title: Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon-glia signaling.
abstract: 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 periaxonal 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 glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate 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 recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive 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 released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.
authors.affiliation: Department of Physiology, The Brody School of Medicine of East Carolina University, Greenville, NC 27858, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Urazaev
authors.forename: R M
authors.initials: RM
authors.lastname: Grossfeld
authors.forename: P L
authors.initials: PL
authors.lastname: Fletcher
authors.forename: H
authors.initials: H
authors.lastname: Speno
authors.forename: B S
authors.initials: BS
authors.lastname: Gafurov
authors.forename: J G
authors.initials: JG
authors.lastname: Buttram
authors.forename: E M
authors.initials: EM
authors.lastname: Lieberman
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D003400:Astacoidea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D002250:Carbon Radioisotopes / Q000378:metabolism; D002268:Carboxypeptidases / Q000187:drug effects / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004151:Dipeptides / Q000096:biosynthesis* / Q000378:metabolism / Q000493:pharmacokinetics; D004558:Electric Stimulation; D043425:Glutamate Carboxypeptidase II; D018698:Glutamic Acid / Q000378:metabolism / Q000493:pharmacokinetics; D005973:Glutamine / Q000378:metabolism; D009420:Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques; D016318:Quisqualic Acid / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014316:Tritium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D003470:Culture Media; D004151:Dipeptides; D005973:Glutamine; D014316:Tritium; C027172:isospaglumic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D016318:Quisqualic Acid; D002268:Carboxypeptidases; D043425:Glutamate Carboxypeptidase II