• The Korean Journal of Physiology and Pharmacology
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• 제1권4호
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• pp.367-376
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• 1997

The effect of an organic peroxide, t-butylhydroperoxide (t-BHP), on glutamate uptake was studied in synaptosomes prepared from cerebral cortex. t-BHP inhibited the $Na^+-dependent$ glutamate uptake with no change in the $Na^+-independent$ uptake. This effect of t-BHP was not altered by addition of $Ca^{2+}$ channel blockers (verapamil, diltiazem and nifedipine) or $PLA_2$ inhibitors (dibucaine, butacaine and quinacrine). However, the effect was prevented by iron chelators (deferoxamine and phenanthroline) and phenolic antioxidants (N,N'-diphenyl-phenylenediamine, butylated hydroxyanisole, and butylated hydroxytoluene). At low concentrations (＜1.0 mM), t-BHP inhibited glutamate uptake without altering lipid peroxidation. Moreover, a large increase in lipid peroxidation by $ascorbate/Fe^{2+}$ was not accompanied by an inhibition of glutamate uptake. The impairment of glutamate uptake by t-BHP was not intimately related to the change in $Na^+-K+-ATPase$ activity. These results suggest that inhibition of glutamate uptake by t-BHP is not totally mediated by peroxidation of membrane lipid, but is associated with direct interactions of glutamate transport proteins with t-BHP metabolites. The $Ca^{2+}$ influx through $Ca^{2+}$ channel or $PLA_2$ activation may not be involved in the t-BHP inhibition of glutamate transport.

• Archives of Pharmacal Research
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• 제21권2호
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• pp.113-119
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• 1998

Changes in the release and uptake of glutamate in cerebellar granule and glial cells of offspring of lead-exposed mothers were determined. In cultured cerebellar granule cells exposed to lead for 5 days, glutamate release was less influenced upon N-methyl-D-aspartate (NMDA) stimulation than that in the control. Although the NMDA-stimulated release of glutamate in cerebellar granule cells prepared from lead-exposed first generation pups was not different from that of the control group, the S-nitroso-N-acetylpenicillamine (SNAP)-stimulated release of glutamate in cerebellar granule cells obtained from lead-treated pups was less elevated than that in the control. Furthermore, in cerebellar granule cells obtained from lead-exposed second generations pups, glutamate release did not respond to both NMDA and SNAP stimulation. In cerebellar glial cells exposed to lead, the basal glutamate uptake was not changed. However, the L-trans-pyrollidine-2,4-dicarboxylic acid (PDC)-blocking effects was significantly reduced. In glial cells obtained from lead-exposed pups, the glutamate uptake was also less blocked by PDC than that in the control. Further decreases in PDC-blocking effects were observed in cerebellar glial cells obtained from lead-treated second generation pups compared to those from the control group. These results indicate that lead exposure induces the changes in the sensitivities of the glutamate release and uptake transporter. In addition, these results suggest that lead exposure might affect the intracellular signalling pathway and transmission in glutamatergic nervous system.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.137-146
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• 1999

Interactions among dexamethasone, dehydroepiandrosterone (DHEA), lipopolysaccharide (LPS), and antimycin A on the glutamate uptake and the polyamine uptake were investigated in primary cultures of rat cerebral cortical astrocytes to examine the effects of dexamethasone and DHEA on the regulatory role of astrocytes in conditions of increased extracellular concentrations of glutamate or polyamines. 1. $[^3H]Glutamate$ uptake: LPS and antimycin A decreased $V_{max},$ but both drugs had little effect on $K_m.$ Dexamethasone also decreased basal $V_{max}$ without any significant effect on $K_m.$ And dexamethasone further decreased the antimycin A-induced decrease of $V_{max}.$ DHEA did not affect the kinetics of basal glutamate uptake and the change by LPS or antimycin A. 2. $[^{14}C]Putrescine$ uptake: LPS increased $V_{max},$ and antimycin A decreased $V_{max}.$ They showed little effect on $K_m.$ Dexamethasone decreased $V_{max}$ of basal uptake and further decreased the antimycin A-induced decrease of $V_{max},$ and also decreased $V_{max}$ to less than control in LPS-treated astrocytes. DHEA did not affect $K_m$ and the change of $V_{max}$ by LPS or antimycin A. 3. $[^{14}C]Spermine$ uptake: Antimycin A decreased $V_{max},$ and LPS might increase $V_{max}.\;K_m$ was little affected by the drugs. Dexamethasone decreased basal $V_{max}$ and might further decrease the antimycin A-induced decrease of $V_{max}.$ And dexamethasone also decreased $V_{max}$ to less than control in LPS-treated astrocytes. DHEA might increase basal $V_{max}$ and $V_{max}$ of LPS-treated astrocytes. 4. $V_{max}$ of glutamate uptake by astrocytes was increased by putrescine (1000 ${\mu}M$ & 2000 ${\mu}M$) and spermidine (200 ${\mu}M,$ 500 ${\mu}M$ & 2000 ${\mu}M$). Spermine, 200 ${\mu}M$ (and 100 ${\mu}M$), also increased $V_{max},$ but a higher dose of 2000 ${\mu}M$ decreased $V_{max}.\;K_m$ of glutamate uptake was not significantly changed by these polyamines, except that higher doses of spermine showed tendency to decrease $K_m$ of glutamate uptake. In astrocytes, dexamethasone inhibited the glutamate uptake and the polyamine uptake in normal or hypoxic conditions, and the polyamine uptake might be stimulated by LPS and DHEA. Polyamines could aid astrocytes to uptake glutamate.

