Aspartic acid (or aspartate) is a non-essential amino acid, meaning that it is readily and naturally synthesized by mammals. It is one of the 20 building-block amino acids of proteins, 3-letter code is ASP, one letter code is D. The DNA codons encoding aspartic acid are GAC and GAU. The IUPAC name is 2-aminobuanedioic acid. ASP is chiral and exists in two enantiomeric forms, l-aspartate and d-aspartate where l-aspartate is the form used in protein biosynthesis (proteinogenic) and neurotransmission. Although aspartic acid (and related derivatives) has pharmacological activity at some glutamate receptors, it is not clear that this has any physiological relevance. N-methyl-d-aspartate is a synthetic analog that is a selective and potent synthetic agonist at the N-methyl-d-aspartate type of glutamate ionotropic receptors. N-acetylaspartate and N-acetylaspatylglutamate are highly abundant in the mammalian CNS but their function is not clearly understood.

Background Individuals with schizophrenia show increased smoking rates which may be due to a beneficial effect of nicotine on cognition and information processing. Decreased amplitude of the P50 and N100 auditory event-related potentials (ERPs) is observed in patients. Both measures show normalization following administration of nicotine. Recent studies identified an association between deficits in auditory evoked gamma oscillations and impaired information processing in schizophrenia, and there is evidence that nicotine normalizes gamma oscillations. Although the role of nicotine receptor subtypes in augmentation of ERPs has received some attention, less is known about how these receptor subtypes regulate the effect of nicotine on evoked gamma activity. Methodology/Principal Findings We examined the effects of nicotine, the α7 nicotine receptor antagonist methyllycaconitine (MLA) the α4β4/α4β2 nicotine receptor antagonist dihydro-beta-erythroidine (DHβE), and the α4β2 agonist AZD3480 on P20 and N40 amplitude as well as baseline and event-related gamma oscillations in mice, using electrodes in hippocampal CA3. Nicotine increased P20 amplitude, while DHβE blocked nicotine-induced enhancements in P20 amplitude. Conversely, MLA did not alter P20 amplitude either when presented alone or with nicotine. Administration of the α4β2 specific agonist AZD3480 did not alter any aspect of P20 response, suggesting that DHβE blocks the effects of nicotine through a non-α4β2 receptor specific mechanism. Nicotine and AZD3480 reduced N40 amplitude, which was blocked by both DHβE and MLA. Finally, nicotine significantly increased event-related gamma, as did AZD3480, while DHβE but not MLA blocked the effect of nicotine on event-related gamma. Conclusions/Significance These results support findings showing that nicotine-induced augmentation of P20 amplitude occurs via a DHβE sensitive mechanism, but suggests that this does not occur through activation of α4β2 receptors. Event-related gamma is strongly influenced by activation of α4β2, but not α7, receptor subtypes, while disruption of N40 amplitude requires the activation of multiple receptor subtypes.

Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of nicotinic agonists improve attention, and nicotinic antagonist exposure impairs it. This study was conducted to investigate the effect of a novel nicotinic receptor agonist at α4β2 nicotinic receptors (AZD3480) on attention and reversal of pharmacologically induced attentional impairment produced by the NMDA glutamate antagonist dizocilpine (MK-801).

In the 1960s, several groups reported the presence of N-acetylaspartylglutamate (NAAG) in the mammalian central nervous system (Curatelo et al. 1965; Miyamoto et al. 1966). In the 1980s, it was clearly demonstrated with immunohistochemistry that NAAG is present throughout the CNS (Anderson et al. 1986; Blakely et al. 1987; Cangro et al. 1987; Forloni et al. 1987; Tsai et al. 1988; Frondoza et al. 1990). NAAG has been shown to be abundant in the central nervous system and it has been proposed to be a neurotransmitter. Additionally it has been proposed to be a selective agonist of the metabotropic glutamate receptor type 3 (mGlu3) (Wroblewska et al. 1997) as well as a selective agonist of native NMDA receptors (NMDARs; Westbrook et al. 1986). A number of studies have suggested that NAAG is a neurotransmitter in the mammalian central nervous system (for review, see Neale et al. 2005). Although indeed there is substantial evidence supporting this hypothesis, and this commentary does not intend to discuss all of those supporting data, neurotransmitters (other than NO or CO), particularly peptide or amino acids, interact with post-synaptic receptors to transduce their signaling. Thus, a key piece in the claim that NAAG is a neurotransmitter, is that it interacts with a receptor and that it is an agonist at mGlu3 receptors. Wroblewska et al. (1997) claimed that NAAG is a highly selective agonist at mGlu3 receptors with an EC50 of 65 μM measured at a chimeric receptor consisting of the extracellular ligand-binding domain of mGlu3 and the transmembrane domain and carboxy terminus of mGlu1a. Two recent publications (Chopra et al. 2009; Fricker et al. 2009) have questioned whether NAAG per se has physiological activity at mGlu3 receptors. These two studies independently showed that NAAG, when purified, is not an agonist at mGlu3 receptors. Both of these groups sought to better understand the role of NAAG as a tool compound and potential neurotransmitter. In their efforts, they discovered that when commercial samples of NAAG were purified of glutamate, activity in functional assays disappeared. In work in our laboratories (Chopra et al. 2009), we assessed two commercial samples of NAAG and found them to have significant glutamate contamination. This work was first motivated by our studies performed at Sibia Neurosciences with human mGlu3 receptors (e.g. Varney et al. 1999) where we had struggled to demonstrate that purified NAAG (100 μM) was a selective agonist at human mGlu3 receptors using several different assay systems. Chopra et al. (2009) demonstrated that unpurified commercial NAAG activated mGlu3 when co-expressed with Gα15 while purified NAAG had no activity in this preparation. Additionally, this study showed that unpurified NAAG could activate rat or human mGlu3 receptors co-expressed with G protein-coupled inwardly-rectifying potassium channel (GIRK) channels in Xenopus oocytes. When NAAG was purified, it no longer had significant activity at mGlu3 receptors. There was trace activity seen in the rat mGlu3 preparation. It should be noted that in the rat and human mGlu3 GIRK assay, the glutamate EC50s were determined to be 28 and 58 nM respectively. A trace contamination of 0.01–0.02% glutamate in 100 μM NAAG sample could explain the residual response seen in the rat mGlu3 preparation; the HPLC analysis indicated that the remaining level of glutamate was in this range. In the study by Fricker et al. (2009), three commercial samples of NAAG were found to have significant glutamate contamination and were purified by cation exchange chromatography with purity demonstrated by HPLC and mass spectrometry. Using this purified NAAG, they assessed its activity in several native and recombinant systems. NAAG failed to activate mGlu2 or mGlu3 receptors expressed in mammalian cells when these receptors were coupled to the GIRK channels, while glutamate did activate both with potency (nanomolar) similar to that shown to activate these receptors in previous studies (e.g. Schweitzer et al. 2000). Interestingly, unpurified NAAG activated GIRK currents in the absence of mGlu2 or mGlu3 receptor co-expression. However, 1 mM NAAG did inhibit the glutamate (100 μM)-induced GIRK current. Perhaps most importantly, this group examined the activity of purified NAAG at native mGlu2/3 receptors in the dentate gyrus of adult hippocampal slices examining field excitatory postsynaptic potential (EPSP). In this preparation, they also employed the NAALADase inhibitor 2-(phosphonomethyl) pentanedioic acid to ensure that NAAG was not hydrolyzed by endogenous NAALADase. With this approach, they demonstrated that NAAG (100 μM) has no effect on the field EPSPs while the mGlu2/3-selective agonist, LY379268, markedly inhibited both components of the field EPSP. This preparation has been shown to have group II mGlu receptor sensitive synaptic transmission (Kew et al. 2002). A previous report, which did not state whether NAAG was purified, has shown that NAAG (50 μM) inhibited long-term potentiation at the same synapse in rat hippocampal slices (Lea et al. 2001). Other groups have also shown that NAAG has failed to effect excitatory postsynaptic currents in circuits where mGlu2/3 receptors have been demonstrated to have activity (Alexander and Godwin 2005; Mateo and Porter 2007). In one autoradiography study in rat brain slices using 10 nM [3H]-NAAG (Shave et al. 2001), binding patterns were observed that were most consistent with group 2 mGlu receptors; binding was not markedly displaced by ionotropic glutamate receptor ligands while it was by displaced by the group II mGlu/NMDAR ligand dicarboxycyclopropyl glycine (DCG-IV). As the ligand in this study is [3H]-NAAG with two 3H on the glutamate moiety ([GLUTAMATE-3,4-3H]-NAAG), and this is a native preparation, it is not clear whether the NAAG has hydrolyzed into glutamate-3,4-3H and acetyl-aspartate and if the study is actually measuring [3H]-glutamate binding. Although it is clear that the conditions of the study are designed to minimize such hydrolysis, the low potency of NAAG in this assay argues again, that only a very minimal hydrolysis is required to explain this observation. Thus, an observation of specific binding of 10 nM [3H]-NAAG is difficult to reconcile with the reported potency of NAAG at mGlu3 in the high micromolar range. Additionally, the displacement of 10 nM [3H]-NAAG by DCG-IV in this study was observed in the micromolar range; this may also be consistent with the reported activity of DCG-IV at NMDARs than at group II mGlu receptors (e.g. Wilsch et al. 1994). Such studies with native receptors, as conducted, are simply not able to reconcile this question particularly when there is not quantitative demonstration that there is not glutamate contamination at the 0.3% level. Interestingly, two independent studies in 2009 sought to examine the role of mGlu3 in pathophysiology and explored whether effects of NAAG previously published could be explained by NAAG with glutamate contamination. These two independent studies (Chopra et al. 2009; Fricker et al. 2009) demonstrated that NAAG, when purified of glutamate contamination, no longer could activate mGlu3 receptors in three different expression systems. The work of Wroblewska et al. (1997) did not describe whether NAAG was purified, additionally; they employed a chimeric mGlu3–mGlu1 receptor, whereas the other two studies utilized fully intact rat or human mGlu3 receptors. In displacement-binding studies (Schweitzer et al. 2000), NAAG was shown to displace [3H]-LY354740 from mGlu2 membranes with a Ki of 236 μM and from mGlu3 membranes with a Ki of 19 μM. This study reported that glutamate had Kis of 1.2 and 0.44 μM respectively which could be explained by a 1–2% glutamate contamination in the NAAG preparation; this study did not describe any purification of the commercial NAAG. Together, the recent two studies, using wild-type rat or human mGlu3 receptors, make a strong case that previous activity of NAAG, using chimeric mGlu3–mGlu1 receptors, can be attributed to trace contamination of glutamate. This is particularly the case for mGlu3 receptors where glutamate has been shown to have such high potency (∼30–300 nM) whereas NAAG is reported to have a low potency of 65 μM. This activity can most simply be explained by a 0.05–0.5% glutamate contamination rather than a direct activation of mGlu3 receptors by NAAG. To clearly demonstrate that NAAG is directly activating mGlu3 receptors, given this very high sensitivity to glutamate, future studies will need to employ highly purified preparations of NAAG. Additionally, given this high sensitivity of mGlu3 to glutamate, to clearly demonstrate that the activity is caused by NAAG, proof that no glutamate is present in the > 10 nM level is needed. Fortunately, present bioanalytical methods can detect glutamate at these levels. Finally, several studies, using molecular docking analysis on mGlu receptors, have presented evidence that the glutamate binding site may not have the space available for larger ligands, including peptides such as NAAG, to bind (Wang and Hampson 2006; Wang et al. 2006). While these studies did not specifically examine the mGlu3 receptor, given that the potency of glutamate at this subtype is by far the highest in the family, one would suspect that the binding pocket would be even less amenable to larger molecules. The view of this commentary is that there is not yet sufficient evidence that NAAG is a selective agonist of mGlu3 receptors. Such a claim is challenged by the high sensitivity of mGlu3 receptors to glutamate and the potential for trace contamination of glutamate in NAAG preparations. Additionally, studies in native preparations would be challenged by the presence of enzymes which can metabolize NAAG into N-acetyl aspartate and glutamate. Unlike other mGlu receptors, mGlu3 has been shown to be expressed perisynaptically and distal to glutamate release. At such sites, it would be expected that only low levels of glutamate would be available even following high levels of excitation. Until stronger evidence is provided, the conservative view should be that glutamate is the only natural agonist demonstrated to activate the mGlu3 receptor. ECJ is a full-time employee of AstraZeneca Pharmaceuticals, Södertälje, Sweden.

Preclinical models of nicotine vaccine pharmacology have relied on i.v. or s.c. administration of nicotine. Models using cigarette smoke inhalation might more accurately simulate nicotine exposure in smokers. Nicotine vaccine effects were examined in rats using two cigarette smoke exposure models: a 10 min nose-only exposure (NSE) producing serum nicotine levels equivalent to the nicotine boost from 1 cigarette in a smoker, and a 2 h whole-body exposure (WBE) producing serum nicotine levels similar to those associated with regular mid-day smoking. Vaccination prior to 10 min smoke NSE reduced nicotine distribution to brain by 90%, comparable to its effect on nicotine administered i.v. Vaccination prior to 2 h smoke WBE reduced nicotine distribution to brain by 35%. The nicotine concentration in broncheoalveolar lavage (BAL) fluid obtained after 2 h WBE was increased by 230% in vaccinated rats but was also increased in rats passively immunized with a nicotine-specific monoclonal antibody, and so was likely due to transfer of antibody from serum rather than local production at the pulmonary mucosa. Nicotine-specific IgA was not detectable in BAL fluid, but titers in serum were appreciable at 21–25% of the IgG titer and could contribute to vaccine efficacy. Both vaccination and passive immunization are effective in reducing nicotine distribution to brain in rats when nicotine is delivered via inhaled cigarette smoke. These data validate results previously obtained in rodents for nicotine vaccines using i.v. or s.c. nicotine dosing and provide a quantitative method for studying aspects of nicotine exposure which are unique to cigarette smoke inhalation.

Metabotropic glutamate receptors (mGluRs), which are coupled to second messenger pathways via G proteins, modulate glutamatergic and GABAergic neurotransmission. Because of their role in modulating neurotransmission, mGluRs are attractive therapeutic targets for anxiety disorders. Previously we showed that mGluR8(-/-) male mice showed higher measures of anxiety in the open field and elevated plus maze than age-matched wild-type mice. In this study, we assessed the potential effects of acute pharmacological modulation of mGluR8 on measures of avoidable and unavoidable anxiety. In addition to wild-type mice, we also tested apolipoprotein E-deficient (Apoe(-/-)) mice, as these mice show increased levels of anxiety-like behaviors and therefore might show an altered sensitivity to mGluR8 stimulation. mGluR8 stimulation with the specific agonist DCPG, or modulation with AZ12216052, a new, positive allosteric modulator of mGluR8 reduced measures of anxiety in both wild-type mice. The effects of mGluR8 positive allosteric modulators, which only affect neurotransmission in the presence of extracellular glutamate, seem particularly promising for patients with anxiety disorders showing benzodiazepine insensitivity.

The peptide N-acetylaspartylglutamate (NAAG) is present in high concentrations in the mammalian central nervous system. Various mechanisms have been proposed for its action, including selective activation of the metabotropic glutamate receptor (mGluR) subtype 3, its action at the N-methyl-d-aspartate receptor, or the production of glutamate by its hydrolysis catalyzed by an extracellular protease. To re-examine its agonist activity at mGluR3, we coexpressed human or rat mGluR3 with G protein inward rectifying channels in Xenopus laevis oocytes. High-performance liquid chromatography analysis of commercial sources of NAAG showed 0.38 to 0.48% glutamate contamination. Although both human and rat mGluR3 were highly sensitive to glutamate, with EC50 values of 58 and 28 nM, respectively, purified NAAG (100 μM) had little activity (7.7% of full activation by glutamate). Only in the millimolar range did it show significant activity, possibly due to residual traces of glutamate remaining in the purified NAAG preparations. In contrast, the unpurified NAAG sample did produce a full agonist response with mGluR3 coexpressed with Gα15, with an EC50 of 120 μM, as measured by a calcium release assay. This response can be explained by the 0.38 to 0.48% glutamate contamination. Our results suggest that NAAG may not have a direct agonist activity at the mGluR3 receptor. Thus, several in vivo and in vitro published results that did not address the issue of glutamate contamination of NAAG preparations may need to be re-evaluated.

