Within only 2-3 min of brain ischemia, extracellular K+ concentrations ([K+])o reach 60 - 80 mM, which implies that the excitotoxic action of glutamate during ischemia is executed at highly elevated [K+]o. The proposed research will study whether such high [K+]o affects mechanisms of glutamate excitotoxicity. The hypothesis """"""""High [K+]o reduces the electrochemical Ca2+ driving force (ECDF) in neurons exposed to glutamate receptor agonists"""""""" will be tested in Aim 1: """"""""Study the effects of various [K+]o on the plasma membrane potential (Em) and the cytoplasmic Ca2+ concentration ([Ca2+]c) in cultured cortical and cerebellar neurons exposed to glutamate receptor agonists"""""""". To this end, neurons will be loaded with Em- and Ca2+-sensitive fluorescent probes. This hypothesis, if true, predicts that high [K+]o may prevent excitotoxicity by decreasing the glutamate mediated Ca2+ influx across the plasma membrane. It is also possible, however, that glutamate may collapse the Na+ and K+ concentration gradients across the plasma membrane and, consequently, set Em close to zero regardless of [K+]o. To test whether this is the case, the cytoplasmic Na+ and K+ concentrations will be measured in parallel experiments in neurons loaded with Na+- and K+-sensitive fluorescent probes. An alternative hypothesis, will be tested in Aim 2: """"""""Determine whether increasing [K+]o may inhibit the glutamate-mediated Ca2+ influx in a manner not related to the plasma membrane depolarization"""""""". To this end, the effects of [K+]o on Ca2+ accumulation stimulated by glutamate receptor agonists will be studied in neurons depolarized by a Na+ and K+ ionophore, gramicidin. Finally, in Aim 3: """"""""Test whether restoration of low [K+]o following ischemia in vitro causes a delayed Ca2+ accumulation and compromises neuronal survival"""""""", it will be studied whether [K+]o during the postischemic period affects Ca2+ homeostasis and is related to improved or compromised neuronal survival during the next 24 hours. These studies may yield a new target for pharmacological intervention to decrease neuronal death following ischemia or hypoglycemia, namely, to prevent excessive K+ loss from the brain during reperfusion.
| Ouardouz, M; Zamponi, G W; Barr, W et al. (2005) Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels. Neuropharmacology 48:566-75 |
