NAAG peptidase inhibitors block cognitive deficit induced by MK-801 and motor activation induced by d-amphetamine in animal models of schizophrenia.

The most widely validated animal models of the positive, negative and cognitive symptoms of schizophrenia involve administration of d-amphetamine or the open channel NMDA receptor blockers, dizocilpine (MK-801), phencyclidine (PCP) and ketamine. The drug ZJ43 potently inhibits glutamate carboxypeptidase II (GCPII), an enzyme that inactivates the peptide transmitter N-acetylaspartylglutamate (NAAG) and reduces positive and negative behaviors induced by PCP in several of these models. NAAG is an agonist at the metabotropic glutamate receptor 3 (mGluR3). Polymorphisms in this receptor have been associated with expression of schizophrenia. This study aimed to determine whether two different NAAG peptidase inhibitors are effective in dopamine models, whether their efficacy was eliminated in GCPII knockout mice and whether the efficacy of these inhibitors extended to MK-801-induced cognitive deficits as assessed using the novel object recognition test. ZJ43 blocked motor activation when given before or after d-amphetamine treatment. (R,S)-2-phosphono-methylpentanedioic acid (2-PMPA), another potent NAAG peptidase inhibitor, also reduced motor activation induced by PCP or d-amphetamine. 2-PMPA was not effective in GCPII knockout mice. ZJ43 and 2-PMPA also blocked MK-801-induced deficits in novel object recognition when given before, but not after, the acquisition trial. The group II mGluR antagonist LY341495 blocked the effects of NAAG peptidase inhibition in these studies. 2-PMPA was more potent than ZJ43 in a test of NAAG peptidase inhibition in vivo. By bridging the dopamine and glutamate theories of schizophrenia with two structurally different NAAG peptidase inhibitors and demonstrating their efficacy in blocking MK-801-induced memory deficits, these data advance the concept that NAAG peptidase inhibition represents a potentially novel antipsychotic therapy.

Ribozyme-mediated reduction of the GABA(A) receptor alpha1 subunit.

As an approach to understanding the role of the alpha1 subunit of the GABA(A) receptor, ribozymes were designed to reduce expression of this subunit protein by hydrolysis of alpha1 subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all known murine GABA(A) receptor subunits at the amino acid and nucleotide sequence level. Two ribozymes were designed and synthesized: one against the extracellular domain and the other against the cytoplasmic domain. These ribozymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro transcribed mRNA. The stable cell line, 122, expressing recombinant human GABA(A) alpha1, beta2 and gamma2S subunits of receptor was stably transfected with the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55-60% reduction of alpha1 mRNA in clones of cells transfected with either the single ribozyme (Cy) or with both ribozymes (EC). The alpha1 protein level was reduced 75% in a stable Cy clone and more than 90% in a stable EC clone when compared with alpha1 expression in 122 cells and the vector transfected (Zeo) cells. Electrophysiological analysis revealed that the GABA(A) receptor properties were very similar in 122 cells and in stable clones in which the subunit protein expression had been greatly reduced. No significant difference was detected in the potentiation of the receptor response by either bretazenil or zolpidem. These data demonstrate the efficacy of the ribozyme approach in dramatically reducing GABA(A) subunit protein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice.

beta-NAAG rescues LTP from blockade by NAAG in rat dentate gyrus via the type 3 metabotropic glutamate receptor.

N-Acetylaspartylglutamate (NAAG) is an agonist at the type 3 metabotropic glutamate receptor (mGluR3), which is coupled to a Gi/o protein. When activated, the mGluR3 receptor inhibits adenylyl cyclase and reduces the cAMP-mediated second-messenger cascade. Long-term potentiation (LTP) in the medial perforant path (MPP) of the hippocampal dentate gyrus requires increases in cAMP. The presence of mGluR3 receptors and NAAG in neurons of the dentate gyrus suggests that this peptide transmitter may inhibit LTP in the dentate gyrus. High-frequency stimulation (100 Hz; 2 s) of the MPP resulted in LTP of extracellularly recorded excitatory postsynaptic potentials at the MPP-granule cell synapse of rat hippocampal slices. Perfusion of the slice with NAAG (50 and 200 microM) blocked LTP. Neither 50 nor 200 microM NAAG produced N-methyl-D-aspartate receptor currents in the granule cells of the acute hippocampal slice. The group II mGluR antagonist ethyl glutamate (100 microM) and a structural analogue of NAAG, beta-NAAG (100 microM), prevented the blockade of LTP by NAAG. Paired-pulse depression of the excitatory postsynaptic potential at 20- and 80-ms interpulse intervals (IPI) was not affected by NAAG or beta-NAAG. beta-NAAG did not affect inositol trisphosphate production stimulated by the agonist glutamate in cells expressing the group I mGluR1alpha or mGluR5. beta-NAAG blocked the decrease in forskolin-stimulated cAMP by the group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) but not the group III mGluR agonist L(+)-2-amino-4-phosphonobutyric acid in cerebellar granule cells. In cells transfected with mGluR3, but not mGluR2, beta-NAAG blocked forskolin-stimulated cAMP responses to glutamate, NAAG, the nonspecific group I, II agonist trans-ACPD, and the group II agonist DCG-IV. We conclude that beta-NAAG is a selective mGluR antagonist capable of differentiating between mGluR2 and mGluR3 subtypes and that the mGluR3 receptor functions to regulate activity-dependent synaptic potentiation in the hippocampus.

NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance.

The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) where it acts to decrease cAMP levels. Rat cortical interneurons express both NAAG and glutamic acid decarboxylase, as well as mGluR3 mRNA. In the presence of ionotropic glutamate receptor antagonists, both NAAG and the group II metabotropic glutamate receptor agonist, DCG-IV, reduced the calcium-dependent, KCl-induced [(3)H]-GABA release from rat cortical neurons by 35%. This release process was unaffected by tetrodotoxin. The group II antagonist, ethyl glutamate, reversed the effects of DCG-IV and NAAG. The mGluR3-selective antagonist, beta-N-acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl-stimulated [(3)H]-GABA-release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H-89, decreased [(3)H]-GABA release while NAAG produced no additional inhibition in the presence of H-89. In contrast, the protein kinase C inhibitor, Ro 31--8220, had no effect on KCl-stimulated release, nor did it affect the inhibition of release produced by NAAG. The L-type calcium channel blocker, nifedipine, also inhibited the release of [(3)H]-GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl-stimulated [(3)H]-GABA release by NAAG is mediated via presynaptic mGluR3 on GABAergic cortical neurons and that this effect is obtained by decreasing cAMP with a consequent decrease in protein kinase A activity and L-type calcium channel conductance.

N-Acetylaspartylglutamate: the most abundant peptide neurotransmitter in the mammalian central nervous system.

In the progress of science, as in life, timing is important. The acidic dipeptide, N-acetylaspartylglutamate (NAAG), was discovered in the mammalian nervous system in 1965, but initially was not considered to be a neurotransmitter candidate. In the mid-1980s, a few laboratories revisited the question of NAAG's role in the nervous system and pursued hypotheses regarding its function that ranged from a precursor for the transmitter pool of glutamate to a direct role as a peptide transmitter. Since that time, NAAG has been tested against nearly all of the established criteria for identification of a neurotransmitter. It successfully meets each of these tests, including a concentrated presence in neurons and synaptic vesicles, release from axon endings in a calcium-dependent manner following initiation of action potentials, and extracellular hydrolysis by membrane-bound peptidase activity. NAAG is the most prevalent and widely distributed neuropeptide in the mammalian nervous system. NAAG activates NMDA receptors with a low potency that may vary among receptor subtypes, and it is a highly selective agonist at the type 3 metabotropic glutamate receptor (mGluR3). Acting through this receptor, NAAG reduces cyclic AMP levels, decreases voltage-dependent calcium conductance, suppresses excitotoxicity, influences long-term potentiation and depression, regulates GABA(A) receptor subunit expression, and inhibits synaptic release of GABA from cortical neurons. Cloning of peptidase activities against NAAG provides opportunities to study the cellular and molecular mechanisms by which synaptic NAAG peptidase activity is controlled. Given the codistribution of this peptide with a spectrum of traditional transmitters and its ability to activate mGluR3, we speculate that one role for NAAG following synaptic release is the activation of metabotropic autoreceptors that inhibit subsequent transmitter release. A second role is the production of extracellular glutamate following NAAG hydrolysis.

