• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.375-379
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• 2003

$Ca^{2+}$ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how $Ca^{2+}$ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in $Ca^{2+}$ mobilization of muscle, and that $Ca^{2+}$ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in $Ca^{2+}$ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a $Ca^{2+}$ imaging system, $Ca^{2+}$ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular $Ca^{2+}$ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular $Ca^{2+}$ increase was completely suppressed by treatment of nifedifine, a L-type $Ca^{2+}$ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular $Ca^{2+}$ increase in myoblasts and then may play an important role in myoblast fusion.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.521-527
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• 1998

An important property of the intestine is the ability to secrete fluid. The intestinal secretion is regulated by a number of substances including vasoactive intestinal peptide (VIP), ATP and different inflammatory mediators. One of the most important secretagogues is adenosine during inflammation. However, the controversy concerning the underlying mechanism of adenosine-stimulated $Cl^-$ secretion in intestinal epithelial cells still continues. To investigate the effect of adenosine on $Cl^-$ secretion and its underlying mechanism in the rabbit colon mucosa, we measured short circuit current ($I_{SC}$) under automatic voltage clamp with DVC-1000 in a modified Ussing chamber. Adenosine, when added to the basolateral side of the muocsa, increased $I_{SC}$ in a dose-dependent manner. The adenosine-stimulated $I_{SC}$ response was abolished when $Cl^-$ in the bath solution was replaced completely with gluconate. In addition, the $I_{SC}$ response was inhibited by a basolateral Na-K-Cl cotransporter blocker, bumetanide, and by apical $Cl^-$ channel blockers, dephenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), glibenclamide. Amiloride, an epithelial $Na^+$ channel blocker, and 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS), a $Ca^{2+}-activated$ $Cl^-$ channel blocker, had no effect. In the mucosa pre-stimulated with forskolin, adenosine did not show any additive effect, whereas carbachol resulted in a synergistic potentiation of the $I_{SC}$ response. The adenosine response was inhibited by 10 ${\mu}M$ H-89, an inhibitor of protein kinase A. These results suggest that the adenosine-stimulated $I_{SC}$ response is mediated by basolateral to apical $Cl^-$ secretion through a cAMP-dependent $Cl^-$ channel. The rank order of potencies of adenosine receptor agonists was $5'-(N-ethylcarboxamino)adenosine(NECA)＞N^6-(R-phenylisopropyl)adenosine(R-$ PIA)＞2-[p-(2-carbonylethyl)-phenyl-ethylamino]-5'-N-ethylcarboxaminoadenosine(CGS21680). From the above results, it can be concluded that adenosine interacts with the $A_{2b}$ adenosine receptor in the rabbit colon mucosa and a cAMP-dependent signalling mechanism underlies the stimulation of $Cl^-$ secretion.

• Journal of Plant Biology
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• v.31 no.2
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• pp.91-100
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• 1988

The effect of Ca2+ and polyamines on the activity $\beta$-glucan synthetase II(GSII) related to cell wall synthesis was studied in carrot suspension cultured cells. The activity of GS II is four times higher than that of $\beta$-glucan synthetase I in carrot suspension cultured cells and in vitro expreiment, the activity of GSII was increased in response to increase in concentration of Ca2+ and polyamines. When carrot suspension cultured cells were incubated together with Ca2+ and polyamines, the GSII activity was high at 0.1mM of Ca2+ and 1mM of putrescine. Also, polycationic poly-L-lysine and poly-L-ornithine increased about 50% the GSII activity than that of the control, respectively. These results may imply that Ca2+ and polyamines were related to the enzyme activity as a polycationic nature. In addition, verapamil as the calcium channel blocker and flunarizine as an antagonist of calcium mechanism in cytoplasm decreased GSII activity ramarkably, Ca2+ and calmodulin stimulated GSII activity as Ca2+ of free ion rather than Ca2+ calmodulin complex. The effect of 2,4-D on the GSII activity in culture medium is shown to be low at 0.1mg per liter and GSII activity increased about 30% more than that of the 0.1mg/l at the range of 0.3-1.0mg per litere. Cummulative results suggest that Ca2+ and polyfamines stimulate the cell wall synthesis by means of the enhancement of GSII activity responsible for synthesizing the cell wall components.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.6
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• pp.489-495
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• 2014

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ${\omega}$-conotoxin GVIA (CgTx), a selective N-type $Ca^{2+}$ channel ($I_{Ca-N}$) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ${\omega}$-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of $I_{Ca-N}$ which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.1
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• pp.55-62
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• 2005

