• Archives of Pharmacal Research
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• 제28권4호
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• pp.413-420
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• 2005

We previously demonstrated the ability of ginseng saponins (active ingredients of Panax ginseng) to enhance $Ca^{2+}$-activated $Cl^{-}$ current. The mechanism for this ginseng saponin-induced enhancement was proposed to be the release of $Ca^{2+}$ from $IP_{3}-sensitive$ intracellular stores through the activation of PTX-insensitive $G\alpha_{q/11}$ proteins and PLC pathway. Recent studies have shown that calmodulin (CaM) regulates $IP_{3}$ receptor-mediated $Ca^{2+}$ release in both $Ca^{2+}-dependent$ and -independent manner. In the present study, we have investigated the effects of CaM on ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current responses in Xenopus oocytes. Intraoocyte injection of CaM inhibited ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement, whereas co-injection of calmidazolium, a CaM antagonist, with CaM blocked CaM action. The inhibitory effect of CaM on ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement was dose- and time-dependent, with an $IC_{50} of 14.9\pm3.5 {\mu}M$. The inhibitory effect of CaM on saponin's activity was maximal after 6 h of intraoocyte injection of CaM, and after 48 h the activity of saponin recovered to control level. The half-recovery time was calculated to be $16.7\pm4.3 h$. Intraoocyte injection of CaM inhibited $Ca^{2+}$-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement and also attenuated $IP_{3}$-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement. $Ca^{2+}$/CaM kinase II inhibitor did not inhibit CaM-caused attenuation of ginseng saponin-induced $Ca^{2+}$-activated $Cl^{-}$ current enhancement. These results suggest that CaM regulates ginseng saponin effect on $Ca^{2+}$-activated $Cl^{-}$ current enhancement via $Ca^{2+}$-independent manner.

• The Korean Journal of Physiology
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• 제28권2호
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• pp.151-157
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• 1994

Intracellular free $Ca^{2+}$ contributes to regulation of various events occurring in vascular smooth muscle cells. One of these events is modulating the membrane iou currents. Single smooth muscle cells were isolated from rabbit mesenteric artery. Three kinds of $Ca^{2+}-activated\;current$ were studied with the patch clamp method. $Ca^{2+}-activated\;K^+\;current$ with a large oscillation was recorded in the depolarized potential range. The single channel conductance of this current was about 250 pS. It was abolished by replacing intracellular $K^+\;with\;Cs^+$. A $Ca^{2+}-activated$ nonselective cation current was observed in both the depolarized and hyperpolarized potential ranges. And it was blocked by replacement of extracellular $Na^+$ with N-methylglucamine (NMG) or extracellular application of $Cd^{2+}$. $Ca^{2+}-activated\;Cl^-\;current$ was revealed in the whole voltage range and was blocked by niflumic acid. These results indicate that at least three kinds of $Ca^{2+}-activated$ ionic currents exist in smooth muscle cells from rabbit superior mesenteric artery.

• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.27-32
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• 2009

The effects of oxidized low-density lipoprotein(OxLDL) and its major lipid constituent lysophosphatidylcholine(LPC) on $Ca^{2+}$ entry were investigated in cultured human umbilical endothelial cells(HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular $Ca^{2+}$ concentration($[Ca^{2+}]_i$), and the increase of $[Ca^{2+}]_i$ by OxLDL or by LPC was inhibited by $La^{3+}$ or heparin. LPC failed to increase $[Ca^{2+}]_i$ in the presence of an antioxidant tempol. In addition, store-operated $Ca^{2+}$ entry(SOC), which was evoked by intracellular $Ca^{2+}$ store depletion in $Ca^{2+}$-free solution using the sarcoplasmic reticulum $Ca^{2+}$ pump blocker, 2, 5-di-t-butyl-l,4-benzohydroquinone(BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased $[Ca^{2+}]_i$ and simultaneously activated non-selective cation(NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, $La^{3+}$ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular $Ca^{2+}$ to 1 ${\mu}M$ activated large-conductance $Ca^{2+}$-activated $K^+(BK_{ca})$ current spontaneously, and this activated $BK_{ca}$ current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates $Ca^{2+}$-permeable $Ca^{2+}$-activated NSC current and $BK_{ca}$ current simultaneously, thereby increasing SOC.

