Inhibition of glutamate/glutamine cycle in vivo results in decreased benzodiazepine binding and differentially regulated GABAergic subunit expression in the rat brain.
OBJECTIVE
The astrocytic enzyme glutamine synthetase (GS) is a key regulator of glutamate and γ-aminobutyric acid (GABA) metabolism in the glutamate/glutamine cycle (GGC). Inhibition of GS results in changes of neurotransmitter release and recycling. However, little is known about the influence of GGC on neurotransmitter receptor expression. In the pentylenetetrazole model of epilepsy, GS becomes nitrated and partially inhibited, and we demonstrated alterations of neurotransmitter receptor expression in the same model. Therefore, we hypothesized similar changes of neurotransmitter receptor expression when GS is inhibited in vivo.


METHODS
Rats were treated with a single dose (100 mg/kg bodyweight) of l-methionine sulfoximine (MSO), an irreversible inhibitor of GS. We used ³H-receptor autoradiography to measure glutamatergic [α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), kainate, N-methyl-D-aspartate (NMDA)], GABAergic (GABA(A) , GABA(B) and GABA(A) -associated benzodiazepine (BZ) binding sites], dopamine D₁, and adenosine A₁ receptor subtypes. In addition, we performed saturation analysis of BZ binding sites on cerebral membrane homogenates and investigated the expression of GABA(A) α₁ and γ₂ subunits (which primarily mediate BZ binding) by western blot analysis.


RESULTS
We demonstrated a significant reduction of BZ binding in the somatosensory, piriform, and entorhinal cortices and in the amygdala, 24 and 72 h after MSO treatment. Saturation analysis revealed decreased BZ binding (B(max)) on cerebral membrane homogenates 72 h after MSO treatment, without changes in binding site affinity (K(D)). Furthermore, we found differential changes of α₁ , γ₂ , and phosphorylated γ₂ subunits following MSO treatment.


CONCLUSIONS
On the basis of our findings, we conclude that the glutamate/glutamine cycle directly influences GABAergic neurotransmission by regulating GABA(A) subunit composition, thereby affecting its modulation by endogenous benzodiazepines.
Cremer
Christian M
CM
Institute of Neuroscience and Medicine (INM-2), Research Center Jülich, Jülich, Germany. c.cremer@fz-juelich.de
Bidmon
Hans-Jürgen
HJ
Görg
Boris
B
Palomero-Gallagher
Nicola
N
Escobar
Jennifer Lopez
JL
Speckmann
Erwin-Josef
EJ
Zilles
Karl
K
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D008297:Male; D008717:Methionine Sulfoximine / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018079:Receptors, GABA / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D021122:Protein Subunits; D012333:RNA, Messenger; D018079:Receptors, GABA; D005973:Glutamine; D014316:Tritium; D001569:Benzodiazepines; D008717:Methionine Sulfoximine; D018698:Glutamic Acid; D005974:Glutamate-Ammonia Ligase