Barbara Wroblewska

N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells

Abstract: 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.

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 NAAGs 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 GABAA 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.

Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery

1 The effect of oestradiol 17β on vascular smooth muscle proliferation was examined in segments of the pig left anterior descending coronary artery (LAD). It was established by cytochemical techniques that out‐growth from the segments was composed of vascular smooth muscle cells. 2 [3H]‐thymidine uptake by pig LAD segments was used as an index of vascular smooth muscle cell proliferation. Nitroprusside and forskolin significantly inhibited [3H]‐thymidine uptake and were used as positive controls. 3 Oestradiol 17β (180–360 nm) inhibited thymidine uptake by pig LAD segments (P < 0.05). The inhibition was observed only in the absence of phenol red, which is a weak oestrogen receptor agonist. The anti‐oestrogens tamoxifen and its more potent metabolite 4‐hydroxytamoxifen, both of which are partial oestrogen receptor agonists, also significantly inhibited thymidine uptake. However, pretreatment with either tamoxifen or 4‐hydroxytamoxifen did not signficantly block oestradiol 17β‐induced inhibition of thymidine uptake. 4 The LAD segments bound [3H]‐oestradiol 17β in a time‐dependent manner and about 20 to 30% was displaced by an excess of unlabelled oestradiol 17β. Autoradiography showed [3H]‐oestradiol 17β was evenly distributed in the cytosol and nuclei of cells in the three layers of the vessel wall. 5 The data suggest that oestradiol 17β inhibits smooth muscle cell proliferation in porcine LAD segments, possibly through an oestrogen receptor mechanism. This in vitro effect suggests an in vivo role for oestradiol 17β in directly protecting coronary arteries against myointimal proliferation in premenopausal women.

NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR.

Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N‐acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea‐based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (Ki = 0.8 nm) and III (Ki = 23 nm) that deactivate NAAG following synaptic release. ZJ43 (100 µm) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP‐induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co‐administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP‐induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.

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. GLIA 24:172–179,1998.

N-Acetylaspartylglutamate Inhibits Forskolin-Stimulated Cyclic AMP Levels via a Metabotropic Glutamate Receptor in Cultured Cerebellar Granule Cells

Abstract: 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 gluta‐mate, NAAG, and the metabotropic receptor agonist frans‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid did not alter the low basal cAMP levels, the application of 300 μM 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 μM NAAG and trans‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid were coapplied. The β‐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. Granule cells in culture expressed very low levels of extracellular peptidase activity against NAAG, converting to glutamate <0.1% of the 10 μM through 1 mM NAAG applied to these cells during 15‐min in vitro assays.

Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model

The peptide neurotransmitter N‐acetylaspartylglutamate (NAAG) acts as an agonist at group II metabotropic glutamate receptors (mGluRs). NAAG is inactivated by extracellular peptidase activity yielding glutamate and N‐acetylaspartate. We recently developed a series of potent NAAG peptidase inhibitors, including ZJ‐11, ZJ‐17 and ZJ‐43. In the present study, we examined the effects of intrathecally administered ZJ‐11 and ZJ‐17 and intravenously administered ZJ‐11 and ZJ‐43 in the rat formalin test (an inflammatory pain model) and in the rat partial sciatic nerve ligation model (a neuropathic pain model). Intrathecal injection of ZJ‐11 or ZJ‐17 or intravenous injection of ZJ‐11 or ZJ‐43 suppressed both phases of the agitation behaviour induced by paw formalin injection. Intrathecal and intravenous injection of ZJ‐11 suppressed the expression of Fos‐like immunoreactivity, induced by paw formalin injection, in laminae I–II in segments L4–L5 of the spinal cord, suggesting an action on sensory spinal transmission. Partial sciatic nerve ligation induced significant mechanical allodynia 7 days after the nerve injury. Intrathecal injection of ZJ‐11 or ZJ‐17 or intravenous administration of ZJ‐11 or ZJ‐43 attenuated the level of mechanical allodynia induced by this nerve ligation. These effects of intrathecally or intravenously administered ZJ compounds in both the formalin test and the partial sciatic nerve ligation model were completely antagonized by pretreatment with LY‐341495, a highly selective group II mGluR antagonist. Thus, elevation of extracellular NAAG, induced by the inhibition of NAAG peptidase, activates group II mGluRs and produces an analgesic effect in neuropathic and inflammatory and pain models. In contrast, peptidase inhibition did not affect the threshold for withdrawal from a noxious mechanical stimulus or from an acute thermal stimulus in the hotplate test.

Molecular Cloning of a Peptidase Against N‐Acetylaspartylglutamate from a Rat Hippocampal cDNA Library

Abstract: 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 β‐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.

NAAG inhibits KCl-induced [3H]-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 [3H]‐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, β‐N‐acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl‐stimulated [3H]‐GABA‐release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H‐89, decreased [3H]‐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 [3H]‐GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl‐stimulated [3H]‐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.

The cloning and characterization of a second brain enzyme with NAAG peptidase activity.

The peptide neurotransmitter N‐acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non‐overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild‐type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar Vmax and Km values (1.4 µm and 54 pmol/min/mg; 3.5 µm and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT‐PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.