Protein kinase A dependent signalling mediates anti-apoptotic effects of the atrial natriuretic peptide in ischemic livers.
OBJECTIVE
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.
Kulhanek-Heinze
Stefanie
S
Department of Pharmacy, Center of Drug Research, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.
Gerbes
Alexander L
AL
Gerwig
Tobias
T
Vollmar
Angelika M
AM
Kiemer
Alexandra K
AK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D019194:Ischemic Preconditioning; D008099:Liver / Q000098:blood supply / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D020935:MAP Kinase Signaling System; D008297:Male; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D051025:bcl-Associated Death Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C494821:Bad protein, rat; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D051025:bcl-Associated Death Protein; D009320:Atrial Natriuretic Factor; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D006152:Cyclic GMP