Characterization of mitochondrial glutaminase and amino acids at prolonged times after experimental focal cerebral ischemia.
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.
Newcomb
R
R
Elan Pharmaceuticals Inc., 3760 Haven Ave., Menlo Park, CA 94025, USA. rnewcomb@neurex.com
Pierce
A R
AR
Kano
T
T
Meng
W
W
Bosque-Hamilton
P
P
Taylor
L
L
Curthoys
N
N
Lo
E H
EH
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D015153:Blotting, Western; D005972:Glutaminase / Q000032:analysis*; D002546:Ischemic Attack, Transient / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D017551:Microdialysis; D008928:Mitochondria / Q000737:chemistry* / Q000201:enzymology; D009474:Neurons / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D000596:Amino Acids; D005972:Glutaminase