Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

Abstract

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5 mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5-10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo-beta-hydroxyaspartic acid were relatively weak inhibitors of PAG (< 25% inhibition) as were the receptor agonists, ibotenic acid and (+-)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40-70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5-10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5-2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG.(ABSTRACT TRUNCATED AT 250 WORDS)

(1R,3S)‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (RS‐ACPD) reduces intracellular glutamate levels in astrocytes

“…If true, accumulating RS‐ACPD would shift the equilibrium between glutamine and glutamate towards less glutamate and more glutamine. This possibility is also supported by previous observations showing that synaptosomal PAG is inhibited by other glutamate analogs at millimolar concentrations (Dawson and Wallace 1993). Moreover, using HPLC, we directly examined the accumulation of RS‐ACPD in astrocytes (Fig.…”

“…It is typically assumed that the majority of glutamate taken up by astrocytes is metabolized by the activity of glutamine synthetase, ignoring the possible contribution of phosphate‐activated glutaminase (PAG). In neurons, however, PAG mediates the production of glutamate from glutamine and t‐ACPD inhibits PAG activity in synaptosomes (Dawson and Wallace 1993). We thus speculated that the reductions in astrocytic [Glu] i by RS‐ACPD may have been mediated by interactions with PAG.…”

“…This suggests that RS‐ACPD and glutamate may each have similar affinity for PAG. Interestingly, at 300 µ m , RS‐ACPD significantly reduced intracellular taurine, which is an endogenous inhibitor of PAG in synaptosomal preparations (Dawson and Wallace 1993). Thus, the actual inhibition of PAG by RS‐ACPD may be underestimated at high concentrations.…”

“…Because neurons express Na + ‐dependent glutamate transporters, it is likely that they also accumulated RS‐ACPD leading to inhibition of neuronal PAG and reduction of [Glu] i . t‐ACPD has been shown to inhibit PAG in synaptosomes (Dawson and Wallace 1993). It may therefore be possible that prolonged incubation with t‐ACPD could lead to suppression of synaptic transmission by either inhibition of PAG, or by inhibition of vesicular glutamate transport (Winter and Ueda 1993).…”