• Journal of Ginseng Research
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• v.44 no.3
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• pp.490-495
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• 2020

Background: Ginsenoside Rk1, a saponin component isolated from heat-processed Panax ginseng Meyer, has been implicated in the regulation of antitumor and anti-inflammatory activities. Although our previous studies have demonstrated that ginsenoside Rg3 significantly attenuated the activation of NMDA receptors (NMDARs) in hippocampal neurons, the effects of ginsenosides Rg5 and Rk1, which are derived from heat-mediated dehydration of ginsenoside Rg3, on neuronal NMDARs have not yet been elucidated. Methods: We examined the regulation of NMDARs by ginsenosides Rg5 and Rk1 in cultured rat hippocampal neurons using fura-2-based calcium imaging and whole-cell patch-clamp recordings. Results: The results from our investigation showed that ginsenosides Rg3 and Rg5 inhibited NMDARs with similar potencies. However, ginsenoside Rk1 inhibited NMDARs most effectively among the five compounds (Rg3, Rg5, Rk1, Rg5/Rk1 mixture, and protopanaxadiol) tested in cultured hippocampal neurons. Its inhibition is independent of the NMDA- and glycine-binding sites, and its action seems to involve in an interaction with the polyamine-binding site of the NMDAR channel complex. Conclusion: Taken together, our results suggest that ginsenoside Rk1 might be a novel component contributable to the development of ginseng-based therapeutic treatments for neurodegenerative diseases.

• Journal of the korean academy of Pediatric Dentistry
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• v.26 no.2
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• pp.399-415
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• 1999

From bacteria to mammalian cells, one of the most important mediators of intracellular signal transduction mechanisms which regulate a variety of intracellular processes is free calcium. In salivary acinar cells, elevation of intracellular calcium concentration ($[Ca^{2+}]_i$) is essential for the salivary secretion induced by parasympathetic stimulation. However, in addition to $[Ca^{2+}]_i$, gap junctions which couple individual cells electrically and chemically have also been reported to regulate enzyme secretion in pancreatic acinar cells. Since the plasma membrane of salivary acinar cells has a high density of gap junctions, and these cells are electrically and chemically coupled with each other, gap junctions may modulate the secretory function of salivary glands. In this respect, I planned to investigate the role of gap junctions in the modulation of salivary secretion and $[Ca^{2+}]_i$, using mandibular salivary glands of rats. In order to measure the salivary flow rate, fluid was collected from the cannulated duct of the isolated perfused rat mandibular glands at 2 min intervals. $[Ca^{2+}]_i$, was measured from the cells loaded with fura-2 by spectrofluorometry. The results obtained were as follows: 1. CCh-induced salivary secretion was reversibly inhibited by 1 mM octanol, a gap junction blocker. 2. CCh-induced increase in $[Ca^{2+}]_i$, was also reversed by the application of 1 mM octanol. 3. Octanol did not block the initial increase in $[Ca^{2+}]_i$ caused by CCh, which suggested that the reduction of $[Ca^{2+}]_i$, caused by gap junction blockade was not resulted from the inhibition of $Ca^{2+}$ release from intracellular $Ca^{2+}$ stores. 4. Addition of octanol during stimulation with $1{\mu}M$ thapsigargin, a potent microsomal ATPase inhibitor, reduced $[Ca^{2+}]_i$, to the basal level. This suggested that inhibition of gap junction permeability closed plasma membrane $Ca^{2+}$ channels. 5. 2,5-di-tert-butyl-1,4-benzohydroquinone (TBQ) generated $[Ca^{2+}]_i$ oscillations resulting from periodic influx of $Ca^{2+}$ via plasma membrane. The TBQ-induced $[Ca^{2+}]_i$ oscillations were stopped by the application of 1mM octanol which implicated that gap junctions modulate the permeability of plasma membrane $Ca^{2+}$ channels. 6. Glycyrrhetinic acid, another well known gap junction blocker, also inhibited CCh-induced salivary secretion from rat mandibular glands. These results suggested that gap junctions play an important role in the modulation of fluid secretion from the rat mandibular glands and this was probably due to the inhibition of $Ca^{2+}$ influx through the plasma membrane $Ca^{2+}$ channels.

