Elevation of endocannabinoids in the brain by synthetic cannabinoid JWH-018: mechanism and effect on learning and memory.

The impairment of learning and memory is a well-documented effect of both natural and synthetic cannabinoids. In the present study, we aimed to investigate the effect of acute administration of JWH-018, a synthetic cannabinoid, on the hippocampal metabolome to assess biochemical changes in vivo. JWH-018 elevated levels of the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The increase of endocannabinoid levels in response to JWH-018 could be inhibited by co-administration of AM251, a CB1 receptor antagonist. Biochemical analyses revealed that this was the result of suppression of two hydrolases involved in endocannabinoid degradation (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL]). Additionally, we showed that JWH-018 causes a reduction in the levels of brain-derived neurotrophic factor (BDNF), which is known to modulate synaptic plasticity and adaptive processes underlying learning and memory. The decrease of BDNF following JWH-018 treatment was also rescued by co-administration of AM251. As both endocannabinoids and BDNF have been shown to modulate learning and memory in the hippocampus, the alteration of their levels in response to JWH-018 may explain the contribution of synthetic cannabinoids to impairment of memory.

Inhibition of Monoacylglycerol Lipase Reduces the Reinstatement of Methamphetamine-Seeking and Anxiety-Like Behaviors in Methamphetamine Self-Administered Rats.

Background: Methamphetamine is a highly addictive psychostimulant with reinforcing properties. Our laboratory previously found that Δ8-tetrahydrocannabinol, an exogenous cannabinoid, suppressed the reinstatement of methamphetamine-seeking behavior. The purpose of this study was to determine whether the elevation of endocannabinoids modulates the reinstatement of methamphetamine-seeking behavior and emotional changes in methamphetamine self-administered rats. Methods: Rats were tested for the reinstatement of methamphetamine-seeking behavior following methamphetamine self-administration and extinction. The elevated plus-maze test was performed in methamphetamine self-administered rats during withdrawal. We investigated the effects of JZL184 and URB597, 2 inhibitors of endocannabinoid hydrolysis, on the reinstatement of methamphetamine-seeking and anxiety-like behaviors. Results: JZL184 (32 and 40 mg/kg, i.p.), an inhibitor of monoacylglycerol lipase, significantly attenuated both the cue- and stress-induced reinstatement of methamphetamine-seeking behavior. Furthermore, URB597 (3.2 and 10 mg/kg, i.p.), an inhibitor of fatty acid amide hydrolase, attenuated only cue-induced reinstatement. AM251, a cannabinoid CB1 receptor antagonist, antagonized the attenuation of cue-induced reinstatement by JZL184 but not URB597. Neither JZL184 nor URB597 reinstated methamphetamine-seeking behavior when administered alone. In the elevated plus-maze test, rats that were in withdrawal from methamphetamine self-administration spent less time in the open arms. JZL184 ameliorated the decrease in time spent in the open arms. Conclusion: We showed that JZL184 reduced both the cue- and stress-induced reinstatement of methamphetamine-seeking and anxiety-like behaviors in rats that had self-administered methamphetamine. It was suggested that a decrease in 2-arachidonoylglycerol in the brain could drive the reinstatement of methamphetamine-seeking and anxiety-like behaviors.

Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs.

Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.

BACKGROUND: Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells. METHODOLOGY/PRINCIPAL FINDINGS: In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters. CONCLUSIONS/SIGNIFICANCE: Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca(2+) in diverse cells enriched of Zn(2+).

Selective inhibition by ethanol of mitochondrial calcium influx mediated by uncoupling protein-2 in relation to N-methyl-D-aspartate cytotoxicity in cultured neurons.

BACKGROUND: We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca(2+) levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca(2+) levels in cultured murine neocortical neurons. METHODOLOGY/PRINCIPAL FINDINGS: In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca(2+) levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca(2+) levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca(2+) levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca(2+) levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca(2+) levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM. CONCLUSIONS/SIGNIFICANCE: Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca(2+) incorporation and cell death after NMDAR activation in neurons.

Myosin VI reduces proliferation, but not differentiation, in pluripotent P19 cells.

BACKGROUND: We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2'-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques. METHODOLOGY/PRINCIPAL FINDINGS: Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca(2+) ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter. CONCLUSIONS/SIGNIFICANCE: These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.

Promotion of both proliferation and neuronal differentiation in pluripotent P19 cells with stable overexpression of the glutamine transporter slc38a1.