Jonsson, M.1; Gurley, D.2; Dabrowski, M.3; Larsson, O.3; Johnson, E. C.2; Eriksson, L. I. Author Information

Background Nondepolarizing neuromuscular blocking agents (NMBAs) are extensively used in the practice of anesthesia and intensive care medicine. Their primary site of action is at the postsynaptic nicotinic acetylcholine receptor (nAChR) in the neuromuscular junction, but their action on neuronal nAChRs have not been fully evaluated. Furthermore, observed adverse effects of nondepolarizing NMBAs might originate from an interaction with neuronal nAChRs. The aim of this study was to examine the effect of clinically used nondepolarizing NMBAs on muscle and neuronal nAChR subtypes.Methods: Xenopus laevis oocytes were injected with messenger RNA encoding for the subunits included in the human alpha(1)beta(1)epsilon delta, alpha(3)beta(2), alpha(3)beta(4), alpha(4)beta(2), and alpha(7) nAChR subtypes. The interactions between each of these nAChR subtypes and atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium were studied using an eight-channel two-electrode voltage clamp setup. Responses were measured as peak current and net charge.Results: All nondepolarizing NMBAs inhibited both muscle and neuronal nAChRs. The neuronal nAChRs were reversibly and concentration-dependently inhibited in the low micromolar range. The mechanism (ie., competitive vs. noncompetitive) of the block at the neuronal nAChRs was dependent both on subtype and the NMBA tested. The authors did not observe activation of the nAChR subtypes by any of the NMBAs tested.Conclusions: The authors conclude that nondepolarizing NMBAs concentration-dependently inhibit human neuronal nAChRs. The inhibition of the presynaptic alpha(3)beta(2) nAChR subtype expressed at the motor nerve ending provides a possible molecular explanation for the tetanic and train-of-four fade seen during a nondepolarizing neuromuscular block.

BACKGROUND:Succinylcholine is one of the most widely used muscle relaxants in clinical anesthesia and emergency medicine. Although the clinical advantages and cardiovascular side effects are well known, its mechanism of action within the human nicotinic cholinergic receptor system remains to be understood. The aim of this study was to investigate the effect of succinylcholine on human muscle and neuronal nicotinic acetylcholine receptor (nAChR) subtypes.METHODS:Xenopus laevis oocytes were injected with human messenger RNA for muscle and neuronal nAChR subunits. Receptor activation, desensitization, and inhibition induced by the natural ligand acetylcholine or by succinylcholine was studied using a multichannel two-electrode voltage clamp setup. Responses were measured as peak current and net charge.RESULTS:Succinylcholine concentration-dependently activated the muscle-type nAChR with an EC50 value of 10.8 microm (95% confidence interval, 9.8-11.9 microm), and after the initial activation, succinylcholine desensitized the muscle-type nAChR. Succinylcholine did not activate the neuronal nAChR subtypes alpha3beta2, alpha3beta4, alpha4beta2, or alpha7 at concentrations up to 1 mm and was a poor inhibitor at these receptor subtypes, with IC50 values above 100 microm.CONCLUSION:Succinylcholine activates the muscle-type nAChR followed by desensitization. The observation that succinylcholine does not inhibit the presynaptic alpha3beta2 autoreceptor at clinically relevant concentrations provides a possible mechanistic explanation for the typical lack of tetanic fade in succinylcholine-induced neuromuscular blockade. Finally, cardiovascular side effects (e.g., tachyarrhythmias) of succinylcholine are not mediated via direct activation of the autonomic ganglionic alpha3beta4 subtype because succinylcholine does not activate the neuronal nAChRs.

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of non-competitive mGlu5 receptor antagonists.

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of noncompetitive mGlu5 receptor antagonists.

Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the group III receptors mGluR4, mGluR6, and mGluR7. [3H]L-AP4 binding was present in human embryonic kidney cells transfected with the high-affinity mGluR4 receptor but not in cells transfected with mGluR6 or the low-affinity mGluR7 receptor. Analysis of mGluR4/mGluR6 receptor chimeras revealed that replacement of the first 35 amino acids of mGluR6 with the first 50 amino acids of mGluR4 was sufficient to impart [3H]L-AP4 binding to mGluR6. Homology models of mGluR4 and mGluR7 were used to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high-affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, whereas the N74K mutation in combination with mutations in residues 258 to 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared with unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the group III mGluRs.