Hepatocyte cytoskeleton during ischemia and reperfusion--influence of ANP-mediated p38 MAPK activation.

AIM: To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischemia and reperfusion. METHODS: For in vivo experiments, animals received ANP (5 microg/kg) intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer+/-ANP (200 nmol/L) +/-SB203580 (2 micromol/L). Livers were then kept under ischemic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hsp27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy. RESULTS: We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observed alterations of the cytoskeleton in hepatocytes of ANP-preconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580. CONCLUSION: This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation.

Protein kinase A dependent signalling mediates anti-apoptotic effects of the atrial natriuretic peptide in ischemic livers.

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 degrees C, 24h). 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.

ANP-induced decrease of iron regulatory protein activity is independent of HO-1 induction.

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.

Atrial natriuretic peptide preconditioning protects against hepatic preservation injury by attenuating necrotic and apoptotic cell death.

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 cGMP) 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.

Kupffer-cell specific induction of heme oxygenase 1 (hsp32) by the atrial natriuretic peptide--role of cGMP.

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 (4 degrees C, 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. CONCLUSION: 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.

Stimulation of p38 MAPK by hormonal preconditioning with atrial natriuretic peptide.

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.