We propose to re-explore the regulation of cerebral glutamine, glutamate and gamma-aminobutyric acid (GABA) metabolism in order to elucidate the mechanisms of neurotoxicity in hepatic encephalopathy (HE). The new impetus is the development of non-invasive assays of enzyme activity and metabolite flux in the brain of living rats, using novel techniques of 15N magnetic resonance (MR) and the more sensitive method of 1H-15N heteronuclear multiple quantum coherence (HMQC) MR spectroscopy (MRS). Both methods depend upon enrichment of metabolic pools with the stable isotope 15N. Specifically, in vivo activities of glutamine synthetase (GS), an astrocyte marker enzyme, and of glutamate decarboxylase (GAD), a marker enzyme of pre-synaptic nerve-terminals, will be measured in the brain of hyperammonemic rats as an animal model of HE, and in thioacetamide-treated rats as a model of fulminant hepatic failure. The GS activity will indicate the in vivo rate of ammonia incorporation into glutamine and of glutamate depletion from the astrocytes. The GAD activity will reflect glutamate concentration in nerve terminals and indicate the rate of GABA synthesis. Finally, the rate of replenishment of the total cerebral glutamate pool will be determined from the rate of transamination from 15N-enriched essential amino acids that readily cross the blood-brain barrier. These experiments will clarify the effect of hyperammonemia on neuronal vs astrocytic pools of glutamate and GABA and contribute to evaluation of three leading hypotheses on the mechanism of neurotoxicity in HE: 1) ammonia per se is neurotoxic; 2) glutamine accumulation contributes to the pathogenesis; and 3) hyperammonemia causes HE through depletion of the neurotransmitter pool of glutamate. In addition, the relative importance of benzodiazepines and GABA in the etiology of brain dysfunction in fulminant hepatic failure will be explored. These experiments will shed light on an increasingly important group of neurological diseases, including hepatic encephalopathy.
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