GABA induces proliferation of immature cerebellar granule cells grown in vitro.

The presence of GABA and its receptors early in rodent nervous system development has lead to speculation on the role of this transmitter system in neuroblast proliferation, migration and differentiation. We studied the effect of GABA and GABA agonists on immature cerebellar granule cell proliferation and survival. Cerebellar granule cell suspensions were obtained from 6-8-day-old rats and grown in culture for up to 7 days in serum-containing or serum-free medium. The addition of GABA (0.1-100 microM) or muscimol (0.01-10 microM) 2 h after inoculation and harvested 22 h later, lead to an increase in 3H-thymidine incorporation over control samples with the correspondent increase in granule cells number assayed 48 h later. The effect on cell proliferation exerted by GABAA agonists was blocked by MgCl2 and nifedipine, as well as by the chloride channel blocker, picrotoxin (50 microM), and the GABAA receptor specific blocker, bicuculline (50 microM). The increase on cell proliferation induced by GABA also was blocked by PD98059 (75 microM), a specific inhibitor of the mitogen-activated protein kinase kinase (MAPKK). GABAA receptor-mediated proliferation was consistently seen in cells inoculated in serum-containing medium supplemented with 25 mM KCl but not seen in serum-free medium, with 5 mM or 25 mM KCl. The presence of serum did not enhance the survival of cerebellar granule cells grown for 7 days in either 5 mM or 25 mM KCl. Additionally, neither GABA nor muscimol applied from day 2 to day 7 in vitro affected cell survival in any culture condition. We conclude that GABA and GABAA receptor agonists influence granule cell proliferation but not survival and that this effect is mediated by a calcium influx via voltage-dependent calcium channel activation, with a subsequent activation of the MAPK cascade.

N-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytes.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide in the mammalian nervous system. NAAG meets the traditional criteria of a neurotransmitter, including localization in synaptic vesicles, depolarization-induced release, low potency activation of some N-methyl-D-aspartate receptors, and highly selective activation of a cAMP-coupled metabotropic glutamate receptor (mGluR) with potency approaching that of glutamate. The peptide is present in cultured cortical glia in high concentration and is hydrolyzed by cell surface peptidase activity. In the present work, we tested the hypothesis that NAAG selectively activates a subclass of metabotropic receptors on cultured rat cerebellar glia, primarily astrocytes. These glial cells express mRNA for mGluR subtypes 1, 3, 4, 5, 6, 7, and 8. We were not able to detect message for mGluR2 in these cells using the reverse transcriptase-polymerase chain reaction. Cerebellar glia responded to NAAG, glutamate, and trans-ACPD with a decrease in forskolin-stimulated cAMP formation. AP4, an agonist of the group III receptors mGluR4, mGluR6, mGluR7, and mGluR8, had little or no effect on stimulated cAMP levels. Treatment with low micromolar NAAG significantly increased uptake of radioactive thymidine by cultured astrocytes through activation of metabotropic glutamate receptors. Antagonists of group II mGluRs prevented the decrease in cAMP and the increase in uptake of thymidine by NAAG. Cultured cerebellar astrocytes expressed 20 pmol NAAG per mg protein, a value that is at least 30-fold lower than that expressed by mixed glial cultures prepared from mouse cortex. We conclude that cerebellar astrocytes respond to NAAG via the mGluR3 receptor and that the peptide may selectively activate this receptor subtype in astrocytes following release from neurons or glia.

Molecular cloning of a peptidase against N-acetylaspartylglutamate from a rat hippocampal cDNA library.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate-specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752-amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by beta-NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5' end, the central region, and the 3' end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5' end of the hippocampal cDNA.

N-acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABA(A) alpha6 subunit in cerebellar granule cells.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells.

In previous studies, we demonstrated that the neuropeptide, N-acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1-6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD) and L-2-amino-4-phosphonobutyrate (L-AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans-ACPD, activated each of the transfected receptors, whereas L-AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans-ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.

Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings.

With the use of the single-cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to alpha 1 and alpha 6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.

Interactions between N-acetylaspartylglutamate and AMPA, kainate, and NMDA binding sites.

The structure of N-acetylaspartylglutamate (NAAG) suggests this neuronal dipeptide as a candidate for interaction with discrete subclasses of ionotropic and metabotropic acidic amino acid receptors. A substantial difficulty in the assay of these interactions is posed by membrane-bound peptidase activity that converts the dipeptide to glutamate and N-acetylaspartate, molecules that will interfere with receptor assays. We have developed two sets of unique receptor assay conditions and applied one standard assay to measure the interactions, under equilibrium binding conditions, of [3H]kainate, [3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), and [3H]CGS-19755 with the three classes (kainate, quisqualate, and N-methyl-D-aspartate) of ionotropic glutamate receptors, while inhibiting peptidase activity against NAAG. Under these conditions, NAAG exhibits apparent inhibition constants (IC50) of 500, 790, and 8.8 microM in the kainate, AMPA, and CGS-19755 receptor binding assays, respectively. Glutamate was substantially more effective and less specific in these competition assays, with inhibition constants of 0.36, 1.1, and 0.37 microM. These data support the hypothesis that, relative to glutamate, NAAG functions as a specific, low potency agonist at N-methyl-D-aspartate subclass of ionotropic acidic amino acid receptors, but the peptide is not likely to activate directly the kainate or quisqualate subclasses of excitatory ionotropic receptors under physiologic conditions.

Comparative distribution of N-acetylaspartylglutamate and GAD67 in the cerebellum and precerebellar nuclei of the rat utilizing enhanced carbodiimide fixation and immunohistochemistry.

The most prevalent peptide in the nervous system, N-acetylaspartylglutamate (NAAG), specifically activates N-methyl D-aspartate (NMDA) receptors and a subclass of metabotropic glutamate receptors. One action of this peptide may be to modulate the release of other neurotransmitters, including gamma-aminobutyric acid (GABA). The present study describes the cellular distribution of NAAG, relative to GABA, in the cerebellum and precerebellar nuclei as a foundation for further physiological investigations. Numerous cells of origin for mossy fibers, including many of the larger neurons of the pontine nuclei, lateral reticular nuclei, vestibular nuclei, reticulotegmental nuclei, and spinal grey, were moderately to strongly stained for NAAG. Many NAAG-labeled fibers were clearly visible in the cerebellar peduncles and central white matter. Mossy fibers and mossy endings were among the most prominent NAAG-immunoreactive elements in the cerebellar cortex. Most neurons in the inferior olive were not stained for NAAG, and only sparse, lightly immunoreactive, climbing fiber-like endings could be identified in restricted regions of the cortical molecular layer. Purkinje neurons ranged from nonreactive to moderately positive, with the great majority being unstained. Cerebellar granule cells did not exhibit any NAAG immunoreactivity. A population of neurons in the deep cerebellar nuclei was highly immunoreactive for NAAG. Additionally, many neurons of the red nucleus were intensely stained for NAAG. Comparisons with staining for the 67 kD form of glutamic acid decarboxylase in serial sections revealed complementary distributions, with NAAG in excitatory pathways and cell groups, and glutamic acid decarboxylase in inhibitory systems. These findings suggest a significant functional involvement of NAAG in the excitatory afferent and efferent projection systems and provide an anatomical basis for investigations into the interactions of NAAG and GABA in the cerebellum.

The regional distribution of N-acetylaspartylglutamate (NAAG) and peptidase activity against NAAG in the rat nervous system.

N-Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N-acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N-acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was > 0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.

N-acetylaspartylglutamate inhibits forskolin-stimulated cyclic AMP levels via a metabotropic glutamate receptor in cultured cerebellar granule cells.

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) fulfills several of the criteria for classification as a neurotransmitter including localization in synaptic vesicles, calcium-dependent release after neuronal depolarization, and low potency activation of N-methyl-D-aspartate receptors. In the present study, the influence of NAAG on metabotropic receptor activation in cerebellar granule cells was examined in cell culture. Stimulation of granule cell adenylate cyclase with forskolin increased cyclic AMP (cAMP) several hundredfold above basal levels within 10 min in a concentration-dependent manner. Although glutamate, NAAG, and the metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid did not alter the low basal cAMP levels, the application of 300 microM glutamate or NAAG or trans-1-amino-1,3-cyclopentanedicarboxylic acid reduced forskolin-stimulated cAMP in granule cells by 30-50% in the absence or presence of inhibitors of ionotropic acidic amino acid receptors, as well as 2-amino-4-phosphonobutyrate. No additivity in the inhibition of cAMP was found when 300 microM NAAG and trans-1-amino-1,3-cyclopentanedicarboxylic acid were coapplied. The beta-analogue of NAAG failed to reduce cAMP levels. Similar effects of NAAG and glutamate were obtained under conditions of inhibition of phosphodiesterase activity and were prevented by pretreatment of the cells with pertussis toxin. These data are consistent with the activation by NAAG of a metabotropic acidic amino acid receptor coupled to an inhibitory G protein. In contrast, the metabotropic acidic amino acid receptor coupled to phosphoinositol turnover in these cells was not activated by NAAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and transport of N-acetylaspartylglutamate in cells of whole murine brain in primary culture.

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 Km = 1.8 microM) 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.

N-acetylaspartylglutamate catabolism is achieved by an enzyme on the cell surface of neurons and glia.

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific, acidic dipeptide which is released from neurons in a manner consistent with a function in synaptic neurotransmission. The hydrolysis of NAAG to produce glutamate and N-acetylaspartate was analyzed in cell cultures prepared from murine brain cells. Peptidase activity against NAAG was found in cultures which contained both neurons and glia, as well as in cultures of glia alone. Several lines of evidence were obtained in support of the hypothesis that this peptidase activity is predominantly bound to the extracellular face of the plasma membranes of these cells. Glutamate released from NAAG accumulated in the extracellular medium. Extracellular application of peptidase inhibitors effectively reduced NAAG hydrolysis. Peptidase activity was not secreted into the cell culture medium by intact cells, and lysed cells did not release detectable peptidase activity beyond that obtained with intact cells. Replacement of extracellular sodium with choline inhibited peptide uptake while stimulating apparent extracellular NAAG hydrolysis by intact cells in culture. Finally, the steady rise in extracellular glutamate as a consequence of NAAG hydrolysis by these brain cells, including glia, supports the conclusion that glutamate uptake is not tightly coupled to peptidase activity and thus that NAAG serves as a significant source of glutamate in the synaptic space following depolarization-induced peptide release.

Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain.

To improve carbodiimide-based immunohistochemistry, carbodiimide-mediated coupling of radiolabeled N-acetylaspartylglutamate (NAAG) to bovine serum albumin was assayed in vitro. Various perfusion protocols, based on assay results, were tested for their ability to improve the immunohistochemical localization of two nervous system-specific molecules, NAAG and N-acetylaspartate (NAA) in the spinal cord, medulla, hippocampus, and cerebral cortex of the rat. Coupling of [3H]-NAAG to BSA in vitro was optimal with 100 mM carbodiimide and 1 mM N-hydroxysuccinimide in water at 37 degrees C. Optimal fixation of tissue was defined as permitting the identification of the NAAG and NAA in neuronal somata, dendritic arborizations, fine axons, and synaptic terminals with minimal diffuse background immunoreactivity. These conditions were obtained at 37 degrees C with 6% carbodiimide, 1 mM N-hydroxysuccinimide, and 5% dimethylsulfoxide perfused transcardially. Strong NAAG and NAA immunoreactivities were co-distributed in the majority of neurons in the spinal cord. Large-diameter spinal sensory afferents were stained for NAAG in the dorsal horn. The dorsal column nuclei were immunoreactive for NAAG and NAA, but only NAA staining was observed in the nucleus of the solitary tract. In cerebral cortex and hippocampus, NAAG and NAA immunoreactivities appeared to be exclusive, with NAAG staining observed in interneurons throughout all cortical layers, and NAA immunoreactivity present in most pyramidal neurons.