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We previously reported that activation of phosphatidylinositol-3-kinase (PI3-kinase) is involved in ischemic preconditioning (PC). Our goal was to determine downstream targets of PI3-kinase. In perfused rat hearts, PC (4 cycles of 5 minutes of ischemia and 5 minutes of reflow) increased phosphorylation of glycogen synthase kinase-3␤ (GSK-3␤), a downstream target of PI3-kinase and protein kinase B (PKB), an effect that was blocked by wortmannin. Because phosphorylation inactivates GSK-3␤, we examined whether PC-induced phosphorylation and inhibition of GSK-3␤ is important in PC by using two inhibitors of GSK-3␤, lithium and SB 216763. Pretreatment of perfused rat hearts with lithium or SB 216763, before ischemia, mimicked the protective effects of PC; hearts treated with either lithium or SB 216763 had improved postischemic function and reduced infarct size. These findings indicate that inhibition of GSK-3␤ is protective and that this PI3-kinase-dependent signaling pathway may play an important role in ischemic preconditioning. Brief intermittent periods of ischemia and reflow, termed ischemic preconditioning (PC), have been shown to protect the myocardium against injury produced by a subsequent sustained period of ischemia. 1 PC has been shown to significantly reduce infarct size, arrhythmias, and postischemic contractile dysfunction. We previously reported that activation of phosphatidylinositol-3-kinase (PI3-kinase) is important in PC. 2 PI3-kinase has been reported to enhance cell survival via phosphorylation of GSK-3␤. 3 Expression of a mutant GSK that cannot be phosphorylated blocked the antiapoptotic action of PI3-kinase. 3 We therefore examined the role of GSK-3␤ in PC. Our results suggest that phosphorylation and subsequent inactivation of GSK-3␤ play an important role in the protection afforded by PC in the heart. Materials and Methods Isolated Rat Heart PreparationAll rats received humane care in accordance with the NIH guidelines (NIH publication No. 8523, revised 1985). Hearts from male Sprague-Dawley rats (200 to 300 g; Taconic, Germantown, NY) were perfused in Langendorff mode under constant pressure (67 mm Hg) as described previously. 2 Experimental ProtocolsThe protocol is illustrated in Figure 1. For studies assessing recovery of postischemic function, the protocol consisted of a 30-minute control period, a treatment period, 20 minutes of global normothermic ischemia, and 30 minutes of reperfusion. Recovery of left ventricular developed pressure (LVDP) was measured after 30 minutes of reflow and expressed as a percentage of the initial LVDP, before PC or drug administration. Group I hearts (control, nϭ19) were perfused with Krebs-Henseleit (KH) buffer. Group II hearts (PC, nϭ11) were preconditioned with four cycles of 5 minutes of ischemia (I) and 5 minutes of reflow (R). Group III hearts (SB, nϭ14) were treated with 3 mol/L SB 216763 for 10 minutes. Group IV hearts (Li, nϭ6) were treated with 3 mmol/L LiCl for 10 minutes. For studies assessing infarct size, the groups (nϭ4 for all) and protocol...

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...