Ischemic brain damage is initiated by elevated levels of the excitatory amino acids (EAAs), glutamate and aspartate, and by sustained activation of EAA receptors. The objective of the proposed study is to evaluate the contribution of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in mediating the pathological release of the EAAs during stroke. Experimental data suggest that volume regulated anion channels (VRACs) represent one of the major pathways contributing to EAA release during ischemia. RNS and ROS are produced during ischemia and reperfusion, and stimulate intracellular signaling cascades thought to activate or modulate VRACs. Therefore, we hypothesize that, during ischemia, RNS and ROS potentiate the release of EAAs via VRAC modulation. To test this hypothesis we have developed three specific aims: (1) To show that endogenous RNS production in cultured astrocytes activates VRACs and EAA release. To test this, VRAC function will be assayed as the release of pre-loaded D-[3H]aspartate from swollen astrocytes. RNS production will be achieved by induction of inducible nitric oxide synthase. (2) To explore the contribution of ROS in triggering pathological EAA release. We will combine selective inhibitors of intracellular signaling with ROS-generating drugs or overexpression of cellular ROS-producing enzymes in cultured astrocytes. (3) To verify the contribution of the ROS and RNS in mediating VRAC activation in vivo, EAA release will be measured using microdialysis during ischemia or when tissue swelling is induced by hypoosmotic medium. The confirmation of our hypothesis will point to additional targets for stroke drug design.
| Rudkouskaya, Alena; Chernoguz, Artur; Haskew-Layton, Renee E et al. (2008) Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes. J Neurochem 105:2260-70 |
| Haskew-Layton, Renee E; Rudkouskaya, Alena; Jin, Yiqiang et al. (2008) Two distinct modes of hypoosmotic medium-induced release of excitatory amino acids and taurine in the rat brain in vivo. PLoS One 3:e3543 |
| Haskew-Layton, Renee E; Mongin, Alexander A; Kimelberg, Harold K (2005) Hydrogen peroxide potentiates volume-sensitive excitatory amino acid release via a mechanism involving Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 280:3548-54 |