• Journal of Ginseng Research
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• 제24권4호
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• pp.168-175
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• 2000

Xenopus oocytes를 이용하여 인삼의 유효 성분으로 알려진 Ginseng total saponin(GTS)의 신호 전달 기작을 two electrode voltage clamp 방법을 이용하여 연구하였다. GTS는 세포 바깥에 처리했을 때 -2OmV보다 더 positive한 voltage에서 커다란 outward current를 유도하였다. 그러나, 세포 안쪽에 GTS를 injection할 경우 아무런 효과가 없는 것으로 나타났다. GTS처리에 의한 outward current유발 효과는 GTS 투여 용량에 의존적인 것으로 나타났다(EC$_{50}$ : 4.4 $\mu\textrm{g}$/ml). GTS의 작용은 $Ca^{2+}$-activated Cl- channel blocker인 niflumic acid에 의하여 차단되었다. 칼슘 chelator인 BAPIA와 IP$_3$ 수용체 길항제인 heparin을 세포내 injection에 의하여 차단되었다. 또한 active phospholipase C inhibitor(PLC)인U-73122를 세포 바깥에 전처리할 경우에도 GTS의 작용이 부분적으로 억제되는 것으로 나타났다. 백일해 독소를 전처리할 경우GTS의 작용은 억제되지 않은 것으로 나타났으나, GTP analog인 GTP${\gamma}$S를 세포내 injection할 경우 GTS의 작용은 억제되는 것으로 나타났다. 이러한 연구 결과는 GTS가 oocytes세포막 성분과 상호 작용에 의하여 $Ca^{2+}$-activated Cl- channel이 열리도록 하고, 이 과정에 PLC활성 및 백일해 독소에 민감하지 않은 G단백질활성 및 IP3에 민감한 세포내 $Ca^{2+}$-activated로부터 칼슘 방출을 유도하는 것으로 나타났다났다

• Environmental Engineering Research
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• 제17권4호
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• pp.197-203
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• 2012

Phosphorus removal during discharge of wastewater is required to achieve in a very high level because eutrophication occurs even at a very low phosphorus concentration. However, there are limitations in the traditional technologies in the removal of phosphorus at very low concentration, such as at a level lower than 0.1 mg/L. Through a series of experiments, a possible technology which can remove phosphate to a very low level in the final effluent of wastewater was suggested. At first Al, Zn, Ca, Fe, and Mg were exposed to phosphate solution by impregnating them on the surface of activated alumina to select the material which has the highest affinity to phosphate. Kinetic tests and isotherm tests on phosphate solution have been performed on four media, which are Ca-impregnated activated alumina, activated alumina, Ca-impregnated loess ball, and loess ball. Results showed that Ca-impregnated activated alumina has the highest capacity to adsorb phosphate in water. Scanning electron microscope image analysis showed that activated alumina has high void volume, which provides a large surface area for phosphate to be adsorbed. Through a continuous column test of the Ca-impregnated activated alumina it was discovered that about 4,000 bed volumes of wastewater with about 0.2 mg/L of phosphate can be treated down to lower than 0.14 mg/L of concentration.

• The Korean Journal of Physiology
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• 제28권2호
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• pp.143-150
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• 1994

The whole-cell mode of the patch clamp technique was used to study $Ca^{2+}-activated\;Cl^-\;current$ $(I_{Cl_{Ca}})$ in gastric antral myocytes. Extracellular application of caffeine evoked $Ca^{2+}-activated\;current$. In order to isolate the chloride current from background current, all known systems were blocked with specific blockers. The current-voltage relationship of caffeine-induced current showed outward rectification and it reversed at around $E_{Cl^-}$. The shift of reversal potential upon the alteration of external and internal chloride concentrations was well fitted with results which were calculated by the Nernst equation. Extracellular addition of N-phenylanthranilic acid and niflumic acid which are known anion channel blockers abolished the caffeine induced current. Intracellular application of a high concentration of EGTA also abolished this current. Application of c-AMP, c-GMP, heparin, or $AIF^-_4$ made no remarkable changes to this current. Sodium replacement with the impermeable cation N-methylglucamine or with $Cd^{2+}$ rarely affected this current. From the above results it is suggested that the caffeine induced current was a $Cl^-$ current and it was activated by intracellular $Ca^{2+}$.

