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Ammonia metabolism in the brain

University of British Columbia

It is known that the functional activity of the nervous system is associated with ammonia formation and that the administration of ammonium salts to experimental animals produces convulsions. Mechanisms, therefore, that control ammonia metabolism in the brain are of importance for brain cell function. The presence of ammonia utilizing mechanisms in the brain maintains the low free cerebral ammonia levels found in vivo. There is, however, a rapid formation of ammonia in the brain on the death of the animal and a further liberation of ammonia takes place when isolated brain cortex is incubated aerobically in the absence of glucose. Studies of these and other aspects of ammonia metabolism form the subject matter of this thesis. The estimation of ammonia in these studies is based on a modification of the diffusion technique of Conway. Ammonia and amino acid analyses have been carried out using the Beckman amino acid analyzer. The rapid rate of cerebral ammonia formation that takes place when the brain is removed from the animal is partially arrested by trichloracetic acid (TCA), presumably by the inactivation of cerebral enzymes. Our results rule out the possibility that glutamine, glutamate, taurine and ATP are significant contributors to the initial or pre-incubation levels of ammonia and the evidence favors the involvement of TCA-insoluble components as precursors of such ammonia. In the presence of glucose the pre-incubation levels of amino acids of cerebral cortex slices of the rat are maintained during subsequent aerobic incubation at 37°C for one hour. In the absence of glucose, however, we have found marked changes in the pre-incubation levels of amino acids of cerebral cortex slices under these experimental conditions. A considerable rise of ammonia also occurs in the absence of glucose and this can be largely accounted for by a net loss of –NH₂-groups of the amino acid pools of brain slices. The significant fall in the cerebral levels of glutamate and glutamine under these conditions indicates that for short periods of incubation (one hour), these amino acids may serve as major sources of ammonia formation by respiring brain cortex slices. Our findings of a marked suppression of ammonia formation by cerebral cortex slices incubated for one hour either anaerobically, or aerobically, in a glucose-free medium in the presence of amytal, D-glutamate or α-methylglutamate, implicate the oxidative deamination of cerebral glutamate as a major mechanism for ammonia liberation. D-glutamate also acts by inhibiting the hydrolysis of glutamine. In the presence of glucose aerobic incubation with 2, 4-dinitrophenol, iodoacetate, malonate, hydroxylamine or D-glutamate, increases the rate of ammonia formation by cerebral cortex slices. This is doubtless due to diminished activity of cerebral glutamine synthetase required for ammonia fixation to occur. We find that the level of ammonia in the brain tissue itself is not markedly affected by the presence of absence of glucose. The increased quantity of ammonia formed by cerebral cortex slices in the absence of glucose is found largely in the incubation medium. This fact points to the formation of ammonia in specific compartment(s), and the retention of ammonia within such compartment(s) up to a certain level. Above this level there is efflux of ammonia into the incubation medium. Such a conclusion helps to explain the apparent high concentration ratio (tissue:medium) of NH₄⁺ obtained either aerobically (viz. 42) or anaerobically (viz. 12) at the end of one hour incubation in the presence of glucose. There is some accumulation of NH₄⁺ ions in rat brain cortex slices against a concentration gradient. Our finding that ouabain stimulates ammonia formation in respiring cerebral cortex slices is in accord with the fact that ouabain inhibits the utilization-of NH₄⁺ for the biosynthesis of glutamine, presumably by affecting the transport of NH₄⁺ to the site of glutamine synthesis. Ouabain has no effect on the cerebral glutaminase of the rat.

Text

2011-06-07

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http://hdl.handle.net/2429/35180

Biochemistry and Molecular Biology

University of British Columbia

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