• Journal of Korean Biological Nursing Science
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• v.1 no.1
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• pp.25-41
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• 1999

Physiological activity of osteoblast including bone formation is known to be closely related to the increase of intracellular $Ca^{2+}$ activity($[Ca^{2+}]_i$) in osteoblast. $Ca^{2+}$ is an important intracellular messenger in diverse cellular functions, and regulation of its level is mediated by the transmembrane $Ca^{2+}$ movement via $Ca^{2+}$ channels, $Na^+-Ca^{2+}$ exchange, and by intracellular $Ca^{2+}$ movement through the intracellular stores. The purpose of this study is to investigate how the intracellular $Ca^{2+}$ is regulated in osteoblast-like cells(OLCs) by measuring $Ca^{2+}$ activity with cell imaging technique. OLCs were isolated from femur and tibia of neonatal rats, and cultured for 7 days. Cultured OLCs were loaded with a $Ca^{2+}$-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored with a cooled CCD camera. The images were processed and analyzed with an image analyzing software. The results were as follows. (1) $[Ca^{2+}]_i$ of OLC decreased as the $Ca^{2+}$ concentration in the superfusing Tyrode solution was lowered. When $Na^+$ concentration in the superfusing solution was decreased, $[Ca^{2+}]_i$ increased.. These suggest that $Ca^{2+}$ flux occurs via the $Na^+-Ca^{2+}$ exchange mechanism. (2) When $Na^+$ in the superfusing solution was removed. a transient $Ca^{2+}$, increase($Ca^{2+}$ spike) was occasionally observed. However, $Ca^{2+}$ spike was not observed after adding 1 ${\mu}M$ thapsigargin. This implies that the generation of $Ca^{2+}$ spike is mediated by the release of $Ca^{2+}$ from endoplasmic reticulum(ER). (3) As the $Ca^{2+}$ concentration in the superfusing solution was raised, the frequency of 0mM $Na^+$-induced $Ca^{2+}$ spike increased, suggesting that $Ca^{2+}$-induced $Ca^{2+}$ release(CICR) mechanism exists. (4) After $[Ca^{2+}]_i$ was decreased with the superfusion of $Ca^{2+}$-free solution containing thapsigargin, the recovery of $[Ca^{2+}]_i$ with reperfusion of 2.5mM $Ca^{2+}$ solution transiently exceeded the control level, suggesting that the depletion of $Ca^{2+}$ in ER induces $Ca^{2+}$ influx from extracellular medium via store-operated $Ca^{2+}$ influx(SOCI) mechanism. (5) $[Ca^{2+}]_i$ was not affected by the superfusion of 25mM $K^+$ Tyrode solution. These results suggest that intracellular $Ca^{2+}$ activity in osteoblast is regulated by transmembrane $Ca^{2+}$ flux via $Na^+-Ca^{2+}$ exchange, $Ca^{2+}$ release from the internal store (ER) via $Ca^{2+}$-induced $Ca^{2+}$ release, and store-operated $Ca^{2+}$ influx across the cell membrane.

• International Journal of Oral Biology
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• v.31 no.4
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• pp.127-133
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• 2006

[ $H_2O_2$ ], a member of reactive oxygen species (ROS), is known to be involved in the mediation of physiological functions in a variety of cell types. However, little has been known about the physiological role of $H_2O_2$ in exocrine cells. Therefore, in the present study, the effect of $H_2O_2$ on cholecystokinin (CCK)-evoked $Ca^{2+}$ mobilization and amylase release was investigated in rat pancreatic acinar cells. Stimulation of the acinar cells with sulfated octapeptide form of CCK (CCK-8S) induced biphasic increase in amylase release. Addition of $30\;{\mu}M\;H_2O_2$ enhanced amylase release caused by 10 pM CCK-8S, but inhibited the amylase release induced by CCK-8S at concentrations higher than 100 pM. An ROS scavenger, $10\;{\mu}M$ Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, increased amylase release caused by CCK-8S at concentrations higher than 100 pM, although lower concentrations of CCK-8S-induced amylase release was not affected. To examine whether the effect of $H_2O_2$ on CCK-8S-induced amylase release was exerted via modulation of intracellular $Ca^{2+}$ signaling, we measured the changes in intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in fura-2 loaded acinar cells. Although $30\;{\mu}M\;H_2O_2$ did not induce any increase in $[Ca^{2+}]_i$ by itself, it increased the frequency and amplitude of $[Ca^{2+}]_i$ oscillations caused by 10 pM CCK-8S. However, $30\;{\mu}M\;H_2O_2$ had little effect on 1 nM CCK-8S-induced increase in $[Ca^{2+}]_i$. ROS scavenger, 1 mM N-acetylcysteine, did not affect $[Ca^{2+}]_i$ changes induced by 10 pM or 1 nM CCK-8S. Therefore, it was concluded that $30\;{\mu}M\;H_2O_2$ enhanced low concentration of CCK-8S-induced amylase release probably by increasing $[Ca^{2+}]_i$ oscillations while it inhibited high concentration of CCK-8S-induced amylase release.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.21-28
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• 2010

