Up-Regulation of Endothelial Nitric Oxide Synthase via Phosphatidylinositol 3-Kinase Pathway Contributes to Ischemic Tolerance in the CA1 Subfield of Gerbil Hippocampus

We here investigated endothelial nitric oxide synthase (eNOS) expression after 10 minutes of forebrain ischemia. Real-time polymerase chain reaction, immunoblots and immunohistochemical studies revealed up-regulation of eNOS expression in the hippocampal CA1 subfield of gerbil. Immunoreactivity of eNOS significantly increased in endothelium but neither in neurons nor astrocytes after 6 to 168 hours of reperfusion. An increased Akt activity preceded the postischemic eNOS up-regulation. Intracerebroventricular injection (i.c.v.) of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI-3K), significantly inhibited the increases in both eNOS mRNA and its protein with concomitant inhibition of Akt activation. The significant increase in the eNOS expression was also evident following preconditioning 2-minute ischemia. Both eNOS up-regulation and acquisition of ischemic tolerance observed at 3 days after preconditioning ischemia were significantly inhibited by pretreatment with wortmannin. Administration (i.c.v.) of N-nitro-L-arginine methyl ester, but not 7-nitroindazole, 30 minutes prior to lethal 10-minute ischemia, significantly abolished the acquired tolerance. Intraperitoneal injections of aminoguanidine at immediately after, 24, and 48 hours after preconditioning had no effects on the tolerance. These results suggest that eNOS expression is up-regulated in the endothelium via PI-3K pathways after transient forebrain ischemia, and that preconditioning-induced eNOS expression plays an important role in neuroprotection in the ischemic tolerance.