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.
Kiemer
Alexandra K
AK
Department of Pharmacy, Center of Drug Research, University of Munich, Germany. kiemer@scripps.edu
Förnges
Anke C
AC
Pantopoulos
Kostas
K
Bilzer
Manfred
M
Andriopoulos
Bill
B
Gerwig
Tobias
T
Kenngott
Silke
S
Gerbes
Alexander L
AL
Vollmar
Angelika M
AM
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D015153:Blotting, Western; D024202:Electrophoretic Mobility Shift Assay; D005293:Ferritins / Q000096:biosynthesis; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis*; D051547:Heme Oxygenase-1; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism; D066298:In Vitro Techniques; D035925:Iron-Regulatory Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D010477:Perfusion; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D015427:Reperfusion Injury / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D035925:Iron-Regulatory Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D009320:Atrial Natriuretic Factor; D005293:Ferritins; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate