• The Korean Journal of Physiology
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• 제28권1호
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• pp.51-59
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• 1994

This study was carried out to elucidate the excitatory mechanisms of Substance P in the antral circular muscle, using isometric contraction recording, conventional microelectrode method and whole-cell patch clamp technique. Substance P produced tonic and phasic contractions in a dose-dependent manner and depolarized membrane potential with increased amplitude of slow waves in muscle strips. Voltage-dependent $Ca^{2+}$ currents were increased by the application of Substance P from a holding potential of -60mV to 50mV in 10mV steps and this effect was blocked by the addition of an antagonist. Also Substance P increased transient and spontaneous oscillatory $K^+$ outward currents. The enhanced outward currents were abolished by apamin in dispersed single cells. These results suggest that the depolarization of membrane potential by Substance P activates voltage-dependent $Ca^{2+}$ channels, which represents an excitatory response in the antral circular muscle and led to an increase in $Ca^{2+}\;activated\;K^+\;currents$.

• Journal of Ginseng Research
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• 제24권1호
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• pp.18-22
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• 2000

앞의 연구에서 우리는 진세노사이드가 신경세포에 존재하는 high-threshold voltage-dependent $Ca^{2+}$ channel을 억제한다는 것을 발표하였다. 그러나, 이러한 연구는 진세노사이드가 여러 칼슘 채널subtypes중 어느 특정 칼슘 채널만을 선택적으로 조절한다는 것을 보여주지는 않았다. 따라서 이 연구에서 우리는 여러 칼슘 채널subtypes에 선택적으로 작용하는 약물 혹은 toxins을 이용하여 진세노사이드가 어느 종류의 칼슘 채널 subtypes를 억제하는가를 bovine chromaffin cell을 이용하여 연구하였다. 사용한 물질은nimodipine(L-type 칼슘 채널 길항제), $\omega$-conotoxin GVIA (N-type $Ca^{2+}$ channel 길항제), $\omega$-agatoxin IVA(P-type 칼슘 채널 길항제)이었다. 연구 결과 진세노사이드는 bovine chromaffin 세포에 존재하는 high-threshold 칼슘 current을 투여 농도별로 억제하였다. $IC_{50}$/은 약 120 $\mu$g/ml인 것으로 나타났다. nimodipine은 진세노사이드에 의한 칼슘 currents억제 작용에 영향을 미치지 않은 것으로 나타났다. 그러나, $\omega$-conotoxin GVIA, $\omega$-agatoxin IVA 및 nimodipine+$\omega$-conotoxin GVIA+$\omega$-agatoxin IVA을 처리한 세포에서는 진세노사이드에 의한 칼슘 currents억제 작용이 현저하게 줄어 들었다. 이러한 연구 결과들은 진세노사이드가 L-type 칼슘 채널은 억제하지 않고, 주로 N-, p-, 및 Q-type칼슘 채널을 억제한다는 것을 보여주고 있다

• 한국의학물리학회지:의학물리
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• 제15권4호
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• pp.220-227
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• 2004

N형 칼슘통로의 비활성화기전에 관하여는 아직까지도 막전압의존성 기전과 칼슘의존성 기전간에 논란이 계속되고 있다. 2003년에 의학물리에 발표한 논문1)에서 본 연구자는 N형 칼슘통로의 비활성화 기전은 2가지 성분 -빠른 성분과 느린 성분을 가지고 있고 빠른 성분은 칼슘의존적이 아니며 오직 느린 성분만이 칼슘의존적일 가능성을 제시하였다. 본 논문에서는 막전압의존성 기전이 옳건 칼슘의존성 기전이 옳건 간에 세포 신호전달 체계로서 비활성화와 연계된 기전이 필요하므로 이러한 맥락에서 인산화 기전을 연구하였다. 흰쥐 경동맥 결절뉴론을 단일 세포로 얻은 후 whole cell patch clamp technique를 사용하여 N형 칼슘전류를 기록하고 대조 세포내액을 사용하였을 때와 phosphatase inhibitor인 okadaic acid를 포함한 세포내액을 사용하였을 때의 차이를 비교하였다. Okadaic acid에 의하여 비활성화정도가 증가되었고 이러한 okadaic acid 효과는 주로 N형 통로를 통하여 영향을 미침을 N형 칼슘통로 억제제인 $\omega$-conotoxin GVIA를 사용함으로써 확인하였다. Okadaic acid에 의한 비활성화 증가 효과는 protein kinase를 비특이적으로 억제하는 staurosporine에 의하여 억제되었고 또한 calmodulin dependent protein kinase의 특이적 억제제인 lavendustin C에 의하여 억제되었으므로 인산화과정이 N형 칼슘통로 비활성화와 관련되어 있고 특히 calmodulin을 통한 인산화과정이 주로 관여함을 확인하였다. 본 연구자가 발표한 선행논문1)에 의해 외부의 2가 양이온에 의해 빠른 비활성화가 진행되며, 본 논문에 의하여 인산화과정에 의해 빠른 비활성화가 촉진된다는 사실이 확인되었다. 그러나 본 연구결과만으로는 인산화과정이 비활성화 자체라고는 볼 수 없으며 단지 인산화과정에 의해 비활성화가 가속되었다고 해석할 수 밖에 없다. 인산화과정이 비활성화자 체인지 여부는 2가 양이온이 칼슘통로에 작용하는 결합부위에 관한 연구 및 인산화 부위가 칼슘통로인지 아니면 다른 조절 부위인지 여부를 확인할 수 있는 연구가 진행되어야 확실히 알 수 있을 것이다.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.26-26
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• 2001

Inactivation of N-type calcium current has been reported to be both voltage dependent and Ca$\^$2＋/ dependent. We have investigated the effects of Ba$\^$2＋/ and Ca$\^$2＋/ on N-channel inactivation in rat superior cervical ganglion neurons using the whole cell configuration of patch clamp technique. Inactivation was larger in Ca$\^$2＋/ than in Ba$\^$2＋/ even with 20 mM BAPTA.(omitted)

• Molecules and Cells
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• 제21권1호
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• pp.121-128
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• 2006

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [$Ca^{2+}$]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular $Ca^{2+}$ chelator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was suppressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly delayed by BAPTA-AM or a ROS scavenger. These results suggest that [$Ca^{2+}$]-dependent generation of intracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and induction of cell death, in human umbilical vein endothelial cells.

• 생명과학회지
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• 제27권3호
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• pp.310-317
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• 2017

본 연구진은 HaCaT-ARE-luciferase 세포를 이용하여 400 여개의 약용식물 에탄올 추출물 중 NRF2/ARE 유도효과가 있는 신규 추출물을 검색하였고 이를 통하여 종대황(Rheum undulatum)과 선복화(Inula japonica)의 주정 추출물이 HaCaT-ARE-luciferase 세포에서 ARE 활성을 강하게 유도하는 것을 관찰하였다. 종대황과 선복화 에탄올 추출물은 HaCaT 세포에서 생존(viability)을 증가시켰고 NRF2/ARE에 의존적인 phase II cytoprotective 효소인 heme oxygenase-1 (HO-1)와 NADPH:quinone oxidoreductase-1 (NQO1)의 전사 및 단백질 발현을 강하게 유도하였다. 또한 종대황과 선복화 추출물은 HaCaT 세포에서 TPA로 유도한 세포 내 활성 산소 및 이를 통하여 생성되는 스트레스 마커인 8-hydroxydeoxyguanosine (8-OH-dG)과 4-hydroxynonenal (4HNE)의 발생을 강하게 억제하였다. 본 연구는 종대황과 선복화의 에탄올 추출물이 인간 피부 세포주인 HaCaT 세포에서 NRF2/ARE에 의존적인 유전자 발현의 유도를 통하여 강력한 항산화 효과를 발휘한다는 것을 증명한다.

• The Korean Journal of Physiology and Pharmacology
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• 제4권5호
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• pp.403-408
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• 2000

The outward currents elicited in hamster eggs by depolarizing pulses were studied. The currents appeared to comprise at least two components, a transient outward component $(I_{to})$ and a steady-state outward component $(I_{\infty}).\;I_{to}$ was transiently followed by the cessation of inward $Ca^{2+}$ current $(I_{Ca}),$ and its current-voltage (I-V) relation was a mirror image of that of $(I_{Ca}).$ Either blockade of $(I_{Ca})$ by $Co^{2+}$ or replacement of $Ca^{2+}$ with $Sr^{2+}$ abolished $I_{to}$ without change in $I_{\infty}.$ Intracellular EGTA (10 mM) inhibited $I_{to}$ but not $I_{\infty}.$ suggesting strongly that generation of $I_{to}$ requires intracellular $Ca^{2+}.$ Apamin (1 nM) abolished selectively $I_{to},$ indicatingthat $I_{to}$ is $Ca^{2+}-dependent\;K^+$ current. On the other hand, $I_{\infty}$ was $Ca^{2+}-independent.$ Both $I_{to}$ and $I_{\infty}$ were completely inhibited by internal $Cs^+$ and external TEA. The estimated reversal potential of $I_{to}$ was close to the theoretical $E_K.$ Taken together, both outward currents were carried by $K^+$ channels. From these results, $I_{to}$ is likely to be a current responsible for the hyperpolarizing responses seen in hamster eggs at fertilization.

• The Korean Journal of Physiology and Pharmacology
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• 제2권4호
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• pp.411-417
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• 1998

The role of phospholipase ($A_2\;PLA_2$) in tumor cell growth was investigated using SK-N-MC human neuroblastoma cells. 4-Bromophenacyl bromide (BPB) and mepacrine (Mep), known $PLA_2$ inhibitors, suppressed growth of the tumor cells in a dose-dependent manner without a significant cytotoxicity. Melittin (Mel), a $PLA_2$ activator, enhanced the cell growth in a concentration-dependent fashion. The growth-enhancing effects of Mel were significantly reversed by the co-treatment with $PLA_2$ inhibitors. In addition, Mel induced intracellular $Ca^{2+}$ release from internal stores like as did serum, a known intracellular $Ca^{2+}$ agonist in the tumor cells. Intracellular $Ca^{2+}$ release induced by these agonists was significantly blocked by $PLA_2$ inhibitors at growth-inhibitory concentrations. Arachidonic acid (AA), a product of the $PLA_2-catalyzed$ reaction, induced cell growth enhancement and intracellular $Ca^{2+}$ release. These effects of AA were significantly blocked by BAPTA/AM, an intracellular $Ca^{2+}$ chelator. Taken together, these results suggest that the modulation of $PLA_2$ activity may be one of the regulatory mechanisms of cell growth in human neuroblastoma cells. Intracellular $Ca^{2+}$ may act as a key mediator in the $PLA_2-induced$ growth regulation.

• The Korean Journal of Physiology and Pharmacology
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• 제14권3호
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• pp.163-167
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• 2010

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase $A_2$ ($PLA_2$) isozyme. $PLA_2$ activity was measured using various $PLA_2$ substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[$^{14}C$]arachidonyl phosphatidylcholine ([$^{14}C$]AA-PC), and [$^3H$]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [$_3H$]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited $cPLA_2/sPLA_2$-induced hydrolysis of [$^{14}C$]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on $cPLA_2$ and $sPLA_2$ appeared to be competitive with inhibition constants ($K_i$) of $3.7{\mu}g/ml$ and $12.6{\mu}g/ml$, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both $Ca^{2+}$-dependent $PLA_2$ such as, $cPLA_2$ and $sPLA_2$. Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to $Ca^{2+}$-dependent $PLA_2$ inhibition.

• The Korean Journal of Physiology and Pharmacology
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• 제2권4호
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• pp.419-425
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• 1998

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ($[Ca^{2+}]_i$) mobilization and cell proliferation were investigated. ATP-induced $[Ca^{2+}]_i$ increased in a dose-dependent manner $(10^{-7}\;M{\sim}10^{-3}\;M)$. ATP-induced $[Ca^{2+}]_i$ increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced $[Ca^{2+}]_i$ increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative $P_{2Y}$ receptor antagonist, dose-dependently weakened ATP-induced $[Ca^{2+}]_i$ mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.