Current therapeutic options for ischemic stroke are limited to tissue plasminogen activator and mechanical clot removal therapies. Diazoxide (DZ) is a mitochondrial ATP-sensitive potassium channel opener and is protective in models of brain ischemia, but the signaling pathways involved are unknown. The mammalian target of rapamycin (mTOR) is a master regulator of protein synthesis and is involved in protection against cerebral ischemia. The neuronal nitric oxide synthase (nNOS) pathway has also been shown to provide protection from ischemic insults. Additionally, mitochondrial respiratory status has not been investigated. I examined the role of the mTOR pathway, the nNOS pathway, and mitochondrial respiration in delayed DZ-induced preconditioning of neurons. I cultured rat primary cortical neurons and simulated ischemic stroke using oxygen-glucose deprivation (OGD) for 3 h followed by re-oxygenation. Viability, mitochondrial membrane potential, reactive oxygen species (ROS) measurements, and western blots were performed. The mTOR pathway was inhibited by rapamycin, Torin-1, and S6K targeted silencing RNA. The NOS pathway was inhibited by L-NAME. NO-donors SNP and DEANONOate (DEANO) were applied to rescue the effects of L-NAME. Mitochondrial oxygen consumption rate (OCR) was measured in intact neurons by serial injections of oligomycin, FCCP, and antimycin/rotenone. OGD decreased viability by 50 percent, depolarized mitochondria, and reduced mitochondrial respiration whereas DZ improved viability to 75 percent and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Diazoxide also increased phosphorylation of protein kinase B, mTOR, and S6K. Rapamycin, Torin-1, and S6K targeted siRNA abolished the protective effects of DZ. Co-application of L-NAME with DZ prevented preconditioning whereas adding SNP or DEANO along with L-NAME and DZ restored protection. Diazoxide increased phosphorylated nNOS. Interestingly, co-application of LNAME with DZ blocked the phosphorylation of nNOS as well as S6K. The ratio of phosphorylated/total Akt and mTOR were not significantly altered with L-NAME co-application. Diazoxide altered OCR 24 and 48 h after the ischemic period. Diazoxide had no acute effect on OCR but increased ECAR significantly. Activation of the mTOR and nNOS pathways is critical for DZ preconditioning in neurons. Furthermore, OCR is modified by the DZ-induced preconditioning of neurons. / acase@tulane.edu
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_27984 |
| Contributors | Dutta, Somhrita (Author), Busija, David (Thesis advisor) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Format | 124 |
| Rights | Copyright is in accordance with U.S. Copyright law |
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