Background/Aims: Preconditioning of livers with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury (IRI) via the particulate guanylate cyclase. Recently, we have shown that ANP affects the p38 MAPK signalling cascade in the liver. Thus, aim of the present study was to elucidate the role of cGMP- and p38 MAPK-dependent signalling pathways in ANP-mediated anti-apoptotic effects.Methods: Rat livers were perfused with KH-buffer with or without ANP, 8-Br-cGMP (+/-kinase inhibitors) and kept in UW solution (4 degreesC, 24 h). Caspase-3-like activity was measured by a fluorometric assay. Expression of cGMP-dependent protein kinases (PKG) in liver tissue was determined by RT-PCR, BAD phosphorylation by Western blot, and cAMP-dependent protein kinase (protein kinase A, PKA) activity by in vitro phosphorylation.Results: Compared to control organs, ANP-preconditioning reduced post-ischemic caspase-3-like activity. Neither perfusion with a p38 MAPK inhibitor nor with a PKG inhibitor abolished the ANP-mediated anti-apoptotic action. The two PKG isoforms were demonstrated not to be expressed in the liver. In contrast, liver perfusion with a selective PKA inhibitor abrogated the anti-apoptotic effect of ANP. Phosphorylation of pro-apoptotic BAD by ANP-activated PKA might inhibit liver cell apoptosis.Conclusions: ANP mediates its anti-apoptotic action during ischemic injury via a crosstalk with the PKA pathway. (C) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Atrial natriuretic peptide (ANP)-preconditioned livers are protected from ischemia-reperfusion injury. ANP-treated organs show increased expression of heme oxygenase (HO)-1. Because HO-1 liberates bound iron, the aim of our study was to determine whether ANP affects iron regulatory protein (IRP) activity and, thus, the levels of ferritin. Rat livers were perfused with Krebs-Henseleit buffer [+/-ANP, 8-bromo-cGMP (8-Br-cGMP), and tin protoporphyrin, 20 min], stored in University of Wisconsin solution (4 degrees C, 24 h), and reperfused (120 min). IRP activity was assessed by gel-shift assays, and ferritin, IRP phosphorylation, and PKC localization were assessed by Western blot. Control livers displayed decreased IRP activity at the end of ischemia but no change in ferritin content during ischemia and reperfusion. ANP-pretreated livers showed reduced IRP activity, an effect mimicked by 8-Br-cGMP. Ferritin levels were increased in ANP-pretreated organs. Simultaneous perfusion of livers with ANP and tin protoporphyrin did not reduce ANP-induced action, arguing against a role for HO-1 in changes in IRP activity. ANP and 8-Br-cGMP decreased membrane localization of PKC-alpha and PKC-epsilon, but this modulation of PKC seems unrelated to inhibition of IRP binding. This work shows the cGMP-mediated attenuation of IRP binding activity by ANP, which results in increased hepatic ferritin levels. This change in IRPs is independent of ANP-induced HO-1 and reduced PKC activation.

Background/Aims: Pretreatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of livers via cGMP. Heme oxygenase-1 (HO-1) is known as a protective mediator in ischemia-reperfusion injury. The aim of this study was to investigate whether ANP affects the expression of HO-1.Methods: Rat livers were perfused with KH-buffer with/without ANP or 8-Br-cGMP, kept in UW solution (4degreesC, 24 h), and reperfused. HO-1 mRNA and protein was determined by Northern and Western blot, in situ hybridization, and immunohistochemistry in livers or isolated liver cells.Results: ANP significantly elevated HO-1 mRNA expression at the end of the preconditioning period and was without effects at the end of ischemia and during reperfusion. 8-Br-cGMP did not affect HO-1 mRNA expression. In situ hybridization as well as immunohistological double-staining revealed that Kupffer cells but not hepatocytes showed HO-1 mRNA and protein expression. Hepatocytes revealed no changes in HO-1 protein whereas Kupffer cells showed a marked increase in HO-1 protein after ANP treatment. Inhibition of HO-1 did not abrogate hepatoprotection conveyed by ANP.Conclusions: Our data show the potency of ANP to specifically induce HO-1 in Kupffer cells independently of cGMP. This increased expression of HO-1 is not involved in hepatoprotection conferred by ANP being in line with the knowledge that ANP mediates hepatoprotection via cGMP. (C) 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Background/Aims: Preconditioning of livers with the atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia-reperfusion injury. Aim of this study was to characterize the influence of ANP preconditioning on necrotic and apoptotic cell death and on proliferation.Methods: Rat livers were perfused with Krebs-Henseleit buffer with or without ANP or its second messenger analogue 8-Bromo cyclic guanosine monophosphate (8-Br cGNIP) for 20 min, stored in cold University of Wisconsin solution (24 h), and reperfused for up to 120 min. Apoptosis and necrosis were determined using biochemical and morphological criteria, proliferation was assessed by Ki67 histochemistry.Results: Apoptosis peaked after 24 h of cold ischemia. Preconditioning with both ANP and 8-Br-cGMP significantly reduced caspase-3-like activity and the number of triphosphate nick-end labelling-positive cells. Reduction of apoptosis was significant for hepatocytes, but not for endothelial cells. After ischemia, degenerative cell changes were clearly reduced in ANP pretreated livers. After reperfusion, ANP preconditioning led to a significant reduction of necrotic hepatocytes and endothelial cells in periportal zones. Cell proliferation was not affected by preconditioning.Conclusions: ANP reduces necrotic and apoptotic cell death without affecting the proliferation status. The protection takes place mainly in the periportal area and seems to be most prominent against necrosis of hepatocytes and endothelial cells during reperfusion. (C) 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

AIM:Stress-activated signaling pathways responsible for hepatic ischemia reperfusion injury and their modulation by protective interventions are widely unknown. Preconditioning of rat livers with Atrial Natriuretic Peptide (ANP) attenuates ischemia reperfusion injury (Gerbes et al. Hepatology 1998, 28:1309-1317). Since ANP has recently been shown to be a regulator of the p38 MAPK pathway in endothelial cells (Kiemer et al. Circ Res 2002, 90:874-881), aim of this study was to investigate activities of MAPK during ischemia and reperfusion and effects of ANP on MAPK.METHODS:Rat livers were perfused with KH-buffer in the presence or absence of ANP for 20 min, kept in cold UW solution for 24 h, and reperfused for up to 120 min. Activities of p38 MAPK and JNK was determined by in vitro phosphorylation assays using MBP and c-jun as substrates. After SDS/PAGE electrophoresis, gels were quantified by phosphorimaging.RESULTS:Activity of p38 MAPK in control organs decreased in the course of ischemia and reperfusion by 85%, whereas ANP increased p38 activity by up to 30-fold. JNK activation of control livers increased in the course of ischemia and reperfusion by up to three-fold. This increase in JNK activity was slightly elevated in ANP preconditioned organs.CONCLUSION:This work represents a systematic investigation of MAPK activation during liver ischemia and reperfusion. Employing ANP, for the first time a pharmacological approach to modulate these central signal transduction molecules is presented.

Background/Aims: The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-kappa B 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-kappa B 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-kappa B 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 I kappa B 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-kappa B were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, mas 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-kappa B 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,Conclusion: ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-kappa B 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.