Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: Implications for axon-glia signaling

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of peria xonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of g lutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant ne rve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiol abeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel re lease was not altered by electrical stimulation of nerve cord loaded with [ C-14]glutamate by bath application or loaded with [14C]glutamate, [H-3]-D-a spartate or [H-3]NAAG by axonal injection. However, when radiolabeled gluta mate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total re covered radiolabel in control conditions. The stimulated increase in radioa ctive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolab eled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [3H]glutamate in the superfusion so lution of nerve incubated with [H-3]NAAG was reduced when beta -NAAG, a com petitive glutamate carboxypeptidase II (GCP II) inhibitor, was present. Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is rel eased from the axon and converted in part to glutamate by GCP Il. A quisqua late-and beta -NAAG-sensitive GCP II activity was detected in nerve cord ho mogenates. These results, together with those in the accompanying paper dem onstrating that NAAG can activate a glial electrophysiological response com parable to that initiated by glutamate, implicate NAAG as a probable mediat or of interactions between the MGA and its periaxonal glia. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.