western blot. mRNA coding for inducible nitric oxide synthase, cyclooxygenase-2, and TNF-alpha was determined by RT-PCR and Northern blot.


RESULTS
After 45 min of reperfusion DNA binding activities of NF-kappaB were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, was activated and in the course of reperfusion the subunit composition of AP-1 changed as assessed by supershift assays. ANP markedly reduced binding activities of both forms of AP-1. 8-Br-cGMP mimicked the effects of ANP on NF-kappaB and AP-1. Neither inducible nitric oxide synthase nor cyclooxygenase-2 mRNA could be detected. In contrast, a profound expression of transcripts coding for TNF-alpha was detected in the course of reperfusion and ANP markedly reduced TNF-alpha mRNA expression.


CONCLUSIONS
ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-kappaB and AP-1 via cGMP. The reduced binding activity of these redox-sensitive transcription factors was accompanied by a diminished mRNA expression of TNF-alpha, a cytokine known to be involved in cellular damage in ischemia reperfusion injury.
Kiemer
A K
AK
Department of Medicine II, Klinikum Grosshadern, University of Munich, Germany. Alexandra.Kiemer@cup.uni-muenchen.de
Vollmar
A M
AM
Bilzer
M
M
Gerwig
T
T
Gerbes
A L
AL
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000008:administration & dosage*; D015536:Down-Regulation / Q000187:drug effects; D007511:Ischemia / Q000378:metabolism*; D008099:Liver / Q000098:blood supply* / Q000378:metabolism*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015424:Reperfusion; D015427:Reperfusion Injury / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D009320:Atrial Natriuretic Factor