Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Abstract

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

“…As the conversion of a-ketoglutarate to glutamate is inhibited almost completely by transaminase inhibitor, the data suggest that glutamate dehydrogenase is not very active in the direction of a-ketoglutarate reductive amination under the conditions used. Several previous reports suggest that glutamate dehydrogenase may not be very active in the oxidative direction (Farinelli and Nicklas, 1992;McKenna et a!., 1993), or in the reductive direction (Yudkoff et al, 1994(Yudkoff et al, , 1996 in rat brain astrocytes, but there is contro- …”

“…Investigations of the regulation of glutamate synthesis and breakdown in astrocytes via pathways other than glutaminase and glutamine synthase have shown that astrocytic glutamate is also metabolized via the citric acid cycle after conversion to a-ketog!utarate (Yu et al, 1982;Farinelli and Nicklas, 1992;Westergaard et al, 1995;McKenna et al, 1996a,b). Thus, astrocyticglutamate can be converted to glutamine and exit the cell, or it can be converted to a-ketoglutarate by one of four reversible astrocytic enzymes: glutamate dehydrogenase (Rao and Murthy, 1993;McKenna et al, 1 996b), aspartate aminotransferase (Rao and Murthy, 1993;McKenna et al, 1996b), branched-chain aminotransferase (Yudkoff et al, 1994(Yudkoff et al, , 1996, or alanine aminotransferase (Rao and Murthy, 1993).…”

The role of astrocytes in Alzheimer's disease

“…Interestingly, a marked increase in astrocytic GFAP levels has also been detected in the brain of AD patients (Haga et al, 1989;Ogata et aL, 1992). One of the sites found to have a significantly high GFAP immunoreactivity is neuritic plaque (Farinelli and Nicklas, 1992). It has been suggested that glial cells surrounding neuritic plaques are also involved in the removal of debris from degenerated neurites (Rozemuller et al, 1989;Wisniewski and Wegiel, 1992), the synthesis and deposition of (3-amyloid (Wisniewski and Wegiel, 1992), or the isolation of P-amyloid from neuropil by astrocytic processes (Wisniewski and Wegiel, 1992).…”

“…A study with radioactive markers showed that most of the exracellular glutamate is taken up by astrocytes rather then by neurons (McLennan, 1976). The glutamate taken up by astrocytes is then converted by a glia cell-specific enzyme, glutamine synthetase, into glutamine which can, in turn, be used by neurons for glutamate synthesis (Farinelli and Nicklas, 1992). The essential role of astrocytes in protection against glutamate neurotoxicity and maintenance of low extracellular levels of glutamate was further supported by in vitro experiments (Rosenberg et al, 1992).…”

“…Extracellular glutamate is taken up rapidly by astrocytes, where it is converted chiefly to glutamine by glutamine synthetase (Farinelli and Nicklas, 1992). The essential role of astrocytes in maintaining low extracellular levels of glutamate was demonstrated clearly in studies showing a significant increase in neuronal susceptibility to glutamate toxicity' under neuron-astrocyte co-culture with relatively few astrocytes (Rosenberg and Aizenman, 1989).…”