BACKGROUND: We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia. METHODOLOGY/PRINCIPAL FINDINGS: The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants. CONCLUSIONS/SIGNIFICANCE: GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.

Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors.

BACKGROUND: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA) and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR) by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. METHODOLOGY/PRINCIPAL FINDINGS: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4ß2 nAChR subtype antagonists dihydro-ß-erythroidine and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice defective of NMDA receptor subunit-1, nicotine was still effective in significantly inhibiting the proliferation. CONCLUSIONS/SIGNIFICANCE: Functional α4ß2 nAChR subtype would be constitutively expressed to play a role in the mechanism underlying the determination of proliferation and subsequent differentiation fate into a neuronal lineage in association with preferential promotion of Math1 expression in undifferentiated neural progenitors of developing rodent neocortex independently of NMDA receptor activation.

Possible neuroprotective property of nicotinic acetylcholine receptors in association with predominant upregulation of glial cell line-derived neurotrophic factor in astrocytes.

The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca(2+) in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line-derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa(2+) chelator. Nicotine significantly increased GDNF in a concentration-dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti-GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca(2+) influx in rat brain in a particular situation.

Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-D-aspartate receptor channels.

We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-d-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca(2+) levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca(2+) levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca(2+) levels in neurons.

Delayed mitochondrial membrane potential disruption by ATP in cultured rat hippocampal neurons exposed to N-methyl-D-aspartate.

Necrotic damage leads to a massive leakage from injured cells of different intracellular constituents such as glutamate (Glu) and ATP, which are believed to play a role in the neuronal survival in the brain. In this study, we evaluated pharmacological properties of ATP, which is shown to be an endogenous inhibitor of N-methyl-D-aspartate (NMDA) receptors, on the neurotoxicity relevant to mitochondrial membrane potential disruption in cultured rat hippocampal neurons. Exposure to Glu or NMDA significantly inhibited cellular viability determined 24 and 48 h later, while simultaneous addition of 1 mM ATP significantly ameliorated the decreased viability in neurons exposed to Glu and NMDA, but not in those exposed to other cytotoxins. Both Glu and NMDA markedly increased intracellular free Ca(2+) levels in a manner sensitive to blockade by the exposure to ATP, but not by that to adenosine. Exposure to ATP significantly delayed the rate of mitochondrial membrane potential disruption induced by Glu and NMDA. These results suggest that extracellular ATP would play a role as an endogenous antagonist endowed to protect rat hippocampal neurons from the excitotoxicity mediated by NMDA receptors in association with the delayed mitochondrial membrane potential disruption after the liberation from adjacent cells under necrotic death.

NR2-reactive antibody decreases cell viability through augmentation of Ca(2+) influx in systemic lupus erythematosus.

OBJECTIVE: Anti-N-methyl-D-aspartate (anti-NMDA) receptor subunit NR2-reactive antibody may play a crucial role in neuronal manifestations of systemic lupus erythematosus (SLE). However, how NR2-reactive antibody acts as a critical modulator of the NMDA receptor is unknown. This study was undertaken to investigate the biologic function of NR2-reactive antibody in patients with SLE. METHODS: The study included 14 patients with SLE, 9 of whom had NR2-reactive antibody. We analyzed the effects of NR2-reactive antibody on cell viability and intracellular Ca(2+) level. We also investigated the efficacy of zinc as a modulator of the intracellular Ca(2+) level in the presence of NR2-reactive antibody. RESULTS: There was a significant inverse correlation between the NR2-reactive antibody titer and cell viability (R(2) = 0.67, P < 0.0001; n = 23), and there was a significant association between the NR2-reactive antibody titer and the intracellular Ca(2+) level in NR1/NR2a-transfected HEK 293 cells (R(2) = 0.69, P < 0.0001). Intracellular Ca(2+) levels were significantly higher in cells incubated with IgG derived from NR2-reactive antibody-positive SLE patients than in those incubated with IgG derived from NR2-reactive antibody-negative SLE patients (P = 0.0002). The addition of zinc decreased the intracellular Ca(2+) level in a dose-dependent manner. NR2-reactive antibody-positive SLE IgG weakened the efficacy of zinc as a negative modulator of the intracellular Ca(2+) level. CONCLUSION: Our findings indicate that NR2-reactive antibody decreases cell viability by Ca(2+) influx in SLE through inhibition of the binding capacity of zinc.

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons.

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl(2) similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.

Requirement of both NR3A and NR3B subunits for dominant negative properties on Ca2+ mobilization mediated by acquired N-methyl-D-aspartate receptor channels into mitochondria.