• Korean Journal of Acupuncture
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• 제17권1호
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• pp.179-190
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• 2000

This study was undertaken to determine whether Juglandis semen extract solution (JLS solution) exerts protective effect against oxidant-induced inhibition of glutamate uptake by synaptosomes. Synaptosome was prepared from rabbit brain cortex. Glutamate uptake increased by incubation time during 10 minutes, which was significantly inhibited by 1mM t-buthylhydroperoxide(t-BHP). JLS solution prevented t-BHP-induced inhibition of glutamate uptake in a dose-dependent manner. t-BHP reduced glutamate uptake in dose-dependent fashion, which was significantly prevented by 2% JLS solution. t-BHP(1mM) and $ascorbate/Fe^{2+}(50/1{\mu}M)$ increased lipid peroxidation in synaptosomes by 5-fold, and it was significantly prevented by 2% JLS solution. $HgCl_2(0.1mM)$ inhibited glutamate uptake and increased lipid peroxidation. These changes were prevented by 2% JLS solution. Synaptosomal Na-K-ATPase activity was inhibited by t-BHP(1mM) and $H_2O_2(50mM)$, which was prevented by 2% JLS solution. The results indicate that JLS solution prevents oxidant-induced inhibition of glutamate by synaptosomes, and this may result from inhibition of lipid peroxidation induced by oxidants.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.211-211
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• 1996

Changes in glutamate release and uptake on cerebellar cells after the chronic exposure to lead were investigated. Rats were received 0.25% lead acetate in drinking water from the beginning of the pregnancy. The control group was given 0.125% sodium acetate in drinking water. The cerebellar cells from 7 or 8 day-old pups were cultured. Amino acid release from cerebellar granule cells and the glutamate uptake into cerebellar glial cells were measured using HPLC-ECD. Basal glutamate release and NMDA-induced glutamate release didn't show significant difference. However, the other amino acids in the granule cells obtained from lead exposed pups were less released than the control after the stimulation by NMDA (50$\mu$M). SNAP-induced (50$\mu$M) glutamate release was significantly reduced in granule cells prepared from lead exposed pups. The basal glutamate uptake in glial cells didn't show any difference. However, the uptake in glial cells prepared from lead exposed pups was significantly less blocked by PDC (24$\mu$M) compared to the control group. These results indicate that lead exposure to the mother might affect the Excitatory amino acid system during the development of the offspring.

• Archives of Pharmacal Research
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• 제19권2호
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• pp.132-136
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• 1996

Exogenously applied oxygen free radical generating agent, pyrogallol, highly elevated extracellular glutamate accumulation and augmented N-methyl-D-aspartate (NMDA)-induced glutamate accumulation in cerebellar granule neuronal cells, but did not in astrocytes. Superoxide dismutase remarkably decreased the pyrogallol-induced glutamate accumulation, but either NMDA or kainate antagonists did not. In addition, pyrogallol did not affect the NMDAinduced intracellular calcium elevation. Pyrogallol partially blocked glutamate uptake into astrocytes. These results suggest that oxygen free radicals elevate extracellular glutamate accumulation by stimulating the release of glutamate as well as blocking the glutamate uptake.

• Archives of Pharmacal Research
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• 제23권5호
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• pp.488-494
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• 2000

Cerebellar granule and glial cells prepared from 7 day-old rat pups were used to investigate the effects of sub-acute nicotine exposure on the glutamatergic nervous system. These cells were exposed to nicotine in various concentrations for 2 to 10 days in situ. Nicotine-exposure did not result in any changes in cerebellar granule and glial cell viability at concentrations of up to 500 $\mu\textrm{M}$. In cerebellar granule cells, the basal extracellular levels of glutamate, aspartate and glycine were enhanced in the nicotine-exposed granule cells. In addition, the responses of N-methyl-D-aspartate (NMDA)-induced glutamate release were enhanced at low NMDA concentrations in the nicotine-exposed granule cells. However, this decreased at higher NMDA concentrations. The glutaminase activity was increased after nicotine exposure. In cerebellar glial cells, glutamate uptake in the nicotine-exposed glial cells were either increased at low nicotine exposure levels or decreased at higher levels. The inhibition of glutamate uptake by L-trans-pyrollidine-2,4-dicarboxylic acid (PDC) was lower in glial cells exposed to 50 $\mu\textrm{M}$ nicotine. Glutamine synthetase activity was lower in glial cells exposed to 100 or 500 $\mu\textrm{M}$ of nicotine. These results indicate that the properties of cerebellar granule and glial cells may alter after subacute nicotine exposure. Furthermore, they suggest that nicotine exposure during development may modulate glutamatergic nervous activity.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• Archives of Pharmacal Research
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• 제20권1호
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• pp.7-12
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• 1997

Glutamate uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxylate (PDC, $20{\mu}M$) elevated basal and N-methyl-D-aspartate (NMDA, $100{\mu}M$)-induced extracellular glutamate accumulation, while it did not augment kainate $100{\mu}M$-induced glutamate accumulation in cultured cerebellar granule neurons. However, pretreatment with PDC for 1 h significantly reduced NMDA-induced glutamate accumulation, but did not affect kainate-induced response. Pretreatment with glutamate $(5{\mu}M)$ for 1 h also reduced NMDA-induced glutamate accumulation, but did not kainate-induced response. Upon a brief application (3-10 min), PDC did neither induce elevation of intracellular calcium concentration $([Ca^{2+}]_i)$ nor modulate NMDA-indLiced $[Ca^{2+}]_1$ elevation. Pretreatment with PDC for 1 h reduced NMDA-induced $[Ca^{2+}]_1$ elevation, but it did not reduce kainate-induced $[Ca^{2+}]_1$ elevation. These results suggest that glutamate concentration in synaptic clefts of neurana cells is increased by prolonged exposure (1 h) of the cells to PDC, and the accumulated glutamate subsequently induces selective desensitization of NMDA receptor.

• 대한약리학회지
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• 제24권1호
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• pp.71-81
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• 1988

Glutamate와 aspartate는 단가 양이온과 칼슘에 대한 세포막의 투과성을 증가시키는 것으로 시사되고 있다. 그러나 칼슘 유입이 voltage에 의존적인 칼슘 통로에 의하여 또는 흥분성 아미노산에 활성적인 통로에 의하여 이루어지는가는 분명하지 않다. 더우기, 신경세포의 칼슘 유입에 미치는 흥분성 아미노산의 영향과 세포의 마그네슘에 대한 이들의 반응이 다른 것으로 추정하고 있다. Synaptosome에서 포타슘에 의한 칼슘 흡수는 세포외 마그네슘에 의존적이었으나 10 mM 농도에서는 그 이하의 농도에서보다 오히려 감소하였다. 소듐이 주된 반응액에서 glutamate와 aspartate에 의한 칼슘 흡수는 마그네슘에 의하여 용량에 비의존적인 양상으로 증가하였다. 그러나 NMDA의 작용은 2 mM 이상의 마그네슘에 의하여 억제되었다. 포타슘과 glutamate에 의한 칼슘 흡수는 2,4-dinitrophenol, chorpromazine과 verapami에 의하여 억제되었으나 tetraethylammonium chloride의 영향은 받지 아니하였다. Tetrodotoxin은 효과적으로 glutamate의 작용을 억제하였으나 $K^+$의 작용에는 영향을 주지 않았다. NMDA의 작용은 2,4-dinitrophenol과 tetrodotoxin에 의하여 억제되었고 verapamil에 의하여 약간 억제되었으며 tetraethylammonium chloride의 영향은 받지 아니하였다. 소듐이 주된 반응액에서 glutamate,, aspartate와 NMDA에 의한 synaptosome의 탈분극은 관찰되지 않았으나 이들은 mitochondria에서 칼슘 유입에 따른 탈분극을 초래하였다. 한편, 흥분성 아미노산은 synaptosoine의 ATPase활성도에 영향을 나타내지 않았다. 이상의 결과로부터 glutamate 또는 NMDA에 의한 synaptosome의 칼슘 흡수는 세포외 마그네슘에 각기 다른 양상을 나타내며 이들에 의한 칼슘 흡수는 포타슘을 제외한 소듐과 칼슘에 대한 세포막 투과성의 증가 그리고 이에 따른 탈분극에 연관이 있을 것으로 시사되있다.