Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning

Background & Aims: 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/e 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-epsilon/delta-dependent signals, PI3K can play a key role in the development of hepatic tolerance to hypoxia/reperfusion.