@ Pergamon Neurochem. Int. Vol.29,No.2,pp.121-128, 1996 Copyright ~ 1996Elsevier ScienceLtd 0197-0186(95)00148-4 PrintedinGreatBritain.Allrightsreserved 01974186/96 $15,00+0,00 PGLUTAMYL PEPTIDES AND RELATED AMINO ACIDS IN RAT HIPPOCAMPUS IN VITRO: EFFECT OF DEPOLARIZATION AND y-GLUTAMYL TRANSPEPTIDASE INHIBITION XIAOYING LI, OWE ORWAR, CAMILLA REVESJO and MATS SANDBERG* Institute of Anatomy and Cell Biology,Universityof Goteborg, Medicinaregatan3-5, S-41390 Goteborg, Sweden (Received 12 June 1995; accepted 7 November 1995) Abstract—The concentrations of y-glutamylglutamate,y-glutamylglutamine,y-glutamylcysteine,glut- amate, aspartate, glutamine, cyst(e)ineand glutathione (includingdisulfides)were determined by HPLC analysisofboth the tissueand the surroundingmediumofincubatedrat hippocampalslices.Highpotassium concentrations (50mM; 2 x 4 rein) increasedthe mediumconcentrationof y-glutamylglutamate(maximal net efflux 0.07tO.06 pmol/mg protein/rein; n = 8~ SD) with a relative time delay compared to the increase in glutamate (maximalnet efflux264t 88 pmol/mg protein/rein). Release of y-glutamylcysteine, the glutathione precursor, demonstrated an immediate response and gradually approached prestimulus levels (maximalnet efflux0.36+0.13 pmol/mgprotein/rein). Addition of acivicin (0.2mM), a y-ghItamyl transpeptidase (EC 2.3.2.2.)blocker, during preincubation for 45 min reduced the tissue concentrations (pmol/mg protein) of y-glutamylglutamate(19.4~8.2 (control) vs. 5.8*3.6 (+acivicin)), y-glutamyl- glutamine (40.3t 6.7 vs. 25.7* 4.2 pmol/mg protein), glutarnine(9.9i 2.0 vs. 4.6t 1.2nmol/mg protein) and cysteine (1.0~ 0.2 vs. 0.56t0.18 nmol/mgprotein). Incubation with acivicin (0.2 mM) reduced the net effluxof y-glutamylglutamine(0.79~ 0.19 vs. 0.21~ 0.07 pmol/mg protein/rein) whereas that of the ghrtathione was increased (4.7+ 1.0 vs. 20+3 pmol/mg protein/rein). The medium concentrations of glutamate in both low and high potassium were unaffectedby acivicin,whilethe high potassium induced increase in y-glutamylglutamatewas blocked. The results demonstrate differential effluxpatterns of y- glutamyl dipeptides from brain slices and show that in oitro the activity of y-glutamyltranspeptidase regulatesextracellularconcentrationsofglutathione,y-glutamylglutamineand y-glutamylglutamate. Copy- right CJ 1996ElsevierScienceLtd. Several of the y-glutamyl dipeptides identified in brain (Sano et al., 1966; Sane, 1970; Reichelt, 1970; Mar- nela et al., 1985; Sandberg et al., 1994) are of potential importance in neurotransmission as they interact with excitatory amino acid (EAA) receptors (Varga et al., 1989, 1992; Mathis et al., 1990; Uemura et al., 1992; Li et al., 1993). The dipeptides are presumably pro- duced in glutathione (GSH) metabolism by y-glu- tamyl transpeptidase (y-GT), a membrane bound enzyme that, among related reactions, catalyzes the transfer of the y-glutamyl part of GSH (or Gin) to an extracellular acceptor amino acid (Hanes et al., 1952; Meister and Tate, 1976; Meisterand Anderson, 1983). The resulting y-glutamyl compound is transported to the cytoplasm of the cell (Sikka et al., 1982). This part of the so called y-glutamyl cycle has been proposed as *To whom all correspondenceshould be addressed. ,m, a complementary amino acid uptake system, par- ticularly in the kidney (Orlowski and Meister, 1970; Griffith et al., 1978; for discussion see Meister, 1989). In brain, y-GT is concentrated to the choroid plexus and blood capillaries, but is also found in cultured astroglial cells and neurons (Okonkwo et al., 1974; Orlowski et al., 1974; Orlowski and Karkowsky, 1976; Kvamme et al., 1985; Reichelt and Poulsen, 1992). Studies employing subfractionation and immu- nohistochemistry indicate a predominant but not exclusive glial localization of y-GT (Shine and Haber, 1981; Raps et al., 1989; Reichelt and Poulsen, 1992). Studies, examining the effects of y-GT inhibition in different brain preparations, are contradictory regard- ing a role of y-GT in uptake of extracellular amino acids such as Glu (Lisy et al., 1983; Johansen et al., 1987; Jankaskova et al., 1992; Zangerle et al., 1992). Similarly, the interplay between y-GT and extra-zyxwvutsrqpon 141