European Journal of Pharmacology, 158 (1988) 263-266 Elsevier EJP 20264 Short communication Depolarization-evoked release of N-acetyl-L-aspartyl-L-glutamate from rat brain synaptosomes Anna Pittaluga, Luis Barbeito, Valrrie Serval, Grrard Godeheu, Fran~oise Artaud, Jacques Glowinski and Andr6 Chrramy * Collbge de France, I N S E R M U 114, Chaire de Neuropharmacologie, 11 place Marcelin Berthelot, 75231 Paris cedex 05, France Received 5 September 1988, accepted 25 October 1988 263 Depolarization by potassium and veratridine stimulated the release of N-acetyl-L-aspartyl-L-glutamate from crude synaptosomes prepared from the rat mesencephalon and diencephalon. The potassium-evoked release of N-acetyl-L- aspartyl-L-glutamatewas calcium-dependent and the stimulatory effect of veratridine was prevented by tetrodotoxin. N-Acetyl-L-aspartyl-L-glutamate; Mesencephalon; Diencephalon; Synaptosomes; (In vitro release, Rat) 1. Introduction N-Acetyl-L-aspartyl-L-glutamate(NAAG) is an endogenous peptide that is unevenly distributed in the brain and which has been shown immunohis- tochemically to be present in the cells of several neuronal pathways (Anderson et al., 1986; Tieman et al., 1987). Complementary studies have sug- gested that NAAG could act as a neurotrans- mitter. NAAG levels are decreased in the striatum or the pyriform cortex following decortication or bulbectomy, respectively (Ffrench-Mullen et al., 1985; Koller et al., 1984). A peptidase that cleaves NAAG into glutamate and N-acetyl-aspartate has been found in the brain; this peptidase is selec- tively inhibited by quisqualate (Robinson et al., 1986). The existence of specific [3H]NAAG bind- ing sites has been proposed (Koller and Coyle, 1985), although this conclusion could be chal- lenged, since the binding experiments were per- formed under conditions in which 3H-NAAG could be rapidly cleaved to [3H]glutamate which could be subsequently bound or transported. Elec- trophysiological evidence to support a neurotrans- mitter role for NAAG has been also obtained in the pyriform cerebral cortex, where the excitatory effects of both NAAG and activation of the lateral olfactory tract were blocked selectively by D,L-2- amino-4-phosphonobutyrate (Ffrench-Mullen et al., 1985). In addition, excitatory responses to NAAG which were blocked by N-methyl-D- aspartate antagonists have also been shown in other brain structures (Sekiguchi et al., 1987). To our knowledge, however, no information is yet available concerning the release of NAAG. In the present study we demonstrate, using crude synaptosomes from the rat mesencephalon and diencephalon, that NAAG is released upon potassium or veratridine depolarization in a calcium-dependent and tetrodotoxin (TTX)-sensi- tive manner, respectively. 2. Materials and methods * To whom all correspondence should be addressed. Crude synaptosomes (P2 fraction) were pre- pared from the mesencephalon and diencephalon 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)