Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning.
OBJECTIVE
Ischemic preconditioning has been proved effective in reducing ischemia/reperfusion injury during liver surgery. However, the mechanisms involved are still poorly understood. Here, we have investigated the role of phosphatidylinositol 3-kinase (PI3K) in the signal pathway leading to hepatic preconditioning.


METHODS
PI3K activation was evaluated in isolated rat hepatocytes preconditioned by 10-minute hypoxia followed by 10-minute reoxygenation.


RESULTS
Hypoxic preconditioning stimulated phosphatidylinositol-3,4,5-triphosphate production and the phosphorylation of PKB/Akt, a downstream target of PI3K. Conversely, PI3K inhibition by wortmannin or LY294002 abolished hepatocyte tolerance against hypoxic damage induced by preconditioning. PI3K activation in preconditioned hepatocytes required the stimulation of adenosine A 2A receptors and was mimicked by adenosine A 2A receptors agonist CGS21680. In the cells treated with CGS21680, PI3K activation was prevented either by inhibiting adenylate cyclase and PKA with, respectively, 2,5-dideoxyadenosine and H89 or by blocking Galphai-protein and Src tyrosine kinase with, respectively, pertussis toxin and PP2. H89 also abolished the phosphorylation of adenosine A 2A receptors. However, the direct PKA activation by forskolin failed to stimulate PI3K. This suggested that PKA-phosphorylated adenosine A 2A receptors may activate PI3K by coupling it with Galphai-protein through Src. We also observed that, by impairing PI3K-mediated activation of phospholypase Cgamma (PLCgamma), wortmannin and LY294002 blocked the downstream transduction of preconditioning signals via protein kinase C (PKC) delta/ isozymes.


CONCLUSIONS
PI3K is activated following hepatocyte hypoxic preconditioning by the combined stimulation of adenosine A 2A receptors, PKA, Galphai protein, and Src. By regulating PKC-/delta-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.
Carini
Rita
R
Dip. Scienze Mediche, Università "A. Avogadro" del Piemonte Orientale, Via Solaroli 17, 28100 Novara, Italy. carini@med.unipmn.it
Grazia De Cesaris
Maria
M
Splendore
Roberta
R
Baldanzi
Gianluca
G
Nitti
Maria Paola
MP
Alchera
Elisa
E
Filigheddu
Nicoletta
N
Domenicotti
Cinzia
C
Pronzato
Maria Adelaide
MA
Graziani
Andrea
A
Albano
Emanuele
E
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D019194:Ischemic Preconditioning / Q000379:methods*; D008297:Male; D023421:Models, Animal; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D043705:Receptor, Adenosine A2A; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat