The goal of this proposal is to provide evidence for the hypothesis that the rate of cerebral ATP synthesis is controlled predominantly by energy- consuming ion movements and especially by the Na+/K+ ATPase. The operation of the latter enzyme is responsible for the preservation of proper ionic balances, and hence neurotransmitter levels, in CNS neurons.
The specific aims are : 1. to determine how much energy the Na+/K+ pump utilizes under both """"""""normal"""""""" resting conditions and during enhanced activity, and what are the sources of ATP, a) when its synthesis is undisturbed and b) when its synthesis becomes limited by lack of either glucose or oxygen, or both. Rat brain synaptosomes will be used as the experimental model because they provide a system in which quantitative evaluation of the relations among various parameters can be made. The Na+/K+ pump activity will be measured as ouabain-sensitive 86 Rb uptake. Its dependence on ATP supplied either by glycolysis or oxidative phosphorylation in the presence of various substrates will be determined by exploring the effect of ouabain on these processes, a) when they function optimally (glucose and O2 plentiful) and b) when their activities are limited either by glucose withdrawal or by inhibition of the electron transport chain. The cellular energy parameters together with [Ca2+]; and leakage of the amino acid transmitters, GABA, aspartate and glutamate will be measured simultaneously to correlate them with the activity of the pump. 2. To determine the extra- and intracellular concentrations of Na+, K+ and Ca2+ using ion-sensitive microelectrodes in rat brain in vivo during hypoxemia and ischemia; conditions that limit ATP synthesis and thus the Na+/K+ pump activity. Hypoxemia will be induced by decreasing the concentration of 02 in the inspired air while ischemia will be produced by vessel occlusion. Comparison will be made between alterations in internal [ion] during hypoxemia/ischemia in various regions of the brain to ascertain whether areas known to be vulnerable to these insults exhibit larger changes. To test the possibility that significant movements of some ions occur via voltage-dependent as well as receptor-operated channels extra- and intracellular [ion] will be measured in animals that have been given drugs which either block the voltage-dependent channels (dihydropyridines) or reduce CNS excitation by interacting with a specific subclass of acidic amino acid receptors (ketamine, phencyclidine, MK-801). The information gained will allow better understanding of changes in ions and neurotransmitter levels during cerebral hypoxia/ischemia and thus permit rational design of future therapies to combat the consequences of these pathologies.
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