Characterization of mitochondrial glutaminase and amino acids at prolongedtimes after experimental focal cerebral ischemia

The mitochondrial enzyme glutaminase is a significant contributor to extrac ellular glutamate after neuronal injury in vitro [R. Newcomb, X. Sun, L. Ta ylor, N. Curthoys, R.G. Giffard, Increased production of extracellular glut amate by the mitochondrial glutaminase following neuronal death, J. Biol. C hem. 272 (1997) 11276-11282.]. As a step towards characterizing the role of the enzyme in neuronal injury in vivo, glutaminase activity was measured i n central and peripheral regions of the ischemic distribution in rat brain at 6, 24, and 48 h after permanent focal ischemia. Although glutaminase act ivity decreases in the central ischemic area, significant activity remains in peripheral areas of evolving damage, even after 24 and 48 h ischemia. We stern blots show no detectable change in glutaminase molecular weight or to tal immunoreactivity, regardless of the degree of inactivation. Significant amounts of glutamine remain in ischemic tissue at prolonged times after fo cal ischemia, while reductions in tissue amounts of glutamate are highly co rrelated with decreases in glutaminase activity. In vivo microdialysis prob es were inserted into the ischemic periphery after 24 h focal ischemia. Glu tamate is significantly elevated in these dialysates. Perfusion of the glut aminase substrate glutamine and the enzyme activator phosphate results in f urther 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 extrac ellular glutamate. Inactivation of the glutaminase in central areas of the ischemic lesion does not involve significant proteolytic degradation, and l ikely involves a specific molecular event. (C) 1998 Elsevier Science B.V. A ll rights reserved.