Conventional N-methyl-D-aspartate (NMDA) receptor (NMDAR) is a heteromeric complex between the essential NR1 subunit and one of NR2A-D subunits toward functional channels permeable to Ca(2+) rather than Na(+) ions. Although recent studies identified dominant negative NR3A and NR3B subunits, whether these subunits inhibit Ca(2+) mobilization through NMDAR channels into mitochondria is not clarified so far. In this study, we investigated Ca(2+) influx across acquired NMDAR channels composed of different NR subunits artificially expressed in HEK293 cells. The addition of NMDA markedly increased intracellular free Ca(2+) levels determined by Fluo-3 in cells transfected with either NR2A or NR2B subunit together with NR1 subunit. Further addition of dizocilpine completely inhibited the increase by NMDA in both types of acquired channels, while the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similar pharmacological profiles were invariably seen with cell death by NMDA. Introduction of both NR3A and NR3B subunits significantly inhibited the increase by NMDA in intracellular free Ca(2+) levels in both acquired channels, while introduction of either NR3A or NR3B alone was ineffective. Co-expression of both NR3A and NR3B subunits was also required for the prevention of increased mitochondrial free Ca(2+) levels determined by Rhod-2, as well as decreased cellular viability, in cells expressing NR1/NR2A or NR1/NR2B subunits upon exposure to NMDA. These results suggest that co-expression of both NR3A and NR3B subunits is essential for the dominant negative properties on Ca(2+) mobilization through acquired functional NMDAR channels into mitochondria.

Preferential inhibition by antidiarrheic 2-methoxy-4-methylphenol of Ca(2+) influx across acquired N-methyl-D-aspartate receptor channels composed of NR1/NR2B subunit assembly.

In our previous studies, particular phenolic ingredients, such as 2-methoxy-4-methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca(2+) influx across native and acquired N-methyl-D-aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca(2+) levels as determined by Fluo-3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca(2+) levels determined by Rhod-2, in addition to intracellular free Ca(2+) levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca(2+) levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA-induced influx of Ca(2+) across acquired NR1/NR2B channels in a concentration-dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca(2+) influx across NMDAR channels composed of NR1/NR2B subunits.

Induced tolerance to glutamate neurotoxicity through down-regulation of NR2 subunits of N-methyl-D-aspartate receptors in cultured rat striatal neurons.

We have previously shown differential vulnerabilities to glutamate (Glu) excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor (NMDAR) between rat cortical and rat hippocampal neurons in culture. In this study, we evaluated the possible induced tolerance to NMDA neurotoxicity in cultured rat striatal neurons with prior sustained activation of NMDAR. Brief exposure to Glu or NMDA for 1 hr led to a significant decrease in cellular vitality determined 24 hr later in cultured rat striatal neurons, whereas no marked loss was seen in cellular survival after exposure to Glu or NMDA in striatal neurons previously cultured with Glu or NMDA. Sustained culture with Glu or NMDA invariably led to a significant decrease in protein levels of NR2, but not NR1, subunits without affecting their mRNA levels. Similar induced tolerance was seen to the excitotoxicity of NMDA in hippocampal neurons in a manner sensitive to an NMDAR antagonist. Prior culture with NMDA induced less effective alterations in both intracellular free Ca(2+) levels and mitochondrial membrane potentials after the addition of NMDA in striatal neurons. However, calpain inhibitor-I significantly prevented the decreased NR2B and NR2C protein levels in striatal neurons cultured with NMDA. These results suggest that prior tonic activation of NMDAR would induce tolerance to the excitotoxicity mediated by NMDAR through a mechanism related to calpain-induced down-regulation of particular NR2 subunits in rat striatal neurons.

Inhibition by 2-methoxy-4-ethylphenol of Ca2+ influx through acquired and native N-methyl-D-aspartate-receptor channels.

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-D-aspartate (NMDA), to modulate Ca(2+) influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca(2+) levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca(2+) levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit-selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca(2+) levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca(2+) influx across NMDAR channels composed of NR1/NR2B, rather than NR1/NR2A, subunits.

Possible protection by notoginsenoside R1 against glutamate neurotoxicity mediated by N-methyl-D-aspartate receptors composed of an NR1/NR2B subunit assembly.

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. We found that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 microM Glu for 1 hr in a dose-dependent manner at concentrations from 0.1 to 10 microM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca(2+) ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl-2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N-methyl-D-aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 microM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 microM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain.