A Metabolomic Approach to Ionotropic Glutamate Receptor Subtype Function: A Nuclear Magnetic Resonance in vitro Investigation

Abstract

A range of behaviours are elucidated via ionotropic glutamate receptors (iGluR). In this work, we examined the acute activation of iGluRs by a range of receptor ligands and effectors to see whether distinguishable metabolic sequelae were elucidated by the activity. We used a guinea-pig brain cortical tissue slice model using targeted receptor ligands ((RS)-(tetrazol-5-yl)glycine (TZG),, as well as compounds (quinolinic acid and kynurenic acid (KynA)) involved in some neuroinflammatory responses. The data were derived using 13 C and 1 H NMR spectroscopy, and analysed by metabolomic approaches and multivariate statistics. The metabolic effects of agonists at the three major classes of iGluR were easily separated from each other using this method. The classical N-methyl-D-aspartate receptor agonist TZG and the antagonist CGS 19755 produced excitatory and inhibitory metabolic responses, respectively, while the blocker MK-801 resulted in a significant decrease in net metabolism and produced the largest decrease in all metabolite pool sizes seen by any glutamatergic ligand we have studied. Quinolinic acid and KynA produced similar acute metabolic responses, which were unlike those to TZG or CGS 19755, but similar to that of D-Ser. D-Ser was highly stimulatory of net flux into the Krebs cycle. These data show that the metabolic response to iGluR perturbation in vitro is a sensitive discriminator of function.

Metabolic Effects of Blocking Lactate Transport in Brain Cortical Tissue Slices Using an Inhibitor Specific to MCT1 and MCT2

“…Addition of 50 lM AMPA to slices resulted in significantly increased net flux and pool sizes, as reported previously [23] (Fig. 1b, 2b).…”

“…Addition of AMPA and the desensitizing agent cyclothiazide produces a different metabolic response to depolarization. Cyclothiazide itself has been shown to have a negligible effect on metabolism [23]. Addition of 10 nM AR-C122982 had little effect on net flux into TCA cycle intermediates.…”

“…Experiment 2: 5 mM D-[1-13 C]glucose, 40 ll DMSO (0.01% v/v), 100 lM cyclothiazide (allosteric modulator of AMPA receptors which potently inhibits receptor desensitisation [22]), 50 lM AMPA (control) plus either 10 or 100 nM AR-C122982. This experiment was performed to activate the AMPA sensitive glutamate receptor and significantly increase flux through the glutamate/glutamine cycle [23]. Experiment 3: 5 mM D-[1-13 C]glucose, 40 ll DMSO (0.01% v/v), 50 lM L-glutamate (control) plus either 10 or 100 nM AR-C122982.…”

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

“…Such movements may occur in response to changes in the activity of glutamatergic synapses and, therefore, activity of L-glu transport could be of importance not only for the normal operation of the synaptic excitation [5] but also as an indicator of local excitatory activity that would have to be matched by local changes in the metabolism and blood flow. Indeed, it has recently been demonstrated that not only pharmacological manipulation of glutamate receptors but, in particular, interference with GluT, can elicit specific metabolic responses in brain tissue [14][15][16][17].…”

A metabonomic study of inhibition of GABA uptake in the cerebral cortex

“…The analysis of the resultant metabolic profiles by multivariate statistics provides an objective means for the identification of differences or similarities in metabolic outcomes. This is a robust and useful approach used successfully to identify novel neurochemical outcomes (Bröer et al 2007;Griffin et al 1998;Moussa et al 2007;Nasrallah et al 2007;Rae et al 2000Rae et al , 2005Rae et al , 2006Stanton et al 2003).…”