Neuronal function in normal brain depends upon a number of astrocyte- neuronal interactions. Under pathological conditions such as ischemia these interactions may be a critical factor determining neuronal survival. A significant cause of neuronal death during ischemia is toxicity mediated by increased levels of extracellular excitatory amino acids (EAAs), chiefly glutamate. Astrocyte functions may have opposing influences on EAA neurotoxicity: while rapid uptake of EAAs may prevent neuronal injury, the production of EAA precursor (glutamine) could exacerbate injury. Our recent data show that acidosis blocks EAA uptake by astrocytes under hypoxic conditions similar to those present during incomplete ischemia. We propose to study the mechanism of this effect with the aim of developing pharmacologic means of preserving glial EAA uptake during ischemia. Primary astrocyte cultures will be used to determine whether blockade of active H+ transport will preserve EAA uptake. Related studies will examine the mechanism by which barbiturates impair glial EAA uptake, an effect that is also increased by acidosis and which may also be due to impaired energy metabolism. Study of the barbiturate effect will likewise have significant clinical relevance as these agents are frequently used in conditions of impaired brain energy metabolism and brain acidosis. While astrocyte EAA uptake is neuroprotective, glutamine export by astrocytes may exacerbate EAA neurotoxicity. Extracellular glutamine serves both as a positive modulator of the NMDA type glutamate receptor and as the immediate precursor for the neurotransmitter pool of glutamate. Inhibition of glial glutamine synthetase reduces brain glutamine and may thereby reduce EAA-mediated neuronal injury under pathological conditions. We have previously shown that the glutamine synthetase inhibitor methionine sulfoximine (MSO) produces a 30% reduction in infarct size in a rat model of stroke, but the mechanism of this effect remains uncertain because MSO also has effects on glycogen metabolism that could alter brain responses to ischemia. MSO also causes seizures, precluding its potential use as a therapeutic agent. Using astrocyte cultures, we will test known inhibitors of glutamine synthetase for a) inhibition of the glial enzyme, b) lack of neuronal and glial cytotoxicity, and c) lack of effects on glycogen levels. The most promising of these agents will be studied in vivo for their ability to lower brain glutamine levels and reduce brain injury in a rat model of focal cerebral ischemia.
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