Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

N-Acetyl-Aspartyl-Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method

Abstract: An isocratic HPLC method to measure endogenous N -acetyl-aspartyl-glutamate (NAAG) and N -acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi-lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.     

Simultaneous determination of N-acetylaspartic acid, N-acetylglutamic acid, and N-acetylaspartylglutamic acid in whole brain of 3-mercaptopropionic acid-treated rats using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

The measurement of N-acetylaspartic acid (NAA), N-acetylglutamic acid (NAG), and N-acetylaspartylglutamic acid (NAAG) in the whole brain of 3-mercaptopropionic acid (3-MPA)-treated rats has been developed using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. The recoveries of these compounds were 90.85 +/- 3.43% for NAA, 91.62 +/- 5.47% for NAG, and 92.29 +/- 4.44% for NAAG. The detection limits for NAA, NAG, and NAAG were 12, 15, and 20 microg/ml, respectively. After administration of 3-MPA, the concentrations of NAA, NAG, and NAAG in the whole brain over 10 min increased 177.25, 134.23, and 127.70%, respectively. These concentrations then decreased over the next 60 min. The simultaneous determination of NAA, NAG, and NAAG using this method was found to be very useful for studies of metabolism of NAA, NAG, and NAAG in biological samples.     

Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

Simultaneous determination method of N-acetyl-l-aspartyl-l-glutamate (NAAG), an endogenous agonist at type 3 metabotropic glutamate receptor, and its degradation product, N-acetyl-l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7+/-0.30 and 2.1 x 10(2)+/-9.2 nmol/mg protein, respectively (n=6), while those in the hippocampus were 6.8+/-0.48 and 1.9 x 10(2)+/-8.5 nmol/mg protein, respectively (n=5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats (p<0.01).     

Functions of N-acetyl-L-aspartate and N-acetyl-L-aspartylglutamate in the vertebrate brain: role in glial cell-specific signaling

N-Acetyl-L-aspartate (NAA) and its derivative N-acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and L-glutamate, hydrolyzed to NAA and L-glutamate by astrocytes, and further hydrolyzed to L-aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell-specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.     

Effects evoked by pentamethylenetetrazol-induced seizures upon N-acetylaspartate and N-acetylaspartylglutamate levels in different regions of the rat neuraxis

We have examined the effects evoked by pentamethylenetetrazol (PTZ)-induced seizures upon the concentration of N-acetylaspartate (NAA) and of N-acetylaspartylglutamate (NAAG) in the forebrain, brainstem and spinal cord of rats. We observed a significant decrease of both NAA and NAAG in each one of the studied regions. These findings are consistent with an inhibitory role proposed in CNS for NAA and NAAG.     

Distinctive response of CNS glial cells in oro-facial pain associated with injury, infection and inflammation

Background

The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is widely expressed throughout the vertebrate nervous system, including the pain processing neuraxis. Inhibitors of NAAG peptidases are analgesic in animal models of pain. However, the brain regions involved in NAAG's analgesic action have not been rigorously defined. Group II metabotropic glutamate receptors (mGluR2/3) play a role in pain processing in the laterocapsular part of the central nucleus of the amygdala (CeLC). Given the high concentration of NAAG in the amygdala and its activation of group II mGluRs (mGluR₃ > mGluR₂), this study was undertaken using the mouse formalin model of inflammatory pain to test the hypothesis that NAAG influences pain processing in the amygdala. Evoked excitatory postsynaptic currents (eEPSCs) were studied in neurons in the CeLC of mouse brain slices following stimulation of the spinoparabrachial amygdaloid afferents.

Results

Application of a NAAG peptidase inhibitor, ZJ43, dose dependently inhibited the amplitude of the eEPSCs by up to 50% in control CeLC demonstrating the role of NAAG in regulation of excitatory transmission at this synapse. A group II mGluR agonist (SLx-3095-1) similarly inhibited eEPSC amplitude by about 30%. Both effects were blocked by the group II mGluR antagonist LY341495. ZJ43 was much less effective than SLx in reducing eEPSCs 24 hours post inflammation suggesting an inflammation induced reduction in NAAG release or an increase in the ratio of mGluR₂ to mGluR₃ expression. Systemic injection of ZJ43 proximal to the time of inflammation blocked peripheral inflammation-induced increases in synaptic transmission of this pathway 24 hrs later and blocked the induction of mechanical allodynia that developed by this time point.

Conclusions

The main finding of this study is that NAAG and NAAG peptidase inhibition reduce excitatory neurotransmission and inflammation-induced plasticity at the spinoparabrachial synapse within the pain processing pathway of the central amygdaloid nucleus.     