Very little research has been carried out on safflower seed for the prevention and treatment of the bone deficiency diseases, including osteoporosis, which are supported by scientific evidences. In the present study, $3{\mu}l$ of 0.1% dried crude extract or $2{\mu}l$ of 0.1% dried aqueous fraction were shown to significantly accelerate the rate of differentiation of osteoblast. Also, the crude extract and aqueous fraction increased the $[Ca^{2+}]_i$ of the cultured osteoblast cells: $3{\mu}l$ of 0.1% dried crude extract and $2{\mu}l$ of 0.1% dried aqueous fraction significantly increased the $[Ca^{2+}]_i$ of the cultured osteoblast cells ($8{\times}10^{-4}$) to the extent that it deserves a considerable attention. Furthermore, the crude extract and aqueous fraction increased the $[Ca^{2+}]_i$ of the cultured osteoblast cells, and $300{\mu}M$ $Cd^{2+}$, specific calcium channel blocker, completely blocked the increase. Therefore, the increased $[Ca^{2+}]_i$ of the cultured osteoblast cells by safflower seed component continued to activate calcium channel.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.19-24
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• 2006

The intermediate conductance $Ca^{2+}-activated$ $K^+$ channels (SK4, IKCa1) are present in lymphocytes, and their membrane expression is upregulated by various immunological stimuli. In this study, the activity of SK4 was compared between Bal-17 and WEHI-231 cell lines which represent mature and immature stages of murine B lymphocytes, respectively. The whole-cell patch clamp with high-$Ca^{2+}$ ($0.8{\mu}M$) KCl pipette solution revealed a voltage-independent $K^+$ current that was blocked by clotrimazole (1 mM), an SK4 blocker. The expression of mRNAs for SK4 was confirmed in both Bal-17 and WEHI-231 cells. The density of clotrimazole-sensitive SK4 current was significantly larger in Bal-17 than WEHI-231 cells ($-11.4{\pm}3.1$ Vs. $-5.7{\pm}1.15$ pA/pF). Also, the chronic stimulation of B cell receptors (BCR) by BCR-ligation (anti-IgM Ab, $3{\mu}g$/ml, 8∼12 h) significantly upregulated the amplitude of clotrimazolesensitive current from $-11.4{\pm}3.1$ to $-53.1{\pm}8.6$ pA/pF in Bal-17 cells. In WEHI-231 cells, the effect of BCR-ligation was significantly small ($-5.7{\pm}1.15$ to $-9.0{\pm}1.00$ pA/pF). The differential expression and regulation by BCR-ligation might reflect functional changes in the maturation of B lymphocytes.

• The Korean Journal of Physiology
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• v.28 no.1
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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.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.25-33
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• 2016

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$)-dependent signaling affects the fate of cancer cells. Here we investigate the role of $Ca^{2+}$-activated $K^+$ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of dif-ferent cell lines; SNU-1076, OSC-19 and HN5. Treatment with $1{\mu}M$ ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of $Ca^{2+}$-activated $Cl^-$ channels (Ano-1) and $Ca^{2+}$-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward $K^+$ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Con-sistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the $Ca^{2+}$ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.177-183
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• 2008

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

• The Journal of Korean Physical Therapy
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• v.14 no.2
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• pp.1-15
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• 2002

Excitation-contraction coupling in skeletal muscle is process by which depolarization of the muscle fiber membrane, elicited by a nerve action potential, triggers the release of $Ca^{2+}$ from the sarcoplasmic reticulum(SR). The resulting rise in intracellular $Ca^{2+}$ concentration$([Ca^{2+}]_i)$ activates the troponin complex, thereby initiating the contraction of the muscle. The question remains as to what factors are involved in the inhibition of SR $Ca^{2+}$ release in fatigued muscle. The purpose of this study was determine whether ATP-sensitive $K^+(K_{ATP})$ channels are activated and contribute to decrease in $[Ca^{2+}]_i$ during fatigue development in the mouse skeletal muscle. To elucidate a role of $K_{ATP})$ in relation to ECC, I measured the modulation effects of $K_{ATP})$ channel blocker(glibenclamide) and opener(pinacidil) on $[Ca^{2+}]_i$ after fatiguing electrical field stimulation(FEFS). Intracellular $Ca^{2+}$ signals were recorded by conforcal laser microscopy(LSM 410) and monitored using the fluorescent $Ca^{2+}$-Sensitive indicator Fluo-3 AM. The results of this study were as followed: 1. The relative [Ca2'li after FEFS in the pre-glibenclamide-treated group was higher than the control. And relative $[Ca^{2+}]_i$ after FEFS in the pre-glibenclamide-treated group was lower than the control. 2. The relative $[Ca^{2+}]_i$ after FEFS for 3 min in the control, pre-glibenclamide-treated group and pre-pinacidil-treated group showed a similar pattern; the gradually significant decrease in $[Ca^{2+}]_i$. But, these decreasing pattern was most significant in the control. These findings suggest a tight relationship between $K_{ATP})$ and $Ca^{2+}$ in ECC during fatigue. Therefore, 1 thought that activation of $K_{ATP})$ channels may be one of mechanisms of the fatigue in skeletal muscle.