Oxidative Stress In The Brain: Effects Of Hydroperoxides And Nitric Oxide On Glyceraldehyde 3-Phosphate Dehydrogenase And Phosphoinositide Cycle Enzymes

In the aerobic cell, oxygen can be converted into a series of reactive metabolites, together called as "reactive oxygen species" (ROS). This large group include both radical and non-radical species such as superoxide anion (02"), hydroxyl radical ("0H), H202, nitric oxide (N0') and lipid hydroperoxides (LOOH). ROS are generated in very small amounts at all stages of aerobic life, and probably have a role in cellular regulation. However, their formation in excess leads to toxicity and damage to tissues. This situation, called 'oxidative stress', is responsible, atleast in part, to the pathophysioiogy of a number of disease states such as inflammation, arthritis, cancer, ageing, ischemia-reperfusion and several neurodegenerative disorders. Compared to other organs in the animal body, brain tissue is more vulnerable to oxidative stress. This is due to three major reasons; (1) brain has a high oxygen consumption (2) high content of polyunsaturated fatty acids and iron, that can promote lipid peroxidation, and (3) low levels of antioxidant enzymes such as catalase and glutathione peroxidase. The inability of neurons to regenerate also contributes to exacerbate an oxidant damage in the brain. The main objective of this investigation was to identify biochemical systems in the brain that are susceptible to ROS, on the following two issues: 1. What are the targets for the action of H2O2 and NO in the glycolytic cycle, the major route for the oxidation of glucose in brain? 2. What are the targets for the action of polyunsaturated fatty acids and their oxidative metabolites among the enzymes of phosphoinositide cycle (PI cycle), the ubiquitous signal transduction event in the brain? Using sheep brain cytosol , it was found that among the various glycolytic enzymes, only glyceraldehyde 3-phosphate dehydrogenase (GAPD) was inhibited by H2O2. The enzyme was purified to homogeneity from sheep brain and its inactivation with H202 was studied in detail. Commercial preparations of rabbit skeletal muscle GAPD was also used in this study. An unusual requirement of glutathione for the complete inactivtion of the enzyme by H2O2 was observed. The H2O2-inactivated GAPD was partially reactivated by prolonged treatment with thiol compounds. Using CD-spectral analysis, a significant change was found in the secondary structure in H2O2-treated GAPD. GAPD was inactivated by NO only in presence of high concentrations of DTT and after prolonged incubation. The N0-inactivated GAPD was partially reactivated by treatment with thiol compounds. A new activity, namely ADP-ribosylation (ADPR) emerged in the NO-treated mammalian, but not in yeast. GAPD, ADPR activity could be generated in GAPD through NO-independent treatments such as incubation with NADPH and aerobic dialysis. During NADPH treatment no loss of dehydrogenase activity occurred. Thus, it was concluded that loss of dehydrogenase activity and emergence of ADPR in NO-treated GAPD were not correlated but coincidental, and that NO treatment yielded small amounts of modified-GAPD that had ADPR activity. In the brain, onset of ischemia is characterized by a significant elevation in free fatty acid (FFA) levels, predominantly, arachidonic acid (AA). It is suggested that AA can be oxidised to its metabolites like prostaglandins and 15-hydroperoxy arachidonic acid (15-HPETE) and some of these might exert toxic effects during reperfusion. Using whole membranes or tissue slices prepared from rat brain, effects of polyunsaturated fatty acids and their oxidative metabolites on five enzymes of PI cycle namely PI synthase, PI and PIP kinases, agonist-stimulated PLC and DG kinase was studied. Hydroperoxides of linoleic- and arachidonic acids inactivated PI synthase selectively among the PI cycle enzymes. Interestingly, AA selectively stimulated DG kinase in neural membranes. Docasahexaenoic acid (DHA) a highly unsaturated fatty acid found in the brain, also stimulated DG kinase activity while saturated, mono-and di-unsaturated fatty acids were ineffective. It was concluded that AA and DHA have a role in modulating neural DG kinase. The data presented in the thesis indicate that ROS have selective targets in cells and the consequent protein modifications can be used to modulate cellular functions under normal and oxidative stress conditions