• 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.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.354-364
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• 2015

Background: Intracellular $Ca^{2+}$($[Ca^{2+}]_i$) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of $[Ca^{2+}]_i$mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the $Ca^{2+}$-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods: We investigated the $Ca^{2+}$-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I ($IP_3RI$) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results: The inhibition of $[Ca^{2+}]_i$ mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-BrcAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)- dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) ($Thr^{197}$) by KRG-TS. The phosphorylation of $IP_3RI$ ($Ser^{1756}$) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-BrcGMPS. These results suggest that the inhibitory effect of $[Ca^{2+}]_i$ mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits $[Ca^{2+}]_i$ mobilization in thrombin-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

• YAKHAK HOEJI
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• v.43 no.6
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• pp.809-817
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• 1999

The effect of 2-(4-cyanophenyl)amino-1,4-naphthalenedione-3-pyridinium perchlorate (PQ5) on pla-telet aggregation and its action mechanisms were investigated with rat platelet. PQ5 inhibited the platelet aggregation induced by collagen ($6{\;}{\mu\textrm{g}}/ml$), thrombin (0.4 U/ml) and A23187 ($3{\mu}M$) in concentration-dependent manner with $IC_{50}$ values of 5.50, 25.89 and $37.12{\;}{\mu}M$, respectively. PQ5 also significantly reduced the thromboxane $A_2$ (TXA2) formation in a concentration dependent manner. The collagen-induced arachidonic acid (AA) release in [-3H]-AA incorporated platelet, an indication of the phospholipase $A_2$ activity, was decreased by PQ5 pretreatment PQ5 significantly inhibited the activity of thormboxane synthase only at high concentration ($100{\mu}M$), but did not affect the cyclooxygenase activity at all. Collagen-induced ATP release was significantly reduced by PQ5. Calcium-induced platelet aggregation experiment suggests that the elevation of intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) by collagen stimulation is decreased by the pretreatment of PQ5, which is due to the inhibition of calcium release from intracellular store and influx from outside of the cell. PQ5 did not showed the effect of anticoagulation as prothrombin time (PT) or activated partial thromboplastin time (APTT). Form these results, it is suggested that PQ5 exerts its antiplatelet activity through the inhibition of the intracellular $Ca^{2+}$ mobilization and the decrease of the $TXA_2$ synthesis.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.2
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• pp.105-111
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• 2010

Inositol 1,4,5-trisphosphate receptors ($InsP_3Rs$) modulate $Ca^{2+}$ release from intracellular $Ca^{2+}$ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block $InsP_3Rs$, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of $Ca^{2+}$ release from intracellular $Ca^{2+}$ stores and $Ca^{2+}$ entry through store-operated $Ca^{2+}$ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the $Ca^{2+}$ entry, but significantly inhibited carbamylcholine (CCh)-induced $Ca^{2+}$ release. In contrast, 2-APB did not block CCh-induced $Ca^{2+}$ release, but remarkably blocked SOC-mediated $Ca^{2+}$ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced $Ca^{2+}$ release, but 2-APB at lower concentration, which effectively blocked $Ca^{2+}$ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of Ins$P_3$-induced $Ca^{2+}$ release and direct stimulation of $Ca^{2+}$ release. Based on the results, we concluded that caffeine is useful as an inhibitor of $InsP_3R$, and 2-APB at lower concentration is considered a blocker of $Ca^{2+}$ entry through SOC channels in the pancreatic acinar cell.

• 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 Pharmacology
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• v.8 no.1
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• pp.67-75
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• 1972

Many studies on the mechanism of the inotropic action of cardiac glycosides have shown the possible intimate relationship between the mobilization of intracellular calcium and inotropic effect. Evidence obtained from recent studies suggests that cardiac glycosides may increase the intracellular $Ca^{++}$ concentration through the release of this ion from cellular or intracellular membrane. It seemed imperative to study the effect of ouabain on the interaction between mitochondria and $Ca^{++}$, because mitochondria are known to have a rather powerful $Ca^{++}$ pump mechanism which may have an important role on the regulation of intracellular $Ca^{++}$ concentration. The present investigations was made into the effect of ouabain on $Ca^{++}$ untake of mitochondria in the presence of ATP and its dependence on $K^+$ and $Na^+$ in the medium. The results are summarized as follows: 1. The rate of rise in the turbidity of superprecipitation was solely influenced by ionic strength of the medium, not by the species of ion, i.e. $Na^+$ or $K^+$. The higher ionic strength suppressed and the lower enhanced the rate of superprecipitation respectively. 2. No effect of ouabain was found on the rate of superprecipitation. 3. Mitochondria depressed the rate of superpretipitation, and the depressed rate of superprecipitation by mitochondria was reversed by ouabain, and the degree of this reversal was almost identical in $Na^+$ and $K^+$ medium. 4. $Ca^{++}$ uptake of mitochondria was inhibited by ouabain in the presence of ATP and the degree of inhibition showed the dose-response manner in terms of concentration of ouabain. 5. In the absence of ATP, mitochondria took or the $Ca^{++}$ in initial period but released it later. Such uptake and release of $Ca^{++}$ was not influenced by ouabain. 6. It is suggested that intracellular calcium mobilization by ouabain through the action upon the mitochondria was due to inhibition on ATP-dependent $Ca^{++}$ uptake by this agent, not to the action upon so called binding.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.103-112
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• 1996

Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic $Ca^{2+}$ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and $H_2O_2$ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of $[Ca^{2+}]_i$ in neutrophils was inhibited by staurosporine, genistein and methyl-2,5-dihydroxycinnamate. Staurosporine inhibited both intracellular $Ca^{2+}$ release and $Mn^{2+}$ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited $Mn^{2+}$ influx induced by PAF, whereas their effects on intracellular $Ca^{2+}$ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of $[Ca^{2+}]_i$ was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and $Ca^{2+}$ mobilization in PAF-stimulated neutrophils. The elevation of $[Ca^{2+}]_i$ appears to be accomplished by intracullular $Ca^{2+}$ release and $Ca^{2+}$ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular $Ca^{2+}$ mobilization.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.2
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• pp.215-223
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• 2018

Intracellular $Ca^{2+}$ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide ($H_2O_2$) on cytosolic $Ca^{2+}$ accumulation in mouse parotid acinar cells. Intracellular $Ca^{2+}$ levels were slowly elevated when $1mM\;H_2O_2$ was perfused in the presence of normal extracellular $Ca^{2+}$. In a $Ca^{2+}-free$ medium, $1mM\;H_2O_2$ still enhanced the intracellular $Ca^{2+}$ level. $Ca^{2+}$ entry tested using manganese quenching technique was not affected by perfusion of $1mM\;H_2O_2$. On the other hand, $10mM\;H_2O_2$ induced more rapid $Ca^{2+}$ accumulation and facilitated $Ca^{2+}$ entry from extracellular fluid. $Ca^{2+}$ refill into intracellular $Ca^{2+}$ store and inositol 1,4,5-trisphosphate ($1{\mu}M$)-induced $Ca^{2+}$ release from $Ca^{2+}$ store was not affected by $1mM\;H_2O_2$ in permeabilized cells. $Ca^{2+}$ efflux through plasma membrane $Ca^{2+}-ATPase$ (PMCA) was markedly blocked by $1mM\;H_2O_2$ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected $H_2O_2-induced$ $Ca^{2+}$ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of $H