• The Korean Journal of Physiology and Pharmacology
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• 제9권1호
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• pp.37-43
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• 2005

Our previous report demonstrated that chick myoblasts are equipped with $Ca^{2+}$-permeable stretchactivated channels and $Ca^{2+}-activated$ potassium channels ($K_{Ca}$), and that hyperpolarization-induced by $K_{Ca}$ channels provides driving force for $Ca^{2+}$ influx through the stretch-activated channels into the cells. Here, we showed that acetylcholine (ACh) also hyperpolarized the membrane of cultured chick myoblasts, suggesting that nicotinic acetylcholine receptor (nAChR) may be another pathway for $Ca^{2+}$ influx. Under cell-attatched patch configuration, ACh increased the open probability of $K_{Ca}$ channels from 0.007 to 0.055 only when extracellular $Ca^{2+}$ was present. Nicotine, a nAChR agonist, increased the open probability of $K_{Ca}$ channels from 0.008 to 0.023, whereas muscarine failed to do so. Since the activity of $K_{Ca}$ channel is sensitive to intracellular $Ca^{2+}$ level, nAChR seems to be capable of inducing $Ca^{2+}$ influx. Using the $Ca^{2+}$ imaging analysis, we were able to provide direct evidence that ACh induced $Ca^{2+}$ influx from extracellular solution, which was dramatically increased by valinomycin-mediated hyperpolarization. In addition, ACh hyperpolarized the membrane potential from $-12.5{\pm}3$ to $-31.2{\pm}5$ mV by generating the outward current through $K_{Ca}$ channels. These results suggest that activation of nAChR increases $Ca^{2+}$ influx, which activates $K_{Ca}$ channels, thereby hyperpolarizing the membrane potential in chick myoblasts.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.463-469
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• 2000

It has been well known that 4-aminopyridine (4-AP) has an excitatory effect on vascular smooth muscle due to causing membrane depolarization by blocking $K^+-channel$. However, we observed that 4-AP had an inhibitory effect on the mesenteric artery of rat. Therefore, we investigated the mechanism of 4-AP-induced vasorelaxation. The mesenteric arcuate artery and its branches were isolated and cut into ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured. 4-AP $(0.1{\sim}10\;mM)$ induced a concentration-dependent relaxation, which was unaffected by NO synthase inhibitor, $N^G-nitro-L-arginine$ methylester $(100\;{\mu}M)$ or soluble guanylate cyclase inhibitor, methylene blue $(100\;{\mu}M).$ Glibenclamide $(100\;{\mu}M)$, ATP-sensitive $K^+$ channel blocker, did not exert any effect on the 4-AP-induced vasorelaxation. 4-AP relaxed the sustained contraction induced by 100 mM $K^+$ or $Ca^{2+}$ ionophore, A23187 $(100\;{\mu}M)$ in a dose-dependent manner. In addition, 4-AP significantly decreased the phasic contractile response to norepinephrine in the absence of extracellular $Ca^{2+}$. However, 4-AP did not block the $^{45}Ca$ influx of rat aorta. From the above results, we suggest that 4-AP may not block the $Ca^{2+}$ influx through $Ca^{2+}-channel,$ but act as a nonspecific vasorelaxant in arterial smooth muscle.

• BMB Reports
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• 제50권6호
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• pp.323-328
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• 2017

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ($[Ca^{2+}]_i$) by releasing $Ca^{2+}$ from intracellular stores and via $Ca^{2+}$ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced $Ca^{2+}$ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated $Ca^{2+}$ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis.

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• The Korean Journal of Physiology
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• 제28권1호
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• pp.37-50
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• 1994

Synthetic potassium channel openers (KCOs) are agents capable of opening K-channels in excitable cells. These agents are known to have their maximal potency in the smooth muscle tissue, especially in the vascular smooth muscle. Much attention has been focused on the type of K-channel that is responsible for mediating the effects of KCOs. As the KCO-induced changes are antagonized by glibenclamide, an $K_{ATP}$ (ATP-sensitive K-channel) blocker in the pancreatic ${\beta}-cell,\;K_{ATP}$ was suggested to be the channel responsible. However, there also are many results in favor of other types of K-channel $$(maxi-K,\;small\;conductance\;K_{Ca,}\; SK_{ATP}) mediating the effects of KCOs. Effects of lemakalim, (-)enantiomer of cromakalim (BRL 34915), on the spontaneous contractions and slow waves, were investigated in the antral circular muscle of the guinea-pig stomach. Membrane currents and the effects on membrane currents and single channel activities were also measured in single smooth muscle cells and excised membrane patches by using the patch clamp method. Lemakalim induced hyperpolarization and inhibited spontaneous contractions in a dose-dependent manner. These effects were blocked by glibenclamide and low concentrations of tetraethyl ammonium (< mM). Glibenclamide blocked the effect of lemakalim on the membrane potential and slow waves. The mechanoinhibitory effect of lemakalim was blocked by pretreatment with glibenclamide. In a whole ceIl patch clamp condition, lemakalim largely increased outward K currents. These outward K currents were blocked by TEA, glibenclamide and a high concentration of intracelIular EGTA (10 mM). Volatage-gated Ca currents were not affected by lemakalim. In inside-out patch clamp experiments, lemakalim increased the opening frequency of the large conductance $Ca^{2+}-activated$ K channels $(BK_{Ca},\;Maxi-K).$ From these results, it is suggested that lemakalim induces hyperpolarization by opening K-channels which are sensitive to internal Ca and such a hyperpolarization leads to the inhibition of the spontaneous contraction.

