Guillermo H. Massieu

Free amino acids and glutamate decarboxylase activity in brain of mice during drug-induced convulsions

Abstract Previous administration of l -glutamic acid-γ-hydrazide (GAH) to mice did not protect them against the convulsant action of thiosemicarbazide, methionine sulfoximine, insulin, or pentylenetetrazol (Metrazol). The concentrations of some free amino acids were measured in the brains from these mice at the moment of convulsions. The changes in free amino acid concentrations produced by the convulsant agents used were in general similar whether GAH had been administered or not. In the first case, however, the changes were exerted on the altered pattern of amino acids obtained by the previous GAH treatment. In all cases when GAH and the convulsant agent were injected and convulsions were produced, a two- to four-fold increase of γ-aminobutyric acid (GABA) concentration was found in brain. In other experiments, it was found that GAH administration increased both free and bound GABA concentrations. When a single convulsant dose of GAH was injected, brain glutamate decarboxylase activity progressively decreased with time. The maximal glutamate decarboxylase inhibition was observed at the onset of convulsions; at this moment GABA levels were increased. γ-Aminobutyric aminotransferase activity was diminished more intensely before the onset than at the occurrence of the convulsive state. Similarly, after the injection of a convulsant dose of amino-oxyacetic acid, glutamate decarboxylase activity was decreased at the onset of convulsions, whereas γ-aminobutyric transferase activity was totally inhibited; GABA levels were significantly increased. Anticonvulsant doses of amino-oxyacetic acid protected mice against convulsions induced by the simultaneous administration of GAH and pyridoxal phosphate. Glutamate decarboxylase activity was found equally diminished in protected and in nonprotected mice in comparison with control animals. It is concluded that the inhibition of glutamate decarboxylase activity, independently of the total concentration of GABA in brain, may be a factor involved in the production of some types of convulsions, and probably the anticonvulsant action of amino-oxyactic acid is not related to its effect on GABA metabolism in brain.

Modifications of brain glutamate decarboxylase activity by pyridoxal phosphate-γ-glutamyl hydrazone

Abstract The administration of pyridoxal phosphate-γ-glutamyl hydrazone to mice had effects identical with those produced by the simultaneous administration of equimolar doses of glutamic acid-γ-hydrazide and pyridoxal phosphate; both treatments produced convulsions, lowered the brain γ-aminobutyric acid concentration, and inhibited the activity of glutamate decarboxylase. The inhibition was completely reversed when pyridoxal phosphate was added in vitro to the incubation mixture. Pyridoxal phosphate-γ-glutamyl hydrazone administration also inhibited the brain pyridoxal kinase activity. On the other hand, the addition of pyridoxal phosphate-γ-glutamyl hydrazone in vitro to brain homogenates resulted in an activation of glutamate decarboxylase similar to that obtained with free pyridoxal phosphate. It is concluded that the effects of the simultaneous administration of glutamic acid-γ-hydrazide and pyridoxal phosphate are due to pyridoxal phosphate-γ-glutamyl hydrazone, which is formed in vivo , and that such effects are possibly mediated by the inhibition of brain pyridoxal kinase. Rupture of pyridoxal phosphate-γ-glutamyl hydrazone at the level of the apoenzyme active site apparently occurs when the substance is added in vitro .

FREE AMINO ACIDS IN BRAIN AND LIVER OF DEOXYPYRIDOXINE-TREATED MICE SUBJECTED TO INSULIN SHOCK*

QUASTEL and WHEATLEY (1932) and KREBS (1935) showed that glutamic acid can replace glucose in 2-itro as oxidizable substrate, and thereafter several authors attempted to find evidence that such utilization occurs also in cico. The decrease of the free glutamic acid level of brain during insulin hypoglycaemia found in the rat by DAWSON (1950) and by CRAVIOTO, MASSIEU and IZQUIERDO (1951) has been interpreted as evidence for the utilization of the amino acid in vir.o as substrate. Several workers have confirmed, both by in vitro and in rico experiments, that the metabolism of glutamic acid is closely related to the tricarboxylic acid cycle, chiefly via transamination and dehydrogenation (WAELSCH, 1940; BELOFF-CHAIN et af., 1955-6; ROBERTS, ROTI~STEIN and BAXTER, 1958; TOWER, 1958). Decarboxylation of glutamic acid produces GABAT which in turn is transaminated and finally converted to succinic acid (BESSMAN, ROSSEN and LAYNE, 1953; ROBERTS and BREGOFF, 1953; ROBERTS et al., 1958). According to MCKHANN el al. (1960), a considerable amount of the free glutamic acid of brain contributes to the tricarboxylic acid cycle through the GABA-succinic acid collateral pathway. If during insulin hypoglycaemia the brain’s free glutamic acid is utilized as substrate, we could expect that the blockade of the pathways of the amino acid towards the Krebs cycle would interfere with such utilization. In the present work we attempted to block some of the PyP-dependent pathways. This objective was partially achieved by treating mice with DOP and submitting them simultaneously to a vitamin B,deficient diet. The animals treated in such a way were subjected to insulin shock, and the levels of glutamic acid, GABA, aspartic acid, and glutamine in the brain were estimated and compared with those found in the brains of animals not subjected to insulin shock. The free amino acid levels of DOP-treated animals were compared with the levels of the control animals. GTD and GOT activities were measured in the brain tissue of both control and vitamin B,-depleted animals. The levels of free glutamic acid, aspartic acid, alanine, and glutamine, as well as the GOT activity, were measured in liver tissue of both control and DOP-treated mice, with and without insulin shock. These estimations were carried out in order to compare the response of liver with that of brain tissue under similar experimental conditions.

Effects in vivo and in vitro of l-glutamic acid-γ-hydrazide on metabolism of some free amino acids in brain and liver

Abstract Further research on the effect of l -glutamic acid-γ-hydrazide (GAH) on free amino acids metabolism in brain is reported. Similar studies of free amino acid metabolism in liver are also presented. The addition of GAH to mice brain homogenates inhibited the activities of γ-aminobutyrate aminotransferase (ABAT), and of glutamate decarboxylase (GAD). The activities of alanine aminotransferase (AAA) and of aspartate aminotransferase (ASA) were not affected by GAH in these conditions. Brain homogenates from GAH-treated mice showed lower GAD, ABAT, AAA, and β-alanine-α-ketoglutaric aminotransferase (βAA) activities than brain homogenates from control mice. GAD activity was inhibited by GAH to a considerably lesser extent in vivo than in vitro . In liver, GAH produced a striking increase in the concentrations of almost all the free amino acids, especially of alanine and β-alanine. The addition of GAH to mice liver homogenates inhibited the βAA activity, but neither AAA nor ASA activity was affected by the same concentration of GAH. Liver homogenates from GAH-treated mice showed diminished βAA and AAA activity in comparison with liver homogenates from control mice. Some differences in the conversion of uniformly labeled 14 C- glucose to liver and brain free amino acids were found between control and GAH-treated mice. In the brain of the latter animals, aspartic and glutamic acids and glutamine exhibited initially a higher specific activity than in control mice. In the case of γ-aminobutyric acid and alanine, opposite results were obtained. In the GAH-treated animals these two amino acids showed no significant decay of the acquired radioactivity. In liver, the decay of the acquired radioactivity of alanine tended to be slower in GAH-treated animals than in control animals.

SOME PROPERTIES OF GLUTAMATE DECARBOXYLASE AND THE CONTENT OF PYRIDOXAL PHOSPHATE IN BRAINS OF THREE VERTEBRATE SPECIES

—Some properties of glutamate decarboxylase (GAD) were studied in the brain of the carp (Carassius auratus), the pigeon (Columbia livia) and the mouse (Mus musculus). The optimum pH for GAD in the three species was 6·3‐6·5. In the three species studied, GAD activity of brain homogenates in water was higher than that of homogenates in buffer. The supernatant from homogenates in Triton‐X‐100 gave an enzyme preparation which showed greater activation by pyridoxal phosphate than those obtained from complete water or buffer homogenates or from the supernatant of Water homogenates.

Study on the effect of 3-acetylpyridine on blood glucose concentration

Abstract It is known that the administration of 3-acetylpyridine (3-AP) to rats induces hyperglycemia. Experiments designed to study the mechanism of this action of 3-AP on blood glucose levels are reported. The hyperglycemia produced by the administration of 3-AP to adult rats was accompanied by a decrease of hepatic and muscular glycogen. The hyperglycemic effect of 3-AP was significantly less in 8-day-old rats than in adult rats. The simultaneous administration of the ganglionic blocking agent mecamylamine inhibited the hyperglycemic effect of 3-AP. Small doses of 3-AP injected intracisternally produced hyperglycemia in short periods of time. The i.p. injection of the same dose of 3-AP did not exhibit such effect. Hyperglycemia was also produced by the intracisternal administration of the analogue of NAD, 3-AP-NAD, at a dose 100-fold less than the intracisternal dose of 3-AP which induced hyperglycemia. The epinephrine content in the adrenal glands did not change significantly after the administration of a hyperglycemic dose of 3-AP. It is concluded that the hyperglycemic effect of 3-AP is due to a release of cathecholamines from the adrenal medulla and that this release is probably secondary to the lesions observed in brain after 3-AP administration. It is also concluded that 3-AP probably acts by forming the analogue 3-AP-NAD in brain.