Ischemic Preconditioning Activates Phosphatidylinositol-3-Kinase Upstream of Protein Kinase C

Abstract

Abstract-The present study is designed to test whether phosphatidylinositol 3-kinase (PI3-kinase) has a role in the signaling pathway in ischemic preconditioning (PC) and whether it is proximal or distal to protein kinase C (PKC). Before 20 minutes of global ischemia, Langendorff-perfused rat hearts were perfused for 20 minutes (control); preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC); treated with either wortmannin (WM) or LY 294002 (LY), each of which is a PI3-kinase inhibitor, for 5 minutes before and throughout PC; treated with 1,2-dioctanoyl-sn-glycerol (DOG), an activator of PKC for 10 minutes ( Key Words: phosphatidylinositol 3-kinase Ⅲ protein kinase C Ⅲ nitric oxide Ⅲ ischemic preconditioning I schemic preconditioning (PC) describes the phenomenon whereby repeated brief episodes of ischemia and reperfusion increase the resistance to myocardial infarction and contractile dysfunction induced by a subsequent sustained episode of ischemia. 1,2 The signaling pathways involved in PC have been investigated intensively. There is strong support for the hypothesis that PC results in protein kinase C (PKC) activation and translocation. 3-9 Downstream targets of PKC include activation of the 12-lipoxygenase pathway of arachidonic acid metabolism 10 and activation of an ATP-sensitive potassium channel K ATP , most likely the mitochondrial K ATP channel. 11,12 The mechanism by which PC activates PKC has not been elucidated; however, NO, 13 reactive oxygen species, 14 and diacylglycerol generated by phospholipase D 15 have been suggested as mediators of PKC activation in PC.Phosphatidylinositol 3-kinase (PI3-kinase) is a key signaling enzyme implicated in cell survival and metabolic control. The PI3-kinases are a subfamily of lipid kinases that catalyze the phosphorylation of the inositol ring of phosphoinositides specifically at the 3 position. Downstream targets of PI3-kinase include protein kinase B (PKB)/Akt, 16,17 p70 S6 kinase, 18 and several isoforms of PKC (ie, PKC⑀, -␦, -, and -). 19 -21 PKB activation in vivo appears to be dependent on the 3-phosphoinositide-dependent protein kinase (PDK1) that phosphorylates and activates PKB. 22,23 PDK1 also phosphorylates several isoforms of PKC. 21,24 It is reported that full activation of PKC requires phosphorylation by PDK1 as well as allosteric activation. 21 Activated PKB phosphorylates and inactivates glycogen synthase (GS) kinase (GSK), and inactivation of GSK leads to activation of GS. 25 Thus, PI3-kinase is a key regulator of glycogen metabolism. PKB also directly activates endothelial NO synthase (eNOS), 26,27 and NO generated by eNOS is proposed to initiate PC. 13 Wortmannin (WM) binds covalently to the 110-kDa subunit of PI3-kinase and has been shown to inhibit PI3-kinase irreversibly (IC 50 ϭ5 nmol/L). 28 LY 294002 (LY) is another selective PI3-kinase inhibitor (IC 50 ϭ1.4 mol/L) that acts on the ATP binding site of the enzyme. 29 Because PI3-kinase is reported to activate PKC and eNOS, which are both involved in PC, and because of...

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