• Nutrition Research and Practice
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• 제12권3호
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• pp.183-190
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• 2018

BACKGROUND/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic ${\beta}-cells$. MATERIALS/METHOD: INS-1 pancreatic ${\beta}-cells$ were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of $Ca^{2+}$ caused by POE treatment, the effect of POE on intracellular $Ca^{2+}$ in INS-1 pancreatic ${\beta}-cells$ was examined using Fluo-2 AM dye. RESULTS: POE at 10 to $200{\mu}g/mL$ significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the $K{^+}_{ATP}$ channel (blocking insulin secretion) and by verapamil (a $Ca^{2+}$ channel blocker). The insulinotropic effect of POE was not observed under $Ca^{2+}$-free conditions in INS-1 pancreatic ${\beta}-cells$. When the cells were preincubated with a $Ca^{2+}$ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular $Ca^{2+}$, which closely correlated with increases in the levels of insulin secretion. CONCLUSIONS: These findings indicate that POE stimulates insulin secretion via a $K{^+}_{ATP}$ channel-dependent pathway in INS-1 pancreatic ${\beta}-cells$.

• The Korean Journal of Physiology and Pharmacology
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• 제15권4호
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• pp.217-239
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• 2011

We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial $Ca^{2+}$ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1~3 Hz) made both the diastolic and systolic [$Ca^{2+}]$ bigger in mitochondria as well as in cytosol. As L-type $Ca^{2+}$ channel has key influence on the amplitude of $Ca^{2+}$ -induced $Ca^{2+}$ release, the relation between stimulus frequency and the amplitude of $Ca^{2+}$ transients was examined under the low density (1/10 of control) of L-type $Ca^{2+}$ channel in model simulation, where the relation was reversed. In experiment, block of $Ca^{2+}$ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial $Ca^{2+}$ transients, while it failed to affect the cytosolic $Ca^{2+}$ transients. In computer simulation, the amplitude of cytosolic $Ca^{2+}$ transients was not affected by removal of $Ca^{2+}$ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [$Ca^{2+}$] in cytosol and eventually abolished the $Ca^{2+}$ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type $Ca^{2+}$ channel to total transsarcolemmal $Ca^{2+}$ flux could determine whether the cytosolic $Ca^{2+}$ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic $Ca^{2+}$ affects mitochondrial $Ca^{2+}$ in a beat-to-beat manner, however, removal of $Ca^{2+}$ influx mechanism into mitochondria does not affect the amplitude of cytosolic $Ca^{2+}$ transients.

• 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

• BMB Reports
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• 제28권2호
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• pp.129-137
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• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

• 대한약리학회지
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• 제32권3호
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• pp.373-380
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• 1996

최근 본 연구실에서는 GS 386인 1-(4'-methoxybenzyl)-6,7-dimethoxy-3,4-dihydroisoquinoline이 적출된 토끼의 심방세포에서 $Ca^{++}$ 채널의 운동성 변화없이 $Ca^{++}$ 채널이 열릴 가능성을 줄임으로써 $Ca^{++}$ 전류의 증폭을 억제한다고 보고하였다. 이번 연구에서는 적출된 쥐의 기관지를 사용하여 GS 386의 작용기전에 대해 연구하였다. GS386은 carbachol $(0.3{\mu}M)$과 높은 농도의 $K^+$ (65.4mM)에 의해 수축된 쥐의 기관지를 용량-의존적으로 이완시켰으며 이때 $IC_{50}$는 5.24와 $5.67\;{\mu}M$이었다. verapamil은 carbachol에 의한 수축시 보다 높은 농도의 $K^+$에 의해 수축된 조직에 더욱 효과적으로 억제하였다. $Ca^{++}$이 없는 상태에서 $Ca^{++}$에 의한 수축은 GS386에 의해 억제되었다. 더욱이 높은 농도의 GS386$(100\;{\mu}M)$은 verapamil과는 다르게 carbachol뿐만 아니라 caffeine에 의한 위상성 수축을 억제 시키므로 GS386은 세포질내로 들어가 sarcoplasmic retuculum과 같은 근육 내부에 2차적인 영향을 나타내었다. 더군다나GS386은 verapamil에 의해 영향을 받지않는 (verapamil-insensitive component)이완을 보였고 쥐 기관지의 평활근에서 cAMP의 양을 증가 시켰다. 이러한 결과는 GS386의 작용기전이 $Ca^{++}$ 길항적인 작용 뿐만 아니라 posphodiesterase억제작용에 기인한다는 사실을 제시한다.