OXIDATIVE STRESS IN THE CENTRAL-NERVOUS-SYSTEM - MONITORING THE METABOLIC RESPONSE USING THE PENTOSE-PHOSPHATE PATHWAY

We propose that monitoring the activity of the pentose phosphate pathw ay (PPP) may provide an opportunity to obtain unique information regar ding the metabolic response to oxidative stress since glutathione pero xidase activity is coupled, via glutathione reductase, to the PPP enzy me glucose-6-phosphate dehydrogenase. PPP activity was quantitated fro m data obtained from gas chromatography/mass spectrometry analysis of released lactate following metabolic degradation of (1,6-C-13(2),6,6-H -2(2))glucose. The feasibility of this approach for in vitro studies i s shown using cultured rat 9L gliosarcoma cells, primary mixed cerebro cortical and primary astrocytic cultures and in vivo using intracerebr al microdialysis. Exposure of 9L gliosarcoma cells to increasing conce ntrations of phenazine methosulfate, diamide and H2O2 correlated with increasing stimulation of the PPP, reveling the coupling of the PPP to the glutathione pathway. In all cultured cell types, the activity of the PPP was stimulated in a concentration-dependent fashion by exposur e to H2O2. In primary mixed and purified astrocytic cultures, PPP acti vity was stimulated with H2O2 from 2.0 to 22.5 and from 5.9 to 66.7%, respectively. H2O2-induced neuronal injury was evident before saturati on of the PPP occurred. H2O2 toxicity was attenuated when neurons were preincubated with the iron chelator, deferoxamine, and did not occur until saturation of the PPP. In vivo measurements of PPP activity in t he conscious rat forebrain revealed basal levels of 4.5%, which was st imulated to 16.9 and 35.7% when 1 mM H2O2 and 500 mu M phenazine metho sulfate were added to the perfusion solution, respectively. We conclud e that monitoring the activity of the PPP both in vitro and in vivo ca n provide a unique perspective from which the metabolic response to ox idative stress can be evaluated.