Phenolic compounds affect intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced $Ca^{2+}$ signaling in PC12 cells using fura-2-based digital $Ca^{2+}$ imaging and whole-cell patch clamping. Treatment with ATP ($100\;{\mu}M$) for 90 s induced increases in $[Ca^{2+}]_i$ in PC12 cells. Pretreatment with octyl gallate (100 nM to $20\;{\mu}M$) for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ response in a concentration-dependent manner ($IC_{50}=2.84\;{\mu}M$). Treatment with octyl gallate ($3\;{\mu}M$) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular $Ca^{2+}$ with nominally $Ca^{2+}$-free HEPES HBSS or depletion of intracellular $Ca^{2+}$ stores with thapsigargin ($1\;{\mu}M$). Treatment for 10 min with the L-type $Ca^{2+}$ channel antagonist nimodipine ($1\;{\mu}M$) significantly inhibited the ATP-induced $[Ca^{2+}]_i$ increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the $[Ca^{2+}]_i$ increase induced by 50 mM KCI. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein ($50\;{\mu}M$) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced $[Ca^{2+}]_i$ increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of $Ca^{2+}$ from extracellular space and P2Y receptor-induced release of $Ca^{2+}$ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced $Ca^{2+}$ responses by inhibiting the secondary activation of voltage-gated $Ca^{2+}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.341-346
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• 2005

The mechanism underlying oxidant-induced intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) increase was studied in cultured bovine aortic endothelial cells (BAECs) using fura-2 AM. In the presence of 2 mM extracellular $Ca^{2+}$, the application of DTBNP ($20{\mu}M$), a membrane-permeable oxidant, caused an increase in $[Ca^{2+}]_i$, and DTT (2 mM) as a reductant completely reversed the effect of DTBNP. The $[Ca^{2+}]_i$ increase induced by DTBNP was also observed in an extracellular $Ca^{2+}$-free/2 mM EGTA solution, indicating the release of $Ca^{2+}$ from intracellular store(s). After endoplasmic reticulum was depleted by an $IP_3$-generating agonist, ATP ($30{\mu}M$) or an ER $Ca^{2+}$ pump inhibitor, thapsigargin ($1{\mu}M$), DTBNP-stressed BAECs showed an increase of $[Ca^{2+}]_i$ in $Ca^{2+}$-free/2 mM EGTA solution. Ratio-differences before and after the application of DTBNP after pretreatment with ATP or thapsigargin were $0.42{\pm}0.15$ and $0.49{\pm}0.07$, respectively (n=7), which are significantly reduced, compared to the control value of $0.72{\pm}0.07$ in a $Ca^{2+}$-free/2 mM EGTA solution. After the protonophore CCCP ($10{\mu}M$) challenge to release mitochondrial $Ca^{2+}$, the similar result was obtained. Ratio-difference before and after the application of DTBNP after pretreatment with CCCP was $0.46{\pm}0.09$ (n=7). Simultaneous application of thapsigargin and CCCP completely abolished the DTBNP-induced $[Ca^{2+}]_i$ increase. The above results together indicate that the increase of $[Ca^{2+}]_i$ by DTBNP resulted from the release of $Ca^{2+}$ from both endoplasmic reticulum and mitochondria.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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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.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.783-796
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• 1997

The relaxation induced by stimulation of the inhibitory non-adrenergic, non-cholinergic (iNANC) nerve is mediated by the release of iNANC neurotransmitters such as nitric oxide (NO), vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP). The mechanisms of NO, VIP or ATP-induced relaxation have been partly determined in previous studies, but the detailed mechanism remains unknown. We tried to identify the nature of iNANC neurotransmitters in the smooth muscle of guinea pig ileum and to determine the mechanism of the inhibitory effect of nitric oxide. We measured the effect of NO-donors VIP and ATP on the intracellular $Ca^{2+}$ concentration$([Ca^{2+}]_i)$, by means of a fluorescence dye(fura 2) and tension simultaneously in the isolated guinea pig ileal smooth muscle. Following are the results obtained. 1. Sodium nitroprusside $(SNP:10^{-5}\;M)$ or S -nitro-N-acetyl-penicillamine $(SNP:10^{-5}\;M)$ decreased resting $[Ca^{2+}]_i$ I and tension of muscle. SNP or SNAP also inhibited rhythmic oscillation of $[Ca^{2+}]_i$ and tension. In 40mM $K^+$ solution or carbachol ($(CCh:10^{-6}\;M)$-induced precontracted muscle, SNP decreased muscle tension. VIP did not change $[Ca^{2+}]_i$ and tension in the resting or precontracted muscle, but ATP increased resting $[Ca^{2+}]_i$ and tension in the resting muscle. 2. 1H-[1,2,4]oxadiazol(4,3-a)quinoxalin-1-one $(ODQ:1\;{\mu}M)$, a specific inhibitor of soluble guanylate cyclase, limited the inhibitory effect of SNP 3. Glibenclamide $(10\;{\mu}M)$, a blocker of $K_{ATP}$ channel, and 4-aminopyridine (4-AP:5 mM), a blocker of delayed rectifier K channel, apamin $(0.1\;{\mu}M)$, a blocker of small conductance $K_{Ca}$ channel had no effect on the inhibitory effect of SNP. Iberiotoxin $(0.1\;{\mu}M)$, a blocker of large conductance $K_{Ca}$ channel, significantly increased the resting $[Ca^{2+}]_i$, and tension, and limited the inhibitory effect of SNP. 4. Nifedipine $(1\;{\mu}M)$ or elimination of external $Ca^{2+}$ decreased not only resting $[Ca^{2+}]_i$ and tension but also oscillation of $[Ca^{2+}]_i$ and tension. Ryanodine $(5\;{\mu}M)$ and cyclopiazonic acid $(10\;{\mu}M)$ decreased oscillation of $[Ca^{2+}]_i$ and tension. 5. SNP decreased $Ca^{2+}$ sensitivity of contractile protein. In conclusion, these results suggest that 1) NO is an inhibitory neurotransmitter in the guinea pig ileum, 2) the inhibitory effect of SNP on the $[Ca^{2+}]_i$ and tension of the muscle is due to a decrease in $[Ca^{2+}]_i$ by activation of the large conductance $K_{Ca}$ channel and a decrease in the sensitivity of contractile elements to $Ca^{2+}$ through activation of G-kinase.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.53-59
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• 2011

The secretion of insulin from pancreatic ${\beta}$-cells is triggered by the influx of $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channels. The resulting elevation of intracellular calcium ($[Ca^{2+}]_i$) triggers additional $Ca^{2+}$ release from internal stores. Less well understood are the mechanisms involved in $Ca^{2+}$ mobilization from internal stores after activation of $Ca^{2+}$ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic ${\beta}$-cell line, INS-1 cells. To measure cytosolic and stored $Ca^{2+}$, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. $[Ca^{2+}]_i$ was repetitively increased by caffeine stimulation in normal $Ca^{2+}$ buffer. However, peak $[Ca^{2+}]_i$ was only observed after the first caffeine stimulation in $Ca^{2+}$ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in $[Ca^{2+}]_i$ were reduced by pretreatment with ruthenium red, as well as by depletion of internal $Ca^{2+}$ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced $Ca^{2+}$ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells,$Ca^{2+}$ release from internal stores was activated by caffeine, $Ca^{2+}$, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in perrneabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic ${\beta}$-cells.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.1
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• pp.57-63
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• 2004

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for $[^3H]-inositol$ phosphates (IPs). Treatment with ATP $(100\;{\mu}M)$ for 2 min induced $[Ca^{2+}]_i$ increases. The ATP-induced $[Ca^{2+}]_i$ increases were significantly decreased by removal of extracellular $Ca^{2+}$ and treatment with the inhibitor of endoplasmic reticulum $Ca^{2+}$ ATPase thapsigargin $(1\;{\mu}M)$. Treatment with fluoxetine for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase concentration-dependently. Treatment with fluoxetine $(30\;{\mu}M)$ for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase following removal of extracellular $Ca^{2+}$ and depletion of intracellular $Ca^{2+}$ stores. While treatment with the L-type $Ca^{2+}$ channel antagonist nimodipine for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM $K^+-induced$ $[Ca^{2+}]_i$ increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced $[^3H]-IPs$ formation. Collectively, we conclude that fluoxetine inhibits ATP-indueed $[Ca^{2+}]_i$ increases in PC12 cells by inhibiting both an influx of extracellular $Ca^{2+}$ and a release of $Ca^{2+}$ from intracellular stores without affecting IPs formation.