Glutamine synthetase activity and ambient L-glutamate in cholesterol-deficient rat brain nerve terminals

Glutamate is not only the predominant excitatory neurotransmitter in the mammalian CNS but also a potent neurotoxin. The excessive activation of glutamate receptors can cause exitotoxicity and cell death both in vitro and in vivo. The low level of ambient glutamate is important for the brain’s spontaneous activity and proper synaptic transmission. The study was focused on the assessment of the effects of glutamine synthetase inhibitor, L-methionine sulfoximine (MSO), on the level of ambient L-[14C]glutamate in rat brain nerve terminals treated with the cholesterol acceptor methyl-b-cyclodextrin (MbetaCD, MbCD) (15 mM), which reduced the synaptosomal cholesterol content by 25.0 ± 3.0 %. It was revealed that the inhibitor per se decreased transporter - mediated uptake of L-[14C]glutamate by synaptosomes in dose-dependent manner. Therefore, we applied the non-transportable competitive inhibitor of glutamate transporters DL-threo-b-benzyloxyaspartate (DL-TBOA) to eliminate the contribution of transporter-mediated glutamate uptake to the value of ambient L-[14C]glutamate during the inhibition of the activity of glutamine synthetase. It was shown that the combined application of 200 mM DL-TBOA and 1.5 mM MSO increased the extracellular L-[14C]glutamate level from 0.193 ± 0.013 nmol/mg protein to 0.52 ± 0.02 nmol/mg protein in control and from 0.282 ± 0.013 nmol/mg protein to 0.62 ± 0.02 nmol/mg protein in MbCD – treated synaptosomes. Thus, the inhibition of glutamine synthetase activity increased the level of ambient L-[14C]glutamate in control and cholesterol-deficient synaptosomes, and this level was higher under condition of cholesterol deficiency than in control irrespective of the presence or absence of glutamine synthetase inhibitor.