Research ReportFormation of extracellular glutamate from glutamine: Exclusion of pyroglutamate as an intermediate
Introduction
The extracellular concentration of glutamate ([GLU]EC) in the normal brain is maintained at a low level to allow a balance between effective signaling and prevention of excitotoxicity [11]. The low micromolar concentration of [GLU]EC is primarily controlled by the action of excitatory amino acid transporters located in astrocytes [13]. Increased [GLU]EC exceeding the reuptake capability of the transporters has been demonstrated to be associated with neuronal cell death in different models of neuronal injury that include hypoxia-ischemia [4], [14], [23], [30], trauma [23], or chronic neurodegenerative diseases [7], [9]. Furthermore, glutamate-induced neuronal injury has been shown to be markedly reduced in the presence of NMDA and AMPA/kainate glutamate-receptor antagonists in several in vitro and in vivo models of neuronal damage, even when the antagonists were administered 1–5 h after the injury [2], [6], [20], [24], [27]. Significant neuroprotection can be achieved in a hippocampal slice model by enzymatic degradation of extracellular glutamate (GLUEC) by glutamate–pyruvate transaminase [26]. The cellular sources of GLUEC after neuronal injury are not completely characterized but include glutamate efflux subsequent to cell swelling [12], reversal of glutamate uptake [39], and Ca+2 dependent vesicular release [34]. The cystine–glutamate exchanger, present on glial cells [36], has also been implicated as a significant source of extracellular glutamate [1]. Glutamine hydrolysis to glutamate in the extracellular space has been proposed to contribute to an increased level of GLUEC in cell culture [15], [31] and in in vivo neuronal injury models [29], [32]. We reported that glutamine administered to rat brain via reverse microdialysis, following direct injection of quinolinic acid to cause neuronal excitotoxicity, was rapidly hydrolyzed to glutamate [29]. Glutamine was also hydrolyzed to glutamate in the untreated brain, but to a lesser extent than in quinolinic acid treated rats. We suggested that either mitochondrial phosphate activated glutaminase (released by cell disruption) or maleate activated glutaminase (especially in the case of the untreated brain) could generate GLUEC by glutamine hydrolysis. Maleate activated glutaminase is a side reaction of the ectoenzyme γ-glutamyl transpeptidase [40], [43]. The current studies address the potential non-enzymatic formation of glutamate from glutamine in the interstitial space, specifically, via pyroglutamate (also referred to as 2-pyrrolidone-5-carboxylic acid and 5-oxoproline) [41]. We examined the conversion of glutamine to glutamate by direct injection of radiolabeled glutamine or pyroglutamate into the extracellular compartment of the rat hippocampus and subsequent recovery via microdialysis of radiolabeled glutamate, glutamine, and pyroglutamate. The in vivo results were compared to the in vitro stability of glutamine, glutamate, and pyroglutamate under comparable physiological conditions.
Section snippets
Materials and methods
Microdialysis supplies were purchased from Bioanalytical Systems, Inc. (West Lafayette, Ind., USA). Chemicals and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO). Radioactive glutamine ([U-14C]-l-glutamine, 8.81 GBq/mmol and glutamate ([U-14C]-l-glutamic acid, 9.4 GBq/mmol were obtained from Amersham Biosciences Corp. (Piscataway, NJ). Radioactive pyroglutamate ([U-14C]-l-pyroglutamic acid, 7.4 GBq/mmol, and α-ketoglutarate ([U-14C]-α-ketoglutarate, 9.25 GBq/mmol) were obtained
GLUEC and GLNEC following reverse microdialysis with glutamine
Reverse microdialysis of rat hippocampus with aCSF containing 5 mM glutamine generated a 4.6-fold increase of glutamate levels in the dialysate compared to the initial 2.5 h baseline period (P < 0.01; Fig. 1). Taurine and aspartate levels were not altered. Also unchanged were the levels of 17 additional amino acids (data not shown). After 10 fractions were collected, the probe was removed from the rat brain and suspended in a vial of sterile aCSF. Perfusion with the 5 mM glutamine solution was
Discussion
Recent reports have suggested that the hydrolysis of glutamine in brain extracellular space results in an increase in extracellular glutamate [29], [32]. Both groups hypothesized enzymatic mechanisms. Newcomb et al. [31], [32] proposed that intramitochondrial PAG, released following cell damage, catalyzes the hydrolysis of glutamine to glutamate. Fragments of mitochondrial membrane with PAG intracellular metabolites and soluble enzymes may leak into the interstitial space after neuronal injury
Acknowledgment
This research was supported in part by NIH grant HD16596.
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