Volume 41, Issue 3, Pages 414-420 (September 2004) Protein kinase A dependent signalling mediates anti-apoptotic effects of the atrial natriuretic peptide in ischemic livers  Stefanie Kulhanek-Heinze, Alexander L. Gerbes, Tobias Gerwig, Angelika M. Vollmar, Alexandra K. Kiemer  Journal of Hepatology  Volume 41, Issue 3, Pages 414-420 (September 2004) DOI: 10.1016/j.jhep.2004.05.017 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Experimental protocol. Livers were just flushed exsanguinous (0′ perfusion) or were perfused with KH buffer before ischemia for 30min (preconditioning) either in the absence or in the presence of 200nM ANP or 50μM 8-Br-cGMP given 20min prior to ischemia and to the storage solution. After preconditioning livers were stored for 24h at 4°C (cold ischemia). In additional experiments, livers were pre-treated by adding different protein kinase inhibitors (2μM SB203580, 1μM Rp-8-Br-pCPT-cGMPS, 1μM Rp-8-Br-cAMP) to the pre-ischemic perfusate in the absence or presence of 200nM ANP. Arrows indicate time points when liver samples were taken. Journal of Hepatology 2004 41, 414-420DOI: (10.1016/j.jhep.2004.05.017) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 p38 MAPK activation is not involved in ANP-mediated anti-apoptotic effects. Either isolated rat livers were flushed exsanguinous (0′ perfusion) with KH buffer or were perfused with KH buffer before ischemia for 30min in the absence (Co) or presence of 200nM ANP±2μM SB203580, which was added 20min prior to ischemia. A: The figure shows the caspase-3-like activity induced by ischemia. Livers were briefly perfused blood-free (0′ perfusion) and caspase-3-like activity was measured as described under Section 2. Activities in sham treated 0′ perfused livers were set equal 100% and rose up to 350mU/mg at the end of 24h ischemia. Columns show mean±SEM of two independent experiments with four to five rat livers. **P<0.01 vs. 0′ perfusion. B: After 24h of cold ischemia livers were snap frozen and caspase-3-like activity was measured as described under Section 2. Data are expressed as percent of enzyme activity of untreated control livers set equal 100%. Columns show mean±SEM of three independent experiments with four to five rat livers. ***P<0.001 vs. control, ##P<0.01 vs. ANP. C: Livers were snap frozen either after 30min perfusion with KH-buffer in the absence or presence of 8-Br-cGMP (50μM) or after 24h ischemia. p38 MAPK activity was determined as described under Section 2. The p38 MAPK activity is expressed as x-fold activity compared to control activity. Columns show mean±SEM of four to five rat livers. Journal of Hepatology 2004 41, 414-420DOI: (10.1016/j.jhep.2004.05.017) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 PKG activation is not involved in the anti-apoptotic action of ANP. A: After 24h of cold ischemia, control livers (Co) and pre-treated livers (200nM ANP)±1μM Rp-8-pCPT-cGMPS (PKG inhibitor) were snap frozen and prepared for fluorogenic measurement of caspase-3-like activity (see Section 2). Data are expressed as percent of enzyme activity of untreated control livers set equal 100%. Columns show mean±SEM of two independent experiments with n=4–5 organs per group. *P<0.05 significantly different from untreated control livers. B: Livers were flushed blood-free and cDNA was prepared as described under Section 2. Brain and liver as control organs were instantly removed, rinsed in PBS and snap frozen in liquid nitrogen. RT-PCR experiments were performed with rat specific primers detecting PKG isoforms I and II. Data show one representative of three independent experiments. Journal of Hepatology 2004 41, 414-420DOI: (10.1016/j.jhep.2004.05.017) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 PKA activation is crucial for ANP-mediated inhibition of apoptosis. A: Livers were perfused either in the absence (Co) or in the presence of 200nM ANP±1μM Rp-8-Br-cAMPS (PKA inhibitor) which was added 20min prior to ischemia (24h, 4°C). After 24h ischemia livers were snap frozen and caspase-3-like activity was measured. Data are expressed as percent of enzyme activity of untreated control livers set equal 100%. Columns show mean±SEM of two independent experiments with n=4–5 organs per group. *P<0.05 significantly different from untreated control livers, #P<0.05 vs. ANP. B: Isolated hepatocytes were left unstimulated (Co) or treated with 200nM ANP, with 200μM of the cell permeable cGMP analogue 8-Br-cGMP or with the positive control Bt2-cAMP (250μM) for 60min. PKA activity assay was performed as described under Section 2. Specific activity is expressed as x-fold increase in comparison to control activity. Columns show means±SEM of three independent experiments with n=3 per group. *P<0.05 represents significant differences in the values between untreated and pretreated hepatocytes. Journal of Hepatology 2004 41, 414-420DOI: (10.1016/j.jhep.2004.05.017) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 ANP-preconditioning leads to BAD phosphorylation. Livers were perfused for 30min in the absence (Co) or presence of 200nM ANP, which was added 20min prior to cold ischemia (24h, 4°C). Western blot analysis (see Section 2) was performed detecting phosphorylated BAD (phospho-BAD) and total BAD (tot-BAD) after 30min reperfusion. Journal of Hepatology 2004 41, 414-420DOI: (10.1016/j.jhep.2004.05.017) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions