N-methyl-D-aspartate Receptor Pathway Research Articles

The dose-dependent effect of 1.5-GHz microwave exposure on spatial memory and the NMDAR pathway in Wistar rats

A certain power of microwave radiation could cause changes in the nervous, cardiovascular, and other systems of the body, and the brain was a sensitive target organ of microwave radiation injury. Studies have shown that microwaves can impair cognitive functions in humans and animals, such as learning and memory, attention, and orientation. The dose-dependent effect of microwave radiation is still unclear. Our study aimed to investigate the effects of 1.5-GHz microwaves with different average power densities on locative learning and memory abilities, hippocampal structure, and related N-methyl D-aspartate receptor (NMDAR) signalling pathway proteins in rats. A total number of 140 male Wistar rats were randomly divided into four groups: S group (sham exposure), L5 group (1.5-GHz microwaves with average power density = 5 mW/cm2), L30 group (1.5-GHz microwaves with average power density = 30 mW/cm2), and L50 group (1.5-GHz microwaves with average power density = 50 mW/cm2). Changes in spatial learning and memory, EEG activity, hippocampal structure, and NMDAR signalling pathway molecules were detected from 6 h to 28 d after microwave exposure. After exposure to 1.5-GHz microwaves, rats in the L30 and L50 groups showed impaired spatial memory, inhibited EEG activity, pyknosis and hyperchromatism of neuron nucleus, and changes in NMDAR subunits and downstream signalling molecules. In conclusion, 1.5-GHz microwaves with an average power density of 5, 30, and 50 mW/cm2 could induce spatial memory dysfunction, hippocampal structure changes, and changes in protein levels in rats, and there was a defined dose-dependent effect.

NMDA Receptor Antagonists Degrade Lipofuscin via Autophagy in Human Retinal Pigment Epithelial Cells.

Background and Objectives: Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Results: Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. Conclusions: This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.

The Role of the NMDA Receptor in the Anticonvulsant Effect of Ellagic Acid in Pentylenetetrazole-Induced Seizures in Male Mice.

Methods In this experimental study, 64 mice were divided into 8 groups and received the following: normal saline; EA at doses of 6.25, 12.5, and 25 mg/kg; NMDA agonist at a dose of 75 mg/kg; NMDA antagonist (ketamine) at a dose of 0.5 mg/kg; an effective dose of EA plus NMDA agonist; and a subeffective dose of EA plus ketamine. We induced seizure using intravenous administration of PTZ. 60 minutes before induction of seizure, drugs were administrated. Duration lasts to seizure-induced was measured. Finally, the gene expression of NMDA receptor subunits (Nr2a and Nr2b) was assessed in the prefrontal cortex. Results Results showed that EA increased the seizure threshold and decreased the expression of Nr2a and Nr2b. We determined that ketamine potentiated and NMDA attenuated the effects of subeffective and effective doses of EA. Conclusion EA probably via attenuation of the NMDA-R pathway possesses an anticonvulsant effect in PTZ-induced seizure in mice.

Pharmacological evidence for the possible involvement of the NMDA receptor pathway in the anticonvulsant effect of tramadol in mice.

Previous studies have shown controversial results regarding the pro- or anticonvulsant effects of tramadol. Additionally, the underlying mechanism of seizure induction or alleviation by tramadol has not been fully understood. In the current study, the effects of tramadol on pentylenetetrazole (PTZ)-induced seizure and the possible involvement of the N-methyl-D-aspartate (NMDA) pathway were assessed in mice. Male Naval Medical Research Institute (NMRI) mice were treated with intravenous infusion of PTZ in order to induce clonic seizures and determine seizure threshold. Tramadol was injected intraperitoneally (0.1-150 mg/kg) 30 minutes prior to elicitation of seizures. The possible effects of intraperitoneal injections of NMDA receptor antagonists, ketamine (0.5 mg/kg) and MK-801 (0.5 mg/kg) on the anticonvulsant property of tramadol were investigated subsequently. Tramadol (1-100 mg/kg) increased PTZ-induced seizure threshold in a dose-dependent, time-independent manner, with optimal anticonvulsant effect at a dose of 100 mg/kg. Acute administration of either ketamine (0.5 mg/kg) or MK-801 (0.5 mg/kg) potentiated the anticonvulsant effect of a subeffective dose of tramadol (0.3 mg/kg). These results suggest a possible role of the NMDA pathway in the anticonvulsant effect of tramadol.

Advancement in research on the role of the transient receptor potential vanilloid channel in cerebral ischemic injury (Review).

Stroke is a common critical disease occurring in middle-aged and elderly individuals, and is characterized by high morbidity, lethality and mortality. As such, it is of great concern to medical professionals. The aim of the present review was to investigate the effects of transient receptor potential vanilloid (TRPV) subtypes during cerebral ischemia in ischemia-reperfusion animal models, oxygen glucose deprivation and in other administration cell models in vitro to explore new avenues for stroke research and clinical treatments. TRPV1, TRPV2 and TRPV4 employ different methodologies by which they confer protection against cerebral ischemic injury. TRPV1 and TRPV4 are likely related to the inhibition of inflammatory reactions, neurotoxicity and cell apoptosis, thus promoting nerve growth and regulation of intracellular calcium ions (Ca2+). The mechanisms of neuroprotection of TRPV1 are the JNK pathway, N-methyl-D-aspartate (NMDA) receptor and therapeutic hypothermia. The mechanisms of neuroprotection of TRPV4 are the PI3K/Akt pathways, NMDA receptor and p38 MAPK pathway, amongst others. The mechanisms by which TRPV2 confers its protective effects are predominantly connected with the regulation of nerve growth factor, MAPK and JNK pathways, as well as JNK-dependent pathways. Thus, TRPVs have the potential for improving outcomes associated with cerebral ischemic or reperfusion injuries. The protection conferred by TRPV1 and TRPV4 is closely related to cellular Ca2+ influx, while TRPV2 has a different target and mode of action, possibly due to its expression sites. However, in light of certain contradictory research conclusions, further experimentation is required to clarify the mechanisms and specific pathways by which TRPVs act to alleviate nerve injuries.

Ferulic acid through mitigation of NMDA receptor pathway exerts anxiolytic-like effect in mouse model of maternal separation stress.

Background Experiencing early-life stress plays an important role in the pathophysiology of anxiety disorders. Ferulic acid is a phenolic compound found in some plants which has several pharmacological properties. N-methyl-D-aspartate (NMDA) receptors are involved in the pathophysiology of mood disorders. In this study we aimed to assess the anxiolytic-like effect of ferulic acid in a mouse model of maternal separation (MS) stress by focusing on the possible involvement of NMDA receptors. Methods Mice were treated with ferulic acid (5 and 40 mg/kg) alone and in combination with NMDA receptor agonist/antagonist. Valid behavioral tests were performed, including open field test (OFT) and elevated plus maze test (EPM), while quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate gene expression of NMDA subunits (GluN2A and GluN2B) in the hippocampus. Results Findings showed that treatment of MS mice with ferulic acid increased the time spent in the central zone of the OFT and increased both open arm time and the percent of open arm entries in the EPM. Ferulic acid reduced the expression of NMDA receptor subunit genes. We showed that administration of NMDA receptor agonist (NMDA) and antagonist (ketamine) exerted anxiogenic and anxiolytic-like effects, correspondingly. Results showed that co-administration of a sub-effective dose of ferulic acid plus ketamine potentiated the anxiolytic-like effect of ferulic acid. Furthermore, co-administration of an effective dose of ferulic acid plus NMDA receptor agonist (NMDA) attenuated the anxiolytic-like effect of ferulic acid. Conclusions In deduction, our findings showed that NMDA, partially at least, is involved in the anxiolytic-like effect of ferulic acid in the OFT and EPM tests.

A Brief Ischemic Postconditioning Protects Against Amyloid-β Peptide Neurotoxicity by Downregulating MLK3-MKK3/6-P38MAPK Signal in Rat Hippocampus.

Oligomeric amyloid-β peptide (Aβ) is associated with dysfunctional neuronal networks and neuronal loss in the development of Alzheimer's disease (AD). Ischemic postconditioning protects against post-ischemic excitotoxicity, oxidative stress, and inflammatory process that have also been implicated in the pathogenesis of AD. Evaluating the roles of ischemic postconditioning in oligomeric Aβ-induced neurotoxicity and underlying signal events may provide potential strategy for medical therapy in AD. The aim of the present study was to explore whether and how a brief ischemic postconditioning protects against Aβ neurotoxicity in rat hippocampus. Oligomeric Aβ25-35 (20 nmol/rat) or Aβ1-42 (5 nmol/rat) was infused by intracerebroventricular injection in adult male Sprague-Dawley rats. Ischemic postconditioning, a brief episode of global brain ischemia (3 min), was conducted at 1, 3, or 7 days after Aβ treatment, respectively. A brief ischemic postconditioning reduced neuronal loss and inhibited the activation of MLK3, MKK3/6, and P38MAPKs in rat hippocampal CA1 and CA3 subfields after Aβ oligomer infusion. An N-methyl-D-aspartate (NMDA) receptor antagonist amantadine, but not non-NMDA receptor antagonist CNQX, reversed the MLK3-MKK3/6-P38MAPK signal events and beneficial effect of ischemic postconditioning on neuronal survival. Such reversion was also realized by NVP-AAM077, a GluN2A-subunit-selective NMDA receptor antagonist. Moreover, posttreatment with low doses of NMDA (5 nmol-40 nmol/rat) suppressed the Aβ-induced P38MAPK signaling and imitated the neuroprotection of ischemic postconditioning against Aβ neurotoxicity. Ischemic postconditioning provides neuroprotection against Aβ neurotoxicity by moderate upregulation of NMDA receptor signaling, especially GluN2A-containing NMDA receptor pathway, and thereafter downregulation of MLK3-MKK3/6-P38MAPK signal events.

Larval metamorphosis in four bivalve species in response to NMDA receptor ligands: The NMDA receptor pathway as potential regulator of bivalve transition to spat

Over the last three decades, a variety of chemical inducers or disruptors for settlement and metamorphosis have been identified for species of clams, oysters, scallops, and mussels. Identifying inducers is important in shellfish aquaculture for increasing metamorphosis rates, and hence, spat (seed) production; however, it also has wider applications, such as in the development of marine anti-fouling agents. Previously, we have suggested the potential importance of an N-Methyl-d-aspartate (NMDA) receptor pathway in regulating bivalve metamorphosis based on preliminary work in Crassostrea gigas. In this study, we further this work by assessing the ability of several known vertebrate NMDA receptor antagonists (MK-801, ifenprodil and chlorpromazine) to induce metamorphosis in a range of bivalve species. In two oyster species (Crassostrea virginica, C. gigas) and two clam species (Mercenaria mercenaria, Mya arenaria) all three NMDA receptor antagonists induced metamorphosis, but with variability in optimal concentrations and effectiveness. Specifically, both clam species were highly responsive to the NMDA receptor antagonists, with M. mercenaria displaying up to 100% induction with both ifenprodil at 10−5 M and 10−6 M and chlorpromazine at 10−7 M to 10−5 M. There is very limited prior work on successful induction using neuroactive compounds for metamorphosis in the hard-shell clam, M. mercenaria, and no prior published work for the soft-shell clam, M. arenaria. Epinephrine and levodopa (L-DOPA), two widely used and effective inducers in Crassostrea species, also induced metamorphosis in both clam species with epinephrine at 10−4 M resulting in 44% and 75% metamorphosis and L-DOPA at 10−5 M resulting in 67% and 98% for M. arenaria and M. mercenaria, respectively. Co-exposures of L-DOPA or epinephrine in conjunction with NMDA receptor antagonists were conducted to identify putative interactions between the proposed NMDA receptor pathway and the dopaminergic or adrenergic pathways by which L-DOPA and epinephrine are believed to operate. In general, co-exposures demonstrated induction of metamorphosis similar or higher than the most effective single treatments. In experiments conducted in 2018 for both oyster species, epinephrine at 10−4 M in conjunction with ifenprodil at 10−6 M, significantly inhibited metamorphosis down to 4% from 12%. The percentage of larvae that underwent metamorphosis was similarly low in response to ifenprodil alone, 2% to 18% metamorphosis, whereas epinephrine alone had a positive inducing effect, 34% to 74% metamorphosis. Results are discussed in relation to the NMDA receptor pathway that appears to be acting independently of previously believed dopaminergic or adrenergic pathways in bivalves.

Activation of the N-methyl-d-aspartate receptor is involved in glyphosate-induced renal proximal tubule cell apoptosis.

Glyphosate-based herbicides have been used worldwide for decades and have been suggested to induce nephrotoxicity, but the underlying mechanism is not yet clear. In this study, we treated a human renal proximal tubule cell line (HK-2) with glyphosate for 24hours at concentrations of 0, 20, 40 and 60μm. Glyphosate was found to reduce cell viability and induce apoptosis and oxidative stress in a dose-dependent manner. Because the chemical structures of glyphosate and those of its metabolite AMPA are similar to glycine and glutamate, which are agonists of the N-methyl-d-aspartate receptor (NMDAR), we investigated the potential role of the NMDAR pathway in mediating the proapoptotic effect of glyphosate on proximal tubule cells. We found that NMDAR1 expression, as well as intracellular Ca2+ ([Ca2+ ]i ) and reactive oxygen species (ROS) levels, increased after glyphosate treatment. Blocking NMDAR attenuated glyphosate-induced upregulation of [Ca2+ ]i and ROS levels as well as apoptosis. Meanwhile, inhibition of [Ca2+ ]i reduced glyphosate-induced ROS and apoptosis, and inhibition of ROS alleviated glyphosate-induced apoptosis. In mice exposed to 400mg/kg glyphosate, the urine low molecular weight protein levels started to increase from day 7. Upregulation of apoptosis and NMDAR1 expression in renal proximal tubule epithelium and an imbalance of oxidant and antioxidative products were observed. These results strongly suggest that activation of the NMDAR1 pathway, together with its downstream [Ca2+ ]i and oxidative stress, is involved in glyphosate-induced renal proximal tubule epithelium apoptosis.

Pharmacological evidence for the relationship between the NMDA receptor and nitric oxide pathway and the antidepressant-like effects of glucagon-like peptide-2 in the mouse forced-swim test.

We previously demonstrated that glucagon-like peptide-2 (GLP-2) exerted antidepressant-like effects in mice. The aim of the present study was to investigate the relationship between N-methyl-D-aspartate (NMDA) receptor-nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway and the antidepressant-like effects of GLP-2 in the forced-swim test (FST) in mice. Intracerebroventricularly administered GLP-2 (3 μg/mouse) decreased the immobility time in the FST. The pretreatment of mice with l-arginine (750 mg/kg, i.p.), a substrate for nitric oxide synthase, sildenafil (5 mg/kg, i.p.), a phosphodiesterase 5 inhibitor, or d-serine (300 mg/kg, i.p.), a NMDA receptor co-agonist, inhibited the antidepressant-like effects of GLP-2 (3 μg/mouse) in the FST. Meanwhile, l-nitroarginine methyl ester (10 mg/kg, i.p.), a non-specific nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (30 mg/kg, i.p.), a neuronal NOS inhibitor, methylene blue (10 mg/kg, i.p.), an inhibitor of both NOS and soluble guanylate cyclase (sGC), ODQ (30 pmol/site, i.c.v.), a sGC inhibitor, or MK-801 (0.05 mg/kg, i.p.), an NMDA receptor antagonist, in combination with a sub-effective dose of GLP-2 (1.5 μg/mouse) also decreased the immobility time in the FST. The present study provided evidence for the synergistic antidepressant-like effects of GLP-2 and inhibition of the NMDA receptor-l-arginine-NO-cGMP pathway in the FST, thereby contributing to our understanding of the mechanisms underlying the antidepressant-like effects of GLP-2.

Annexin 1 inhibits remifentanil-induced hyperalgesia and NMDA receptor phosphorylation via regulating spinal CXCL12/CXCR4 in rats

Chemokines related neuroinflammation and N-methyl-d-aspartate receptor (NMDAR) mediated nociceptive transmission are pivotal determinants in the pathogenesis of opioid-induced hyperalgesia (OIH), but little is known about specific mechanism and treatment. Chemokine CXCL12 with its receptor CXCR4 is implicated in different pathological pain, moreover, neurotoxicity of CXCL12 is associated with NMDAR activation. Recent studies recapitulate the anti-nociception of Annexin 1 (ANXA1) in inflammatory pain. This study examined whether ANXA1 prevented remifentanil-caused OIH through modulating CXCL12 and NMDAR pathway in rats. Acute exposure to remifentanil induced mechanical allodynia and thermal hyperalgesia, which was accompanied by the increase of spinal ANXA1 and CXCL12/CXCR4 expression. Central injection of Anxa12-26 attenuated behavioral OIH in a dose-dependent manner, facilitated ANXA1 production, and inhibited up-regulation of CXCL12/CXCR4 level and NR2B-containing NMDAR phosphorylation. Moreover, pretreatment with AMD3100 reduced hyperalgesia and NR2B-containing NMDAR phosphorylation. Also, exogenous CXCL12 elicited pain hypersensitivity and NMDAR activation in naïve rats, which was reversed by the supplemental delivery of Anxa12-26. These current findings indicate the participation of spinal CXCL12/CXCR4 and NR2B-containing NMDAR pathway in anti-hyperalgesic action of ANXA1 in OIH.

GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth

SummaryGenetic linkage analysis previously suggested that GKAP, a scaffold protein of the N-methyl-D-aspartate receptor (NMDAR), was a potential modifier of invasion in a mouse model of pancreatic neuroendocrine tumor (PanNET). Here, we establish that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. Combining genetic knockdown of GKAP and pharmacological inhibition of NMDAR, we implicate as downstream effectors FMRP and HSF1, which along with GKAP demonstrably support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells. Furthermore, we distilled genome-wide expression profiles orchestrated by the NMDAR-GKAP signaling axis, identifying transcriptome signatures in tumors with low/inhibited NMDAR activity that significantly associate with favorable patient prognosis in several cancer types.

Plasma cyclic guanosine 3′,5′-monophosphate levels: A marker of glutamate-nitric oxide-guanyl cyclase activity?

Remifentanil-based anesthesia can lead to acute opioid tolerance and/or hyperalgesia. A low-dose intraoperative infusion of the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine did not result in reduced postoperative morphine consumption after remifentanil-based anesthesia in adolescents. This study investigates the potential role of the glutamate-nitric oxide-cyclic guanosine 3'5'-monophosphate (cyclic GMP) pathway in the failure of low-dose ketamine to prevent remifentanil-induced acute opioid tolerance and/or hyperalgesia. Prospective, double-blind, placebo-controlled randomized clinical trial at a university teaching hospital. PATIENTS, PARTICIPANTS, AND INTERVENTIONS: Thirty-four adolescents receiving remifentanil-based anesthesia for surgical correction of idiopathic scoliosis were randomly assigned to receive either intraoperative ketamine administered as a bolus dose of 0.5 mg/kg in 10 mL of normal saline and a continuous intravenous infusion of 4.0 microg/kg/min or an equal volume of saline. Blood samples were collected before and after the administration of ketamine for analyzing the concentrations of cyclic GMP, ketamine, and norketamine. Blood samples were analyzed using high-performance liquid chromatography and an enzyme immunoassay. The median (interquartile range) value of the concentration of plasma cyclic GMP decreased from 23.7 (17.4-26.7) to 14.8 (14.0-17.3) nmol/L after ketamine infusion (p < 0.005) and from 23.9 (16.3-29.2) to 163 (14.5-18.6) nmol/L after saline infusion (p < 0.005). The median value of the concentration of plasma cyclic GMP at the end of ketamine infusion did not differ significantly when compared with that after saline infusion (p = 0.07). The concentration of plasma cyclic GMP was inversely correlated with the concentration of plasma ketamine (r = -0.61). This study suggests that the low dose of intraoperative ketamine infused was insufficient to suppress the NMDA receptor pathway. The concentrations of plasma cyclic GMP may serve as an indirect biological marker of ketamine-induced NMDA receptor antagonism.

Pharmacological evidence for the involvement of the NMDA receptor and nitric oxide pathway in the antidepressant-like effect of lamotrigine in the mouse forced swimming test

Lamotrigine is an anticonvulsant agent that shows clinical antidepressant properties. The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) synthesis in possible antidepressant-like effect of lamotrigine in forced swimming test (FST) in mice. Intraperitoneal administration of lamotrigine (10mg/kg) decreased the immobility time in the FST (P<0.01) without any effect on locomotor activity in the open-field test (OFT), while higher dose of lamotrigine (30mg/kg) reduced the immobility time in the FST (P<0.001) as well as the number of crossings in the OFT. Pretreatment of animals with NMDA (75mg/kg), l-arginine (750mg/kg, a substrate for nitric oxide synthase [NOS]) or sildenafil (5mg/kg, a phosphodiesterase [PDE] 5 inhibitor) reversed the antidepressant-like effect of lamotrigine (10mg/kg) in the FST. Injection of l-nitroarginine methyl ester (l-NAME, 10mg/kg, a non-specific NOS inhibitor), 7-nitroindazole (30mg/kg, a neuronal NOS inhibitor), methylene blue (20mg/kg, an inhibitor of both NOS and soluble guanylate cyclase [sGC]), or MK-801 (0.05mg/kg), ketamine (1mg/kg), and magnesium sulfate (10mg/kg) as NMDA receptor antagonists in combination with a sub-effective dose of lamotrigine (5mg/kg) diminished the immobility time of animals in the FST compared with either drug alone. None of the drugs produced significant effects on the locomotor activity in the OFT. Based on our findings, it is suggested that the antidepressant-like effect of lamotrigine might mediated through inhibition of either NMDA receptors or NO-cGMP synthesis.

Interaction between NMDA glutamatergic and nitrergic enteric pathways during in vitro ischemia and reperfusion

Nitric oxide (NO) and glutamate, via N-methyl-d-aspartate (NMDA) receptors, participate to changes in neuromuscular responses after ischemic/reperfusion (I/R) injury in the gut. In the present study we investigated the existence of a possible interplay between nitrergic and NMDA receptor pathways in the guinea pig ileum after in vitro I/R injury, resorting to functional and biomolecular approaches. In normal metabolic conditions NMDA concentration-dependently enhanced both glutamate (analyzed by high performance liquid chromatography with fluorimetric detection) and NO (spectrophotometrically quantified as NO2− and NO3−) spontaneous overflow from isolated ileal segments. Both effects were reduced by the NMDA antagonists, (-)-AP5 (10µM) and 5,7-diCl-kynurenic acid (10µM, 5,7-diCl-KYN). Nω-propyl-l-arginine (1µM, NPLA) and 1400W (10µM), respectively, nNOS and iNOS inhibitors, reduced NMDA-stimulated glutamate overflow. After in vitro I/R, glutamate overflow increased, and returned to control values in the presence of NPLA and 1400W. NO2− and NO3− levels transiently increased during I/R and were reduced by both (-)-AP5 and 5,7-diCl-KYN. In longitudinal muscle myenteric plexus preparations, iNOS mRNA and protein levels increased after in vitro I/R; both parameters were reduced to control values by (-)-AP5 and 5,7-diCl-KYN. Both antagonists were also able to reduce ischemia-induced enhancement of nNOS mRNA levels. Protein levels of GluN1, the ubiquitary subunit of NMDA receptors, increased after I/R and were reduced by both NPLA and 1400W. On the whole, this data suggests the existence of a cross-talk between NMDA receptor and nitrergic pathways in guinea pig ileum myenteric plexus, which may participate to neuronal rearrangements occurring during I/R.

Prevention of postoperative fatigue syndrome in rat model by ginsenoside Rb1 via down-regulation of inflammation along the NMDA receptor pathway in the hippocampus.

Postoperative fatigue syndrome (POFS) is a common complication which decelerates recovery after surgery. The present study investigated the anti-fatigue effect of ginsenoside Rb1 (GRb1) through the inflammatory cytokine-mediated N-methyl-D-aspartate (NMDA) receptor pathway. A POFS rat model was created by major small intestinal resection and assessed with an open field test. Real-time quantitative polymerase chain reaction, western blot analysis, high performance liquid chromatography and a transmission electron microscopic analysis were used to determine typical biochemical parameters in the hippocampus. Our results showed that POFS rats exhibited fatigue associated with an increased expression of inflammatory cytokines and NMDA receptor 1, higher (kynurenine)/(tryptophan) and (kynurenine)/(kynurenic acid) on postoperative days 1 and 3, and an increased expression of indoleamine 2,3-dioxygenase (IDO) on postoperative day 1. Degenerated neurons were found in the hippocampus of POFS rats. The NMDA receptor antagonist MK801 had a significant effect on central fatigue on postoperative day 1. GRb1 had no effect on IDO or tryptophan metabolism, but exhibited a significant effect on POFS by inhibiting the expression of inflammatory cytokines and NMDA receptor 1. These data suggested that inflammatory cytokines could activate tryptophan metabolism to cause POFS through the NMDA receptor pathway. GRb1 had an anti-fatigue effect on POFS by reducing inflammatory cytokines and NMDA receptors.

Src kinase as a mediator of convergent molecular abnormalities leading to NMDAR hypoactivity in schizophrenia.

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia: yet molecular underpinnings for such dysregulation are largely unknown. In the postmortem dorsolateral prefrontal cortex, we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2), which is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be due to downregulation of NMDA receptors since MK-801 binding and NMDA receptor complexes in the PSD were in fact increased in schizophrenia cases. At the post-receptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity, which in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms impacting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their impacts on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased RPTPa and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome wide association study results, incorporating 13,394 cases and 34,676 controls, which yielded no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a wider protein-protein interaction based network centered on Src, showed significant enrichment of gene-level associations with schizophrenia compared to other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the post-receptor level and propose Src as a nodal point of convergent dysregulations impacting NMDA receptor pathway via protein-protein associations.

Activation of N-methyl-d-aspartate receptor downregulates inflammasome activity and liver inflammation via a β-arrestin-2 pathway.

Activation of the cytosolic inflammasome machinery is responsible for acute and chronic liver inflammation, but little is known about its regulation. The N-methyl-d-aspartate (NMDA) receptor families are heterotetrameric ligand-gated ion channels that are activated by a range of metabolites, including aspartate, glutamate, and polyunsaturated fatty acids. In the brain NMDA receptors are present on neuronal and nonneuronal cells and regulate a diverse range of functions. We tested the role of the NMDA receptor and aspartate in inflammasome regulation in vitro and in models of acute hepatitis and pancreatitis. We demonstrate that the NMDA receptor is present on Kupffer cells, and their activation on primary mouse and human cells limits inflammasome activation by downregulating NOD-like receptor family, pyrin domain containing 3 and procaspase-1. The NMDA receptor pathway is active in vivo, limits injury in acute hepatitis, and can be therapeutically further activated by aspartate providing protection in acute inflammatory liver injury. Downregulation of inflammasome activation by NMDA occurs via a β-arrestin-2 NF-kβ and JNK pathway and not via Ca(2+) mobilization. We have identified the NMDA receptor as a regulator of inflammasome activity in vitro and in vivo. This has identified a new area of immune regulation associated by metabolites that may be relevant in a diverse range of conditions, including nonalcoholic steatohepatitis and total parenteral nutrition-induced immune suppression.

In Vitro Stretch Injury Induces Time- and Severity-Dependent Alterations of STEP Phosphorylation and Proteolysis in Neurons

Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP(61) expression progressively increases after sublethal stretch with no change in calpain-mediated STEP(33) formation, while lethal stretch injury results in STEP(33) formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP(33) formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP(33) formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP(33) should be further examined as an early marker of neuronal fate after stretch injury.

Exposure of Sebastiscus marmoratus embryos to pyrene results in neurodevelopmental defects and disturbs related mechanisms

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants, which are known to be carcinogenic and teratogenic. These compounds cause a range of macroscopic malformations, particularly to the craniofacial apparatus and cardiovascular system during vertebrate development. However, little is known concerning microscopic effects, especially on the sensitive early life stages or on the molecular basis of developmental neurotoxicity. Using the rockfish (Sebastiscus marmoratus), we explored the neurodevelopmental defects caused by early-life exposure to environmentally relevant concentrations of pyrene, a 4-ring PAH. The results showed that pyrene substantially disrupted the cranial innervation pattern and caused deficiency of motor nerves. The expression of a protein associated with axon growth, growth associated protein 43, was decreased in the central nervous system after treatment with pyrene. N-methyl-d-aspartate receptor (NMDAR) plays a vital role in a variety of processes, including neuronal development, synaptic plasticity, and neuronal survival and death. Our results showed that the expression of Ca2+/calmodulin dependent kinase II and cAMP-response element-binding, which belong to the NMDAR pathway, were increased in a dose-dependent manner after exposure to pyrene. Acetylcholine, an important neurotransmitter which is known to suppress retinal cells neurite outgrowth, was increased by pyrene exposure. Nitric oxide (NO) acts as an activity-dependent retrograde signal that can coordinate axonal targeting and synaptogenesis during development. The level of NO was decreased in a dose-dependent manner following exposure to pyrene. Taken together, the defects in neurodevelopment and the damage to related mechanisms provided the basis for a better understanding of the neurotoxic effects of pyrene.