Z Gastroenterol 2005; 43 - 3_18
DOI: 10.1055/s-2005-862255

Pretreatment with Atrial Natriuretic Peptide Leads to Inhibition of Caspase–3 in Vivo – Involvement of PI 3-Kinase-Pathway and Bad

M Keller 1, U Grützner 2, S Zahler 1, AK Kiemer 3, AL Gerbes 2, AM Vollmar 1
  • 1Ludwig-Maximilians-University Munich, Department of Pharmacy, Munich
  • 2Medizinische Klinik II, Klinikum Großhadern, Universität München, München
  • 3The Scripps Research Institute, Department of Molecular and Experimental Medicine, , USALa Jolla

Aims: Pretreatment with Atrial Natriuretic Peptide (ANP) reduces ischemia-reperfusion injury in isolated perfused rat livers by prevention of apoptotic and necrotic cell death (Gerwig et al., J. Hepatol. 2003, 39: 341–48). A central role in antiapoptotic signalling in the liver was previously assigned to the phosphatidylinositol–3-kinase (PI 3-kinase)-pathway and protein kinase B/Akt (Hong et al., Biochem. Biophys. Res. Commun. 2000, 279: 974–79, Imose et al., Liver Int. 2003, 23(5): 386–96).

Aim of study: To investigate whether ANP preconditioning mediates antiapoptotic effects in vivo and to elucidate a possible involvement of the PI 3-kinase-pathway.

Methods: Rats were infused with NaCl, ANP (5µg/kg), Wortmannin (WM, 16µg/kg), or a combination of ANP and WM for 20 min. Livers were stored in UW solution (4°C) for 24h, transplanted and reperfused for up to 2h. Apoptotic cell death was examined by measurement of caspase–3 activity. Phosphorylation of Akt and Bad was determined by Western Blotting, cellular localization of phospho-Akt and apoptotic cells by histological analysis.

Results: Pretreatment with ANP for 20 min results in a decrease of caspase–3 activity and TUNEL positive cells after 24h cold ischemia indicating antiapoptotic effects of ANP also in vivo. However, ANP did not affect necrotic tissue damage as monitored by measurement of GOT, GPT, and LDH. Regarding the signalling responsible for the antiapoptotic effect of ANP, preconditioning leads to phosphorylation of Akt and Bad. Pretreatment with the PI 3-kinase inhibitor WM abrogates the reduction of caspase–3 activity by ANP indicating an essential role of Akt in ANP-induced cytoprotection. Interestingly, analysis of liver tissue by confocal microscopy suggests a translocation of phosphorylated Akt to the plasma membrane of hepatocytes evoked by ANP.

Conclusion: ANP activates the PI 3-kinase pathway in the liver in vivo leading to phosphorylation of Bad – an event, which is considered to trigger an antiapoptotic signalling cascade.

Key words

ANP - PI 3-kinase - antiapoptotic

References

Gerwig et al., J. Hepatol. 2003 Sep; 39:341-348 Hong et al., Biochem. Biophys. Res. Commun. 2000 Dec 29; 279(3):974-979 Imose et al., Liver Int. 2003 Oct; 23(5):386-396