The concentrations of
gamma-glutamylglutamate,
gamma-glutamylglutamine,
gamma-glutamylcysteine,
glutamate,
aspartate,
glutamine,
cyst(e)ine and
glutathione (including
disulfides) were determined by HPLC analysis of both the tissue and the surrounding medium of incubated rat hippocampal slices. High
potassium concentrations (50 mM; 2 x 4 min) increased the medium concentration of
gamma-glutamylglutamate (maximal net efflux 0.07 +/- 0.06 pmol/mg
protein/min; n = 8 +/- SD) with a relative time delay compared to the increase in
glutamate (maximal net efflux 264 +/- 88 pmol/mg
protein/min). Release of
gamma-glutamylcysteine, the
glutathione precursor, demonstrated an immediate response and gradually approached prestimulus levels (maximal net efflux 0.36 +/- 0.13 pmol/mg
protein/min). Addition of
acivicin (0.2 mM), a
gamma-glutamyl transpeptidase (EC 2.3.2.2.) blocker, during preincubation for 45 min reduced the tissue concentrations (pmol/mg
protein) of
gamma-glutamylglutamate (19.4 +/- 8.2 (control) vs. 5.8 +/- 3.6 (+
acivicin)),
gamma-glutamylglutamine (40.3 +/- 6.7 vs. 25.7 +/- 4.2 pmol/mg
protein),
glutamine (9.9 +/- 2.0 vs. 4.6 +/- 1.2 nmol/mg
protein) and
cysteine (1.0 +/- 0.2 vs. 0.56 +/- 0.18 nmol/mg
protein). Incubation with
acivicin (0.2 mM) reduced the net efflux of
gamma-glutamylglutamine (0.79 +/- 0.19 vs. 0.21 +/- 0.07 pmol/mg
protein/min) whereas that of the
glutathione was increased (4.7 +/- 1.0 vs. 20 +/- 3 pmol/mg
protein/min). The medium concentrations of
glutamate in both low and high
potassium were unaffected by
acivicin, while the high
potassium induced increase in
gamma-glutamylglutamate was blocked. The results demonstrate differential efflux patterns of gamma-glutamyl
dipeptides from brain slices and show that in vitro the activity of
gamma-glutamyl transpeptidase regulates extracellular concentrations of
glutathione,
gamma-glutamylglutamine and
gamma-glutamylglutamate.