• The Korean Journal of Physiology and Pharmacology
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• 제10권2호
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• pp.95-99
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• 2006

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of $Ca^{2+}$ -activated $K^+$ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward $K^+$ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of $K^+$ current was increased by NS1619, a specific large-conductance $Ca^{2+}$-activated $K^+$ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance $Ca^{2+}$-activated $K^+$ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various $Ca^{2+}$-activated $K^+$ channel modulators were added into culture media for 1∼3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance $Ca^{2+}$-activated $K^+$ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.

• The Korean Journal of Physiology and Pharmacology
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• 제11권5호
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• pp.215-219
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• 2007

Small and large conductance $Ca^{2+}$-activated $K^+(SK_{Ca}\;and\;BK_{Ca})$ channels are implicated in the modulation of neuronal excitability. We investigated how changes in peripheral $K_{Ca}$ channel activity affect mechanical sensitivity as well as the afferent fiber type responsible for $K_{Ca}$ channel-induced mechanical sensitivity. Blockade of $SK_{Ca}$ and $BK_{Ca}$ channels induced a sustained decrease of mechanical threshold which was significantly attenuated by topical application of capsaicin onto afferent fiber and intraplantar injection of 1-ethyl-2-benzimidazolinone. NS1619 selectively attenuated the decrease of mechanical threshold induced by charybdotoxin, but not by apamin. Spontaneous flinching and paw thickness were not significantly different after $K_{Ca}$ channel blockade. These results suggest that mechanical sensitivity can be modulated by $K_{Ca}$ channels on capsaicin-sensitive afferent fibers.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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• pp.109-117
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• 1998

Role of $Ca^{2+}$/calmodulin complex in intracellular $Ca^{2+}$ mobilization in neutrophils has not been clearly elucidated. In this study, effects of chlorpromazine, trifluoperazine and imipramine on the intracellular $Ca^{2+}$ mobilization, including $Ca^{2+}$ influx, in C5a-activated neutrophils were investigated. Complement C5a- stimulated superoxide production and myeloperoxidase release in neutrophils were inhibited by chlorpromazine, trifluoperazine and imipramine, except no effect of imipramine on myeloperoxidase release. A C5a-elicited elevation of [$Ca^{2+}$]i in neutrophils was inhibited by chlopromazine, trifluoperazine, imipramine, staurosporine, genistein, EGTA, and verapamil but not affected by pertussis toxin. The intracellular $Ca^{2+}$ release in C5a-activated neutrophils was not affected by chlorpromazine and imipramine. Chlorpromazine and imipramine inhibited $Mn^{2+}$ influx by C5a-activated neutrophils. Thapsigargin-evoked $Ca^{2+}$ entry was inhibited by chlorpromazine, trifluoperazine, imipramine, genistein, EGTA and verapamil, while the effect of staurosporine was not detected. The results suggest that $Ca^{2+}$/calmodulin complex is involved in the activation process of neutrophils. The depressive action of calmodulin inhibitors on the elevation of cytosolic $Ca^{2+}$ level in C5a-activated neutrophils appears to be accomplished by inhibition of $Ca^{2+}$ influx from the extracellular medium.

• Molecules and Cells
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• 제44권2호
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• pp.88-100
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• 2021

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca2+-activated ion channel and Ca2+-activated phospholipid scramblase activities, requiring a high intracellular Ca2+ concentration (e.g., half-maximal effective Ca2+ concentration [EC50] of [Ca2+]i > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca2+-activated chloride channel exhibiting higher Ca2+ sensitivity (EC50 of 1 μM) than ANO6, suggested that a homologous Ca2+-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca2+ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca2+-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6-1-6 showed higher sensitivity to Ca2+ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca2+ interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca2+-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca2+ sensitivity, implying direct interactions of Ca2+ with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca2+ sensitivity between ANO1 and ANO6.