• Biomolecules & Therapeutics
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• v.15 no.2
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• pp.73-77
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• 2007

N,N-Dimethyl-D-ribo-phytosphingosine (DMPH) is an N-methyl derivative of sphingosine. In the present paper, we studied effects of DMPH on intracellular Ca$^{2+}$ concentration, pH, glutamate uptake, and cell viability in human 1321N1 astrocytes. DMPH increased intracellular Ca$^{2+}$ concentration and cytosolic pH significantly in a dose-dependent manner. DMPH also inhibited glutamate uptake by 1321N1 astrocytes. Finally, treatment of cells with DMPH for 24 h reduced viability of cells largely and concentration-dependently. In summary, DMPH increased intracellular Ca$^{2+}$ concentration and pH, inhibited glutamate uptake and evoked cytotoxicity in 1321N1 astrocytes. Our observations with DMPH in the 1321N1 astrocytes would enhance understanding of DMPH actions in the brain.

• Natural Product Sciences
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• v.10 no.6
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• pp.289-295
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• 2004

Myristica fragrans seed from Myristica fragrans Houtt (Myristicaceae) has various pharmacological activities peripherally and centrally. The present study aims to investigate the effect of the methanol extract of Myristica fragrans seed (MF) on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in primary cultured rat cerebellar granule neuron. MF, over a concentration range of 0.05 to $5\;{\mu}g/ml$, inhibited NMDA (1 mM)- induced neuronal cell death, which was measured by trypan blue exclusion test and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. MF $(0.5\;{\mu}g/ml)$ inhibited glutamate release into medium induced by NMDA (1 mM), which was measured by HPLC. Pretreatrnent of MF $(0.5\;{\mu}g/ml)$ inhibited NMDA (1 mM)-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that MF prevents NMDA-induced neuronal cell damage in vitro.

• Journal of Chest Surgery
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• v.28 no.8
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• pp.742-746
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• 1995

The present study was aimed at investigating possible transmitter mechanisms in the endothelial cell layer in regulating the tone of the vascular smooth muscle. The thoracic aorta was isolated from the anesthetized male white rabbits and its helical strips were prepared. Electrical field stimulation was delivered to platinum wire electrodes positioned parallel to the vessel segment preconstricted with phenylephrine [3.5x10-6 mol/L at a distance of 1.5-2.0 mm. The electrical stimulation [70 V, 5 msec, 0.5-200 Hz caused either relaxation only [34% or a biphasic response [prolonged relaxation following a weak and transient contraction, 66% . The relaxation response was frequency- dependent, and at 200 Hz a complete relaxation was noted. Mechanical rubbing of the endothelial layer abolished or greatly attenuated the relaxation. The relaxation was also markedly attenuated in the presence of NG-nitro- L-arginine methyl ester [10-3mol/L or procaine hydrochloride [3.5x10-4mol/L . Tetrodotoxin,guanethidine, atropine or indomethacin failed to block or enhance the relaxation response to electrical field stimulation. It is concluded that the vascular endothelium in the aorta contains diffusible substances that regulates the function of the smooth muscle layer, in which relaxation is more prominent than contraction. Their release by the electrical stimualtion in vitro may not involve classic neuronal transmitter release mechanisms or metabolism of arachidonic acids by cyclooxygenase. The release of the relaxing agents may require an increase in cytosolic calcium level. The chemical nature of the relaxant may be, to a large extent, nitric oxide.

• Natural Product Sciences
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• v.9 no.4
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• pp.249-255
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• 2003

Zizypus is one of the herbs widely used in Korea and China due to CNS calming effect. The present study aims to investigate the effect of the methanol extract of Zizyphi Jujube Semen (ZJS) on kainic acid (KA)-induced neurotoxicity in cultured rat cerebellar granule neuron. ZJS, over a concentration range of 0.05 to $5\;{\mu]g/ml$, inhibited KA $(500\;{\mu}M)-induced$ neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. Pretreatment of ZJS $(0.5\;{\mu}g/ml)$ inhibited KA$(50\;{\mu}M)$-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). ZJS $(0.5\;{\mu}g/ml)$ inhibited glutamate release into medium induced by KA $(500\;{\mu}M)$, which was measured by HPLC. These results suggest that ZJS prevents KA-induced neuronal cell damage in vitro.

• The Korea Journal of Herbology
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• v.20 no.2
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• pp.115-125
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• 2005

Objectives : Polygalae Radix (PR) from Polygalae tenuifolia (Polygalaceae) has been clinically used as a sedative, anti-inflammatory, and anti-bacterial agent. To extend pharmacological effects of PR in the central nervous system (CNS) on the basis of its CNS protective effect, the present study was conducted to identify the effect of PR, whether it shows the neuroprotective action against excitatory neurotoxicity. Methods : To identify the protective effect of PR to excitatory neuro-toxic agent, the present study was focused on the PR effect on cell death, that was caused by applying NMDA to nerve cell, elevation of $(Ca^{2+})_i$, releasement of glutamate, and ROS generation. Result : 1. PR methanol extract, at the concentration range of 0.05 to 5 g/ml, significantly inhibited NMDA (1 mM)-induced neuronal cell death as well as MK-801 (non competitive NMDA antagonist). 2. PR methanol extract $(0.5\;{\mu}g/ml)$ inhibited NMDA (1 mM)-induced elevation of cytosolic calcium concentration $[Ca^{2+}]_i$. NMDA application in the presence of MK-801 $(10\;{\mu}M)$ failed to produce the increase of $[Ca^{2+}]_i$ through all the measurement time. 3. PR methanol extract $(0.5\;{\mu}g/ml)$ inhibited the NMDA-induced elevation of glutamate release. Also, MK-801 showed similar protective effects. 4. PR methanol extract $(0.5\;{\mu}g/ml)$ inhibited the NMDA-induced elevation of ROS generation. Also, MK-801 showed similar protective effects. Conclusion : The present study provides the availability of PR to exert its protective effect on the neuronal cell death in various neurodegenerative pathophysiological conditions.

• Korean Journal of Medicinal Crop Science
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• v.11 no.4
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• pp.298-305
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• 2003

Polygalae Radix (PR) from Polygala tenuifolia. (Polygalaceae) is traditionally used in China and Korea, since this herb has a sedative, antiinflammatory, and antibacterial agent. To extend pharmacological actions of PR in the CNS on the basis of its CNS inhibitory effect, the present study examined whether PR has the neuroprotective action against kainic acid (KA) -induced cell death in primarily cultured rat cerebellar granule neurons. PR, over a concentration range of 0.05 to $5{\mu}g/ml$ inhibited KA $(500\;{\mu}M)$-induced neuronal cell death, which was measured by a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl-tetrazolium bromide (MTT) assay. PR $(0.5{\mu}g/ml)$ inhibited glutamate release into medium induced by KA $(500\;{\mu}M)$, which was measured by HPLC. Pretreatment of PR $(0.5{\mu}g/ml)$ inhibited KA $(500\;{\mu}M)$-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$ which was measured by a fluorescent dye, Fura 2-AM, and generation of reactive oxygen species (ROS). These results suggest that PR prevents KA-induced neuronal cell damage in vitro.

• Korean Journal of Medicinal Crop Science
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• v.13 no.2
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• pp.95-102
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• 2005

Caulis Bambusae in Taenia is widely used in Korea and China due to its various pharmacological activity. The present study aims to investigate the effect of the methanol extract of Caulis Bambusae in Taenia (CB) from Phyllostachys nigra Munro var. henonis Stapf (Gramineae) on amyloid ${\beta}$ protein (25-35) $(A{\beta}\;(25-35))$, a synthetic 25-35 amyloid peptide, -induced neurotoxicity using cultured rat cortical neurons. CB, over a concentration range of $10-50{\mu}g/{\mu}l$, inhibited the $A{\beta}\;(25-35)\;(10\;{\mu}M)$-induced neuronal cell death, as assessed by a 3-[4,5-dimethyIthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. CB $(50\;{\mu}g/{\mu}l)$ inhibited glutamate release into medium induced by $10\;{\mu}M\;A{\beta}$, (25-35) which was measured by HPLC. Pretreatment of CB $(50\;{\mu}g/{\mu}l)$ inhibited $10{\mu}M\;A{\beta}$ (25-35)-induced elevation of cytosolic calcium concentration $([Ca^{2+}]_c)$, which was measured by a fluorescent dye, fluo-4 AM, and generation of reactive oxygen species. These results suggest that CB prevents $A{\beta}$ (25-35)-induced neuronal ell damage in vitro.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.401-408
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• 2009

$K^+$-$Cl^-$-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase $A_2$ ($PLA_2$)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced $K^+$ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7 -dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1Hinden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activatorinduced $K^+$ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calciumindependent $PLA_2$ ($iPLA_2$), whereas it was not significantly altered by arachidonyl trifluoromethylketone ($AACOCF_3$) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic $PLA_2$ ($cPLA_2$) and the secretory $PLA_2$ ($sPLA_2$), respectively. NEM increased AA liberation in a doseand time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas $AACOCF_3$ and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that $iPLA_2$-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.45-45
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• 2003

We isolated a novel ankyrin-repeat containing protein, rSIAP (rSlo Interacting Ankyrin-repeat Protein), as an interacting protein to the cytosolic domain of the alpha-subunit of rat large-conductance Ca$\^$2+/-activated K$\^$+/ channel (rSlo) by yeast two-hybrid screening. Affinity pull-down assay showed the direct and specific interaction between rSIAP and rSlo domain. The channel-binding proteins can be classified into several categories according to their functional effects on the channel proteins, i.e. signaling adaptors, scaffolding net, molecular tuners, molecular chaperones, etc. To obtain initial clues on its functional roles, we investigated the cellular localization of rSIAP using immunofluorescent staining. The results showed the possible co-localization of rSlo and rSIAP protein near the plasma membrane, when co-expressed in CHO cells. We then investigated the functional effects of rSIAP on the rSlo channel using electrophysiological means. The co-expression of rSIAP accelerated the activation of rSlo channel. These effects were initiated at the micromolar [Ca$\^$2+/]$\_$i/ and gradually increased as [Ca$\^$2+/]$\_$i/ raised. Interestingly, rSIAP decreased the inactivation kinetics of rSlo channel at micromolar [Ca$\^$2+/]$\_$i/, while the rate was accelerated at sub-micromolar [Ca$\^$2+/]$\_$i/. These results suggest that rSIAP may modulate the activity of native BK$\_$Ca/ channel by altering its gating kinetics depending on [Ca$\^$2+/]$\_$i/. To localize critical regions involved in protein-protein interaction between rSlo and rSIAP, a series of sub-domain constructs were generated. We are currently investigating sub-domain interaction using both of yeast two-hybrid method and in vitro binding assay.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.233-239
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• 2017

Intracellular calcium ($Ca^{2+}$) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide ($H_2O_2$) on intracellular $Ca^{2+}$ accumulation in mouse pancreatic acinar cells. Perfusion of $H_2O_2$ at $300{\mu}M$ resulted in additional elevation of intracellular $Ca^{2+}$ levels and termination of oscillatory $Ca^{2+}$ signals induced by carbamylcholine (CCh) in the presence of normal extracellular $Ca^{2+}$. Antioxidants, catalase or DTT, completely prevented $H_2O_2$-induced additional $Ca^{2+}$ increase and termination of $Ca^{2+}$ oscillation. In $Ca^{2+}$-free medium, $H_2O_2$ still enhanced CCh-induced intracellular $Ca^{2+}$ levels and thapsigargin (TG) mimicked $H_2O_2$-induced cytosolic $Ca^{2+}$ increase. Furthermore, $H_2O_2$-induced elevation of intracellular $Ca^{2+}$ levels was abolished under sarco/endoplasmic reticulum $Ca^{2+}$ ATPase-inactivated condition by TG pretreatment with CCh. $H_2O_2$ at $300{\mu}M$ failed to affect store-operated $Ca^{2+}$ entry or $Ca^{2+}$ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial $Ca^{2+}$ uniporter blocker, failed to attenuate $H_2O_2$-induced intracellular $Ca^{2+}$ elevation. These results provide evidence that excessive generation of $H_2O_2$ in pathological conditions could accumulate intracellular $Ca^{2+}$ by attenuating refilling of internal $Ca^{2+}$ stores rather than by inhibiting $Ca^{2+}$ extrusion to extracellular fluid or enhancing $Ca^{2+}$ mobilization from extracellular medium in mouse pancreatic acinar cells.