Glutamate와 NMDA에 의한 Synaptosome에서의 칼슘 유입과 이들의 작용의 차이

이정수(Chung-Soo Lee),심재건(Jae-Keon Sim),신용규(Yong-Kyoo Shí,n),이광수(Kwang-Soo Lee) 대한약리학회 1988 대한약리학잡지 Vol.24 No.1

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. Glutamate and aspartate may evoke an increase in membrane permeability to monovalent cations and Ca⁺⁺. However, it is uncertain whether Ca⁺⁺ influx is mediated by voltage dependent Ca⁺⁺ channels or by excitatory amino acid activated channels. In addition, the influences of excitatory amino acids on Ca⁺⁺ uptake by neuronal tissues as well as the responses of their actions to extracellular Mg⁺⁺ concentration are different. K⁺ induced Ca⁺⁺ uptake by synaptosomes was dependent on extracellular Mg⁺⁺ up to 5 mM and at concentration of 10 mM, Ca⁺⁺ influx was rather reduced. In Na⁺ rich media, glutamate-and aspartate-induced Ca⁺⁺ uptake was increased by Mg⁺⁺ in a dose independent manner. However, the response for NMDA was inhibited by Mg⁺⁺ at concentrations above 2 mM. K⁺-and glutamate-induced Ca⁺⁺ influx s were inhibited by 2,4-dinitrophenol, chlorprom-azine and verapamil but not by tetraethylammonium chloride. Tetrodotoxin effectively inhibited the action of glutamate but did not affect that of K⁺. The response for MNDA was inhibited by 2, 4-dinitrophenol and tetrodotoxin, slightly inhibited by verapamil, and not affected by tetraethylammonium chloride. In Na⁺⁺ rich medium, depolarizing action of glutamate, aspartate and MNDA on synaptosomes was not demonstrated, whereas these agents stimulated Ca⁺⁺ uptake and caused Ca⁺⁺ influx induced depolarization at mitochondria. On the other hand, the activities of synaptosomal ATPases were not affected by excitatory amino acids at 5 mM. The results suggest that glutamate or NMDA induced Ca⁺⁺ influx at synaptosomes exhibits different responses for extracellular Mg⁺⁺ Ex citatory amino acids induced Ca⁺⁺ influx at synaptosomes may be associated with increased permeability of membrane for Na⁺⁺ and Ca⁺⁺ except K⁺⁺ and membrane depolarization due to increased ionic permeability.

The role of inversely operating glutamate transporter in the paradoxical analgesia produced by glutamate transporter inhibitors

Kim, W.Mo.,Chae, J.W.,Heo, B.H.,Park, K.S.,Kim, H.S.,Lee, H.G.,Choi, J.I.,Yoon, M.H. North-Holland 2016 European journal of pharmacology Vol.793 No.-

<P>Controlling extracellular glutamate level in a physiological range is important to maintain normal sensory transmission. Here, we investigated the paradoxical action of glutamate transporters in the rat formalin test to elucidate a possible role of inversely operating transporters in its analgesic mechanism. The effects of glutamate transporter inhibitor on formalin-induced pain behavior were examined. Then we performed a microdialysis study to clarify the differential change in extracellular glutamate concentration by intrathecal administration of transportable and non-transportable blockers. And we further investigated the mechanism pharmacologically via pretreatment with antagonists of various receptors and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Intrathecally-injected glutamate transporter inhibitors, non-transportable DL-threo-I3-benzyloxyaspartat (TBOA) and transportable trans-pyrrolidine-2,4dicarboxylic acid (t-PDC), produced paradoxical antinociception in the formalin test. In normal rats, inhibition of the glutamate transporter increased extracellular glutamate. In the formalin model rats, TBOA suppressed while t-PDC enhanced glutamate release. When tPDC was pretreated 30 min prior to formalin injection, glutamate release was blocked. Blocking alpha-2 adrenergic receptors reversed the tPDC analgesia. Increased apoptosis was not apparent in the spinal dorsal horn of tPDC-treated rats compared to the control group. These data suggest that glutamate transporters in a formalin-induced pain state work in a reverse mode and can be blocked from releasing glutamate by TBOA and preloaded tPDC. The analgesic mechanism of TBOA may be related to the blockade of inversely operating transporter, and that of tPDC may be associated with the activation of noradrenergic neurotransmission but not with dorsal horn neurotoxicity.</P>

Glutamate-and NMDA-induced calcium influx at synaptosomes and the difference of their actions

이정수,심재건,신용규,이광수,Lee, Chung-Soo,Sim, Jae-Keon,Shin, Yong-Kyoo,Lee, Kwang-Soo The Korean Society of Pharmacology 1988 대한약리학잡지 Vol.24 No.1

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. Glutamate and aspartate may evoke an increase in membrane permeability to monovalent cations and $Ca^{++}$. However, it is uncertain whether $Ca^{++}$ influx is mediated by voltage dependent $Ca^{++}$ channels or by excitatory amino acid activated channels. In addition, the influences of excitatory amino acids on $Ca^{++}$ uptake by neuronal tissues as well as the responses of their actions to extracellular $Mg^{++}$ concentration are different. $K^{+}$ induced $Ca^{++}$ uptake by synaptosomes was dependent on extracellular $Mg^{++}$ up to 5 mM and at concentration of 10 mM, $Ca^{++}$ influx was rather reduced. In $Na^{+}$ rich media, glutamate-and aspartate-induced $Ca^{++}$ uptake was increased by $Mg^{++}$ in a dose independent manner. However, the response for NMDA was inhibited by $Mg^{++}$ at concentrations above 2 mM. $K^+$-and glutamate-induced $Ca^{++}$ influx s were inhibited by 2,4-dinitrophenol, chlorprom-azine and verapamil but not by tetraethylammonium chloride. Tetrodotoxin effectively inhibited the action of glutamate but did not affect that of $K^+$. The response for MNDA was inhibited by 2, 4-dinitrophenol and tetrodotoxin, slightly inhibited by verapamil, and not affected by tetraethylammonium chloride. In $Na^{++}$ rich medium, depolarizing action of glutamate, aspartate and MNDA on synaptosomes was not demonstrated, whereas these agents stimulated $Ca^{++}$ uptake and caused $Ca^{++}$ influx induced depolarization at mitochondria. On the other hand, the activities of synaptosomal ATPases were not affected by excitatory amino acids at 5 mM. The results suggest that glutamate or NMDA induced $Ca^{++}$ influx at synaptosomes exhibits different responses for extracellular $Mg^{++}$ Ex citatory amino acids induced $Ca^{++}$ influx at synaptosomes may be associated with increased permeability of membrane for $Na^{++}$ and $Ca^{++}$ except $K^{++}$ and membrane depolarization due to increased ionic permeability.

Molecular Cloning and Characterization of a Large Subunit of Salmonella typhimurium Glutamate Synthase (GOGAT) Gene in Escherichia coli

정태욱,이동익,김동수,진언호,박춘,김종국,김민곤,하상도,김근성,이규호,김광엽,정덕화,김철호 한국미생물학회 2006 The journal of microbiology Vol.44 No.3

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%. Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

Quercetin ameliorates glutamate toxicity-induced decrease in parvalbumin expression and rise in intracellular calcium in HT22 cells

Ju-Bin Kang,Dong-Ju Park,Phil-Ok Koh 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Glutamate is the main excitatory neurotransmitter in neurons. However, excessive glutamate causes excitatory toxicity and increases intracellular calcium, leading to neuronal cell death. Parvalbumin is a calcium binding protein that regulates calcium homeostasis. Quercetin is a polyphenol that present in plant flavonoids and has neuroprotective effects against neurodegenerative diseases. We investigated whether quercetin regulates apoptosis through the expression of pavabumin in neuronal damage caused by glutamate. Glutamate was treated in hippocampal-derived cell lines, and quercetin and/or vehicle was treated 1 h before glutamate exposure. Cells were collected for experimental procedure 24 h after glutamate treatment. Glutamate exposure reduced cell viability and increased intracellular calcium concentration. Quercetin treatment alleviated these changes and exerts neuroprotective effects against glutamate toxicity. Moreover, glutamate exposure reduced parvalbumin expression and quercetin treatment attenuated the decrease in paralbumin expression by glutamate. These changes were confirmed using Western blot and immunocytochemical techniques. Glutamate treatment increased caspase-3 expression, quercetin attenuated this increase caused by glutamate toxicity in both parvalbumin siRNA transfected and non-transfected cells. However, the alleviative effect of quercetin was significant in non-transfected conditions. Caspase-3 is a representative apoptosis associated protein. Glutamate also induced a decrease in bcl-2 and increase in bax, quercetin alleviated glutamate-induced these changes. Bcl-2 expression in siRNA transfected cells was lower than in non-transfected cells. However, the expression of bax was high in siRNA transfected cells. Maintenance of proper calcium concentration is important for cell survival because calcium overload causes cell death. These results indicate that parvalbumin contributes to the prevention of apoptosis and quercetin modulates parvalbumin expression in glutamate-exposured cells. Maintenance of proper calcium concentration is important for cell survival because calcium overload causes cell death. Thus, the results of this study suggest that quercetin can perform neuroprotective function against glutamate toxicity by attenuating intracellular calcium overload and modulating bcl-2 family proteins and caspase-3 through regulating of pavalbumin expression. This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF- 2018R1D1A1B07044074).

Peroxiredoxin 5 Inhibits Glutamate-Induced Neuronal Cell Death through the Regulation of Calcineurin-Dependent Mitochondrial Dynamics in HT22 Cells

Kim, Mi Hye,Lee, Hong Jun,Lee, Sang-Rae,Lee, Hyun-Shik,Huh, Jae-Won,Bae, Yong Chul,Lee, Dong-Seok American Society for Microbiology 2019 Molecular and cellular biology Vol.39 No.20

<P>Glutamate is an essential neurotransmitter in the central nervous system (CNS). However, high glutamate concentrations can lead to neurodegenerative diseases. A hallmark of glutamate toxicity is high levels of reactive oxygen species (ROS), which can trigger Ca<SUP>2+</SUP> influx and dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Peroxiredoxin 5 (Prx5) is a well-known cysteine-dependent peroxidase enzyme.</P><P>Glutamate is an essential neurotransmitter in the central nervous system (CNS). However, high glutamate concentrations can lead to neurodegenerative diseases. A hallmark of glutamate toxicity is high levels of reactive oxygen species (ROS), which can trigger Ca<SUP>2+</SUP> influx and dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Peroxiredoxin 5 (Prx5) is a well-known cysteine-dependent peroxidase enzyme. However, the precise effects of Prx5 on glutamate toxicity are still unclear. In this study, we investigated the role of Prx5 in glutamate-induced neuronal cell death. We found that glutamate treatment induces endogenous Prx5 expression and Ca<SUP>2+</SUP>/calcineurin-dependent dephosphorylation of Drp1, resulting in mitochondrial fission and neuronal cell death. Our results indicate that Prx5 inhibits glutamate-induced mitochondrial fission through the regulation of Ca<SUP>2+</SUP>/calcineurin-dependent dephosphorylation of Drp1, and it does so by scavenging cytosolic and mitochondrial ROS. Therefore, we suggest that Ca<SUP>2+</SUP>/calcineurin-dependent mitochondrial dynamics are deeply associated with glutamate-induced neurotoxicity. Consequently, Prx5 may be used as a potential agent for developing therapies against glutamate-induced neurotoxicity and neurodegenerative diseases where it plays a key role.</P>

Chlorobium limicola f. thiosulfatophilum NCIB 8327에서의 광수소발생 조절 기작에 대하여

나종욱,강사욱 한국미생물학회 1992 미생물학회지 Vol.30 No.6

녹색황세균인 Chlorobium limicola f.thiosulfaphilum NCIB 8327 의 성장은 암모늄, glutamine, glutamate 와 질소가스를 각각의 질소원으로 사용하여 배양한 것 중에서 질소가스를 제외하고는 거의 일정하였고, 황화수소만 있을 경우보다 티오황산을 첨가하였을 때 좀 더 잘자랐고, 아세트산을 더 첨가하였을 때 매우 잘 자랐다. 4가지 서로 다른 질소원으로 키운 세포들 중에서 glutamine synthetase 의 specific activity 는 glutamate 를 질소원으로 키운 세포의 파쇠액에서 최고 높았지만, glutamate synthase 의 경우는 거의 일정하였다. Glutamate 에서 키운 세포의 파쇠액 중에서 반응액의 암모늄의 농도가 높아진 경우, Glutamine synthetase 의 활성은 낮아지고, glutamate synthase 의 활성은 일정하며, glutamate dehydrogenase 의 활성은 높아졌다. 암모늄의 농도를 달리하여 키운 세포의 파쇄액들 중에서 반응액의 암모늄이온의 농도가 높아짐에 따라 높은 농도의 암모늄이온에서 키운 세포의 파쇄액에서의 glutamine synthetase 의 활성이 비교적 덜 불활성화 되엇다. Glutamine synthetase 는 methionine sulfoximine 의 농도라 높아짐에 다라 더 빨리 불활성화되었다. Glutamine synthetase 는 methionine sulfoximine 의 농도가 높아짐에 따라 더 빨리 불활성화되었다. Glutamine synthetase 는 빛에 있을 경우 활성이 증가하였고, 어두운 곳에서는 활성이 점차 낮아졌다. 온정한 세포에서의 수소발생은 빛에 의존하였고, 첨가된 암모늄 이온에 의해 저해되지만, netguibube sulfoximine 에 의해 곧바로 회복되었다. 수소발생이 빛에 의존하고, 암모늄이온에 의해 쉽게 저해되었다. Methionine sulfoximine 에 의해 빠르게 회복되는 것으로 보아, 본 균주는 nitrogenase 에 의해 수소밸생이 일어나며 glutamine synthetase 의 간접적인 조절을 받는 것으로 추정된다. Chlorobium liimicola f. thiosulfatophilum NCIB 8327 was grown on modified Pfennig's medium using ammonium chloride. glutamine. glutamate, or dinitrogen gas as nitrogen sources. Except for the case of dinitrogen gas. the extent of gro\\1h was almost the s~me. The specific activity of glutamine synthetase in crude extracts is the highest in the cells which were grown on the medium containing glutamate. hut that of glutamate synthase is uniform for all four nitrogen sources. When the concentration of ammonium ions increases in the reaction mixture. the specific activity of glutamine synthetase in crude extract from the cells grown on glutamate decreases. hut that of glutamate dehydrogenase increases. whereas that of glutamate synthase remains unchanged. When the concentration of methionine sulfoximine increases, the activity of glutamine synthetases decreases rapidly. On the other hand. when the concentration of ammonium ions increases in the reaction mixture gradually. the activity of glutamine synthetase from the cells grown on higher concentration of ammonium ions less decreases. In the presence of light. the activity of glutamine synthetase increases. hut in the dark it decreases gradually. The production of hydrogen in intact cells depends on light. It is inhihited by adding ammonium ions. hut restores immediately hy adding methionine sulfoximine. The produclion of hydrogen in this strain can he mediated by nitrogenase only. and regulated hy glutamine synthetase.

Inhibitory Effects of Ginsenosides on Glutamate-Induced Swelling of Cultured Astrocytes

Seong, Yeon-Hee,Koh, Sang-Bum,Kim, Hack-Seang The Korean Society of Ginseng 2000 Journal of Ginseng Research Vol.24 No.3

Effects of ginsenosides (Rb$_1$, Rb$_2$, Rc, Re, Rg$_1$, Rf) on L-glutamate (glutamate)-induced swelling of cultured astrocytes from rat brain cerebral cortex were studied. Following the exposure to 0.5mM glutamate for 1 hr, the intracellular water space (as measured by [$^3$H]O-methyl-D-glucose uptake) of astrocytes increased by about two-fold. Simultaneous addition of ginsenosides Rb$_2$ and Rc with glutamate reduced the astrocytic swelling in a dose-dependent manner. These ginsenosides at 0.5 mg/ml did not affect the viability of astrocytes for up to 24 hr which was determined by a colorimetric assay (MTT assay) for cellular growth and survival. These ginsenosides at 0.3 mg/ml inhibited the increase of intracellular Ca$\^$2+/ concentration ([Ca$\^$2+/]$\_$i/) induced by glutamate. These data suggest ginsenosides Rb$_2$ and Rc prevent the cell swelling of astrocytes induced by glutamate, maybe via inhibition of Ca$\^$2+/ influx. 흰쥐 대뇌겉질로부터 별아교세포를 배양하여 흥분성아미노산인 L-glutamate에 의하여 유발되는 세포종창(astrocytic swelling)에 대한 ginsenosides의 억제효과를 검토하였다. Glutamate(0.5 mM)를 세포에 가하고 1시간동안 배양하면 swelling을 일으켜, 세포내의 물의 용적([$^3$H]OMG의 uptake량으로 측정)은 대조세포에 비하여 약 2배의 증가를 나타냈다. Glutamate와 함께 ginsenosides Rb$_2$와 Rc를 가하고 배양하면 glutamate에 의한 astrocytic swelling이 용량의존적으로 감소하였다. 세포는 Rb$_2$와 Rc(0.5 mg/ml)에 24시간까지 노출시켜도 MTT reduction이 감소하지 않는 것으로 보아 이 ginsenosides에 의한 swelling의 억제효과는 세포막의 손상에 의한 것이 아님을 알 수 있었다. Rb$_2$와 Rc는 glutamate에 의한 세포내 $Ca^{2+}$농도의 상승을 억제하였다. 따라서 Rb$_2$와 Rc는Ca$^{2+}$의 유입을 억제하므로서 glutamate에 의한 astrocytic swelling을 억제하는 것으로 생각된다.

Effects of clonidine on the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes

우재희,한종인,백희정,이희승 대한마취통증의학회 2012 Korean Journal of Anesthesiology Vol.62 No.3

Background: Clonidine has been shown to be a potent neuroprotectant by acting at α2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate. Methods: EAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 μM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 × 10-7 M clonidine. Results: Clonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50× 10-7 M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate. Conclusions: These results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine. Background: Clonidine has been shown to be a potent neuroprotectant by acting at α2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate. Methods: EAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 μM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 × 10-7 M clonidine. Results: Clonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50× 10-7 M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate. Conclusions: These results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine.

육군자탕(六君子湯)이 Glutamate에 의한 C6 신경교세포의 Apoptosis에 미치는 영향

장원석,신용진,고석재,하예진,권영미,신선호,Jang, Won-Seok,Shin, Yong-Jeen,Ko, Seok-Jae,Ha, Ye-Jin,Kwon, Young-Mi,Shin, Sun-Ho 대한한방내과학회 2010 大韓韓方內科學會誌 Vol.31 No.3

Objective : The water extract of Yukgunja-tang(YGJT) has been traditionally used in treatment of qi deficiency and phlegm in Oriental medicine. However, little is known about the mechanism by which YGJT protects neuronal cells from injury damages. Therefore, this study was designed to evaluate the protective effects of YGJT on C6 glial cells by glutamate-induced cell death. Methods : The present study describes glutamate, which is known as an excitatory neurotransmitter, related with oxidative damages, and YGJT, which shows protective effects against glutamate-induced C6 glial cell death. One of the main mediators of glutamate-induced cytotoxicity was known on the generation of reactive oxygen species(ROS) via activation of NADPH oxidase (NOX). The protective effects of antioxidant(NAC) and NOX inhibitor(apocynin) on the glutamate-induced C6 glial cells were determined by a MTT reduction assay. Result : YGJT inhibited glutamate-induced ROS generation via inhibition of NOX expression on glutamate-stimulated C6 glial cells. Furthermore, YGJT attenuated glutamate-induced caspase activation. These results suggest that YGJT could be a new potential candidate against glutamate-induced oxidative stress and cell death. Conclusion : These findings indicate that in C6 glial cells, ROS plays an important role of glutamate-induced cell death and that YGJT may prevent cell death from glutamate-induced cell death by inhibiting the ROS generation.