; The long term objective of the proposed studies is to determine mechanisms that dynamically regulate ascorbic acid (Asc) compartmentalization in the brain. Asc functions as an antioxidant and free radical scavenger to protect the brain from reactive species generated during aerobic metabolism. Preliminary data suggest that the anoxic injury-resistant turtle brain may synthesize Asc, suggesting a protective value of the vitamin. The planned experiments provide basic information about Asc regulation in the brain including: (1) uptake, (2) release and (3) utilization. These will be compared in hippocampal slices from the guinea pig, which, like humans, cannot synthesize Asc, and the turtle.
Specific aims are: (1) To characterize Asc uptake in brain tissue using HPLC for tissue analysis. Experiments will determine Michaelis-Menten uptake parameters, ionic dependence and energy requirements. Whether turtle brain can synthesize Asc will also be verified. (2) To determine the mechanisms of Asc release from brain cells. [Asc]o will be monitored using carbon fiber microelectrodes and high-speed voltammetry. Release and extracellular volume change will be distinguished by co-monitoring the extracellular marker tetramethylammonium using ion-selective microelectrodes. (3) To examine Asc utilization during depolarization and hypoxia/reoxygenation by measuring tissue Asc and its oxidation product, dehydroascorbate, using HPLC. [Asc]o will be monitored. The effects of therapeutic agents known to affect free radical production will be tested to evaluate the mechanisms of Asc action as a protective agent. The protective action of Asc in brain is important during normal aerobic metabolism, but may be crucial during the pathological conditions of stroke or ischemia. The strength of this proposal lies in the estimation of intra- and extracellular compartmentalization of Asc under various conditions by coupling total tissue analysis with direct extracellular Asc measurements.
