Increased production of extracellular glutamate by the mitochondrial glutaminase following neuronal death

Abstract

Elevated extracellular concentrations of the excitatory transmitter glutamate are an important cause of neuronal death in a variety of disorders of the nervous system. The concentrations and rates of clearance and production of extracellular glutamate were measured in the medium of primary cultures from mouse neocortex containing neurons, astrocytes, or both cell types. Measurements were performed in the presence and absence of 2 mM glutamine with or without neuronal injury caused by 5-h exposure to hypoxia or 500 μM N-methyl-D-aspartate or a freeze-thaw cycle. High rates of glutamate generation (0.5-0.8 μM/min in the 0.4-ml culture well) occurred if neurons were both damaged and exposed to glutamine. Intact neurons or glia exposed to glutamine generated only small amounts of glutamate (0.03 μM/min). Glutamate generation by damaged neurons was dependent on the presence of glutamine, activated by phosphate, and inhibited by 6-diazo-5- oxo-L-norleucine and p-chloromercuriphenylsulfonic acid (pCMPS), strongly implicating the mitochondrial glutaminase. Following 5-h exposure to 500 μM N-methyl-D-aspartate, the glutaminase was localized to fragments of damaged neurons and was accessible to inhibition by the membrane-impermeant pCMPS. The glutaminase activity from damaged neurons is sufficient to account for the neurotoxic concentrations of glutamate in hypoxic mixed neuronal-glial cultures exposed to 2 mM glutamine. Finally, pCMPS is neuroprotective and also prevents the increased rate of generation of glutamate observed in neuronal cultures after prolonged exposure to glutamine. The cumulative data indicate the following: 1) excitotoxic neuronal death activates the hydrolysis of extracellular glutamine by the mitochondrial glutaminase, and 2) the glutaminase in damaged neurons is sufficient to cause neuronal death in in vitro models of neuronal injury.