Comparison of Inter Subject Variability and Reproducibility of Whole Brain Proton Spectroscopy

The aim of these studies was to provide reference data on intersubject variability and reproducibility of metabolite ratios for Choline/Creatine (Cho/Cr), N-acetyl aspartate/Choline (NAA/Cho) and N-acetyl aspartate/Creatine (NAA/Cr), and individual signal-intensity normalised metabolite concentrations of NAA, Cho and Cr. Healthy volunteers underwent imaging on two occasions using the same 3T Siemens Verio magnetic resonance scanner. At each session two identical Metabolic Imaging and Data Acquisition Software (MIDAS) sequences were obtained along with standard structural imaging. Metabolite maps were created and regions of interest applied in normalised space. The baseline data from all 32 volunteers were used to calculate the intersubject variability, while within session and between session reproducibility were calculated from all the available data. The reproducibility of measurements were used to calculate the overall and within session 95% prediction interval for zero change. The within and between session reproducibility data were lower than the values for intersubject variability, and were variable across the different brain regions. The within and between session reproducibility measurements were similar for Cho/Cr, NAA/Choline, Cho and Cr (11.8%, 11.4%, 14.3 and 10.6% vs. 11.9%, 11.4%, 13.5% and 10.5% respectively), but for NAA/Creatine and NAA between session reproducibility was lower (9.3% and 9.1% vs. 10.1% and 9.9%; p <0.05). This study provides additional reference data that can be utilised in interventional studies to quantify change within a single imaging session, or to assess the significance of change in longitudinal studies of brain injury and disease.     

Dynamic or inert metabolism? Turnover of N‐acetyl aspartate and glutathione from d‐[1‐13C]glucose in the rat brain in vivo

The rate of (13)C-label incorporation into both aspartyl (NAA C3) and acetyl (NAA C6) groups of N-acetyl aspartate (NAA) was simultaneously measured in the rat brain in vivo for up to 19 h of [1-(13)C]glucose infusion (n = 8). Label incorporation was detected in NAA C6 approximately 1.5 h earlier than in NAA C3 because of the delayed labeling of the precursor of NAA C3, aspartate, compared to that of NAA C6, glucose. The time courses of NAA were fitted using a mathematical model assuming synthesis of NAA in one kinetic compartment with the respective precursor pools of aspartate and acetyl coenzyme A (acetyl-CoA). The turnover rates of NAA C6 and C3 were 0.7 +/- 0.1 and 0.6 +/- 0.1 micromol/(g h) with the time constants 14 +/- 2 and 13 +/- 2 h, respectively, with an estimated pool size of 8 micromol/g. The results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. Several hours after starting the glucose infusion, label incorporation into glutathione (GSH) was also detected. The turnover rate of GSH was 0.06 +/- 0.02 micromol/(g h) with a time constant of 13 +/- 2 h. The estimated pool size of GSH was 0.8 micromol/g, comparable to the cortical glutathione concentration. We conclude that NAA and GSH are completely turned over and that the metabolism is extremely slow (< 0.05% of the glucose metabolic rate).     

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.     

N-Acetylaspartylglutamate Selectively Inhibits Neuronal Responses to N-Methyl-d-Aspartic Acid In Vitro

Abstract: Canavan's disease is an autosomal recessive disorder characterized by a deficiency of aspartoacylase and accumulation of N -acetylaspartic acid (NAA), leading to a severe leukodystrophy and spongy degeneration of the brain. N -Acetylaspartylglutamate (NAAG), the presumed product of NAA, also accumulates in this disease. The endogenous dipeptide NAAG has been suggested to have low potency at NMDA receptors. Here we have tested the actions of NAAG and NAA on NMDA-evoked responses in cultured cerebellar granule cells. In differentiating granule cells grown in low-K⁺ medium, NAAG negated the survival-promoting effects of NMDA but not K⁺ depolarization. Neither NAAG nor NAA alone promoted cell survival in low-K⁺ medium. The modest trophic action of 50 µ M kainic acid in low-K⁺ medium was reinforced by the NMDA receptor antagonist dizocilpine maleate and by NAAG. In K⁺-differentiated granule cells, NAAG raised the threshold of NMDA neurotoxicity but not that of kainate. The observed activities of NAAG were overcome by excess NMDA and were not mimicked by NAA. These data raise the possibility that disruption of NMDA receptor processes by NAAG may be of pathophysiological relevance.     

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N-acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N-acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon-glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58-83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLUR(II). No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.     

Production and characterization of monoclonal antibodies to N-acetyl-aspartyl-glutamate

N-acetyl-aspartyl-glutamate (NAAG) is a putative neuromodulator/neurotransmitter in the mammalian nervous system. Immunohistochemical studies with polyclonal NAAG antisera have revealed immunoreactive neurons and processes in several brain regions. However, these antisera crossreact to some degree with N-acetyl-aspartate (NAA), which is present in mM concentrations in brain, prompting the development of monoclonal antibodies (MAb) more specific for NAAG. By fusing spleen lymphocytes obtained from BALB/c mice pre-immunized with NAAG covalently linked to bovine serum albumin by carbodiimide with SP2/0-Ag 14 mouse myeloma cells, we produced three IgG2a (kappa) MAb which specifically reacted with NAAG. These MAb exhibited negligible crossreactivity with NAA or with structurally similar peptides, as shown by solid-phase radioimmunoassay. Antibody activity was absorbed out selectively by both NAAG-thyroglobulin conjugate and free NAAG. These MAb stained many nuclei of the medulla-pons and midbrain, mitral cells in the olfactory bulb, pyramidal neurons in sensorimotor cortex, locus ceruleus, and several cholinergic cranial nuclei. The staining pattern strongly correlated with NAAG levels determined by HPLC. Monoclonal antibodies significantly enhanced sensitivity of staining, allowing visualization of dorsal horn neurons in spinal cord, which were not readily detectable with polyclonal antiserum. Availability of these MAb now facilitates further clarification of the role of NAAG in the brain.     

Magnetic resonance spectroscopy: an in vivo tool for monitoring cerebral injury in SIV-infected macaques

The purpose of our study was to demonstrate the feasibility of using in vivo proton Magnetic Resonance Spectroscopy (MRS) to monitor the brain manifestations of SIV infection in the macaque model of AIDS. Previous spectroscopy work on macaque brain tissue and in vivo work in humans is reviewed to provide the motivation and context for this study. We collected 34 MRS data sets on 14 uninfected rhesus macaques. From this data, we demonstrate that we are capable of detecting changes similar to those observed in human MRS studies for most metabolites using less than 10 animals. The juvenile macaques utilized in this study demonstrate age-related changes in the levels of N-acetyl aspartate (NAA), a neuronal marker. The quantity and distribution of neurochemicals in the macaque are found to be slightly, but significantly, different than in the human.     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

Localization and Transport of N-Acetylaspartylglutamate in Cells of Whole Murine Brain in Primary Culture

Abstract: N -Acetylaspartylglutamate (NAAG) is the most abundant neuropeptide in the mammalian nervous system. Considerable data support the hypothesis that NAAG is synaptically released in a manner consistent with neurotransmission. Primary murine brain cultures containing neurons and glia expressed 1.2-3.5 nmol of NAAG/mg of protein. In contrast to conclusions drawn from immunohistochemistry, pure glial cultures also expressed high levels of NAAG (0.6-2.11 nmol/mg of protein). These data suggest that although a subpopulation of neurons contains very high NAAG levels, micromolar concentrations of the peptide also are present in glia. Both culture types demonstrated robust extracellular peptidase activity when incubated with NAAG, as well as peptide transport. Uptake of [³H]NAAG was both temperature and sodium dependent, yet relatively insensitive to the presence of extracellular glutamate. These results indicate that synaptically released NAAG, as well as that which may be released from glia, is removed from the extracellular space by direct uptake as well as the robust enzymatic degradation of the peptide. A kinetic analysis of this NAAG transport (estimated K _m= 1.8 μ M ) suggests a high-affinity NAAG transport system. The balance of the two processes of direct peptide uptake and peptide hydrolysis would markedly influence the sequence of receptor-mediated events that follow NAAG release.     

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.     

Competitive Inhibition of N-Acetylated-α-Linked Acidic Dipeptidase Activity by N-Acetyl-L-Aspartyl-β-Linked L-Glutamate

The endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-alpha-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAALADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu-3H]NAAG were determined first (Km = 0.40 +/- 0.05 microM; Vmax = 155 +/- 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki = 1.9 +/- 0.3 microM), and competitively by N-acetyl-L-aspartyl-beta-linked L-glutamate (beta-NAAG; Ki = 0.70 +/- 0.05 microM). To determine whether glutamate-containing dipeptides, such as NAAG, beta-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-gamma-linked L-glutamate, were substrates of NAALADase, rat brain membranes were immobilized on a C-8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o-phthaldialdehyde/reverse-phase HPLC detection procedure. beta-NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and beta-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.     

Molecular Characterization of N-Acetylaspartylglutamate Synthetase

The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system.