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Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.

Background and Purpose: Increased activation of excitatory amino acid (EAA) receptors is considered a major cause of neuronal damage. Possible sources and mechanisms of ischemia-induced EAA release were investigated pharmacologically with microdialysis probes placed bilaterally in rat striatum., Methods: Forebrain ischemia was induced by bilateral carotid artery occlusion and controlled hypotension in halothane-anesthetized rats. During 30 minutes of ischemia, microdialysate concentrations of glutamate and aspartate were measured in the presence of a nontransportable blocker of the astrocytic glutamate transporter GLT-1, dihydrokinate (DHK), or an anion channel blocker, 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS), administered separately or together through the dialysis probe., Results: In control striata during ischemia, glutamate and aspartate concentrations increased 44+/-13 (mean+/-SEM) times and 19+/-5 times baseline, respectively, and returned to baseline values on reperfusion. DHK (1 mmol/L in perfusate; n=8) significantly attenuated EAA increases compared with control (glutamate peak, 9. 6+/-1.7 versus control, 15.4+/-2.6 pmol/ microL). EAA levels were similarly decreased by 10 mmol/L DHK. DNDS (1 mmol/L; n=5) also suppressed EAA peak increases (glutamate peak, 5.8+/-1.1 versus control, 10.1+/-0.7 pmol/ microL). At a higher concentration, DNDS (10 mmol/L; n=7) further reduced glutamate and aspartate release and also inhibited ischemia-induced taurine release. Together, 1 mmol/L DHK and 10 mmol/L DNDS (n=5) inhibited 83% of EAA release (glutamate peak, 2.7+/-0.7 versus control, 10.9+/-1.2 pmol/ microL)., Conclusions: These findings support the hypothesis that both cell swelling-induced release of EAAs and reversal of the astrocytic glutamate transporter are contributors to the ischemia-induced increases of extracellular EAAs in the striatum as measured by microdialysis.

Dopaminergic control over striatal targets appears to be retained in rats sustaining lesions of the nigrostriatal dopamine (DA) system as long as 5-10% of that projection remains. Similarly, during postnatal development, dopaminergic control over striatal neurons matures well before the innervation of striatum by the nigrostriatal bundle is attained. These observations suggest that enhanced efficacy of dopaminergic transmission may compensate for hypoinnervation of striatum after lesions or during development. To examine this hypothesis, striatal slices were superfused with Krebs bicarbonate buffer and effluent was collected and analyzed for endogenous DA. Electrical field stimulation (2 Hz) continuously delivered to slices prepared from intact adult rats increased DA efflux to 3-5 times the prestimulation rate within 10 min. Efflux then fell to approximately twice the basal rate over the next 20 min. DA efflux was also examined using slices prepared from adult animals given 6-hydroxydopamine 2-3 weeks earlier, and from 7-10-d-old rat pups. In each group, striatal DA levels were 10-40% of adult control values. Nevertheless, stimulated DA efflux from these slices attained the same rate as that observed with intact, adult slices. Thus, fractional DA efflux from these slices was several times higher than the control rate by the end of the stimulation period. This increased DA efflux appeared to be a consequence of both increased release and decreased reuptake of DA, as the fractional DA efflux from control striatal slices could not be increased to the rate seen in hypoinnervated slices using nomifensine (10 microM), an inhibitor of DA efflux.(ABSTRACT TRUNCATED AT 250 WORDS)