Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Abstract

Astrocytes in primary cultures contain a relatively high activity of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and -insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

“…These results strongly indicate that the transmitter pool of glutamate is indeed affected by reduced numbers of synaptic vesicles in the synapsin DKO mice and that this finding most likely was overshadowed by the large metabolic pool of glutamate when studied in cerebrum extracts. Previous studies have shown that PAG, the enzyme catalyzing the conversion of glutamine to glutamate, is inhibited by even small increases in the intracellular levels of glutamate (Kvamme et al 1982;Hogstad et al 1988), similar to the inhibition of GAD by increased cytosolic GABA concentrations. The tendency towards a less pronounced increase in [4,[5][6][7][8][9][10][11][12][13] C]glutamate compared to [4,[5][6][7][8][9][10][11][12][13] C]glutamine in cerebrum extracts from synapsin DKO mice could indicate a decrease in the conversion of glutamine to glutamate, possibly by feedback inhibition of PAG.…”

Glutamine

“…56 Preliminary studies have also shown a similar attenuation of the MPT, as well as reduction in ROS formation by histidine in cultured astrocytes treated with ammonia. 62 While PAG is clearly present in cultured astrocytes, [63][64][65] its localization in astrocytes in vivo is a matter of controversy. Some immunohistochemical studies have not shown PAG in glia 60,66 ; yet, other immunohistochemical, 67 enzyme histochemical studies, 68 as well as studies using bulk-isolated astrocytes, 69 have all shown a glial localization of PAG.…”

Brain glutaminases

“…Primary cultures of astrocytes displayed strong GA activity (63)(64)(65) and GA mRNA transcripts (66), but these in vitro results have been questioned claiming that GA could be induced by culture conditions (4). By contrast, contradictory results appear in the literature about the expression of GA in astrocytes: immunohistochemical studies have shown expression of GA protein and GA activity in rat brain astrocytes (32,67); however, no GA was found in astrocytes from rat cerebellum via post-embedding immunocytochemistry with colloidal gold (33).…”