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Characterization of mitochondrial glutaminase and amino acids at prolonged times after experimental focal cerebral ischemia
Authors:Robert Newcomb  Allen R Pierce  Tsuneo Kano  Wei Meng  Prince Bosque-Hamilton  Lynn Taylor  Norman Curthoys  Eng H Lo
Institution:aElan Pharmaceuticals Inc., 3760 Haven Ave., Menlo Park, CA 94025, USA;bDepartment of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA;cNeuroprotection Research Laboratory, Departments of Neurology and Radiology, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA 02129, USA
Abstract:The mitochondrial enzyme glutaminase is a significant contributor to extracellular glutamate after neuronal injury in vitro R. Newcomb, X. Sun, L. Taylor, N. Curthoys, R.G. Giffard, Increased production of extracellular glutamate by the mitochondrial glutaminase following neuronal death, J. Biol. Chem. 272 (1997) 11276–11282.]. As a step towards characterizing the role of the enzyme in neuronal injury in vivo, glutaminase activity was measured in central and peripheral regions of the ischemic distribution in rat brain at 6, 24, and 48 h after permanent focal ischemia. Although glutaminase activity decreases in the central ischemic area, significant activity remains in peripheral areas of evolving damage, even after 24 and 48 h ischemia. Western blots show no detectable change in glutaminase molecular weight or total immunoreactivity, regardless of the degree of inactivation. Significant amounts of glutamine remain in ischemic tissue at prolonged times after focal ischemia, while reductions in tissue amounts of glutamate are highly correlated with decreases in glutaminase activity. In vivo microdialysis probes were inserted into the ischemic periphery after 24 h focal ischemia. Glutamate is significantly elevated in these dialysates. Perfusion of the glutaminase substrate glutamine and the enzyme activator phosphate results in further and specific elevations in dialysate glutamate. In sum, significant mitochondrial glutaminase activity remains in the periphery of the ischemic lesion at 24 and 48 h, where it can contribute directly to elevated extracellular glutamate. Inactivation of the glutaminase in central areas of the ischemic lesion does not involve significant proteolytic degradation, and likely involves a specific molecular event.
Keywords:Stroke  Ischemia  Excitotoxicity  Neurodegeneration  Glutaminase  Delayed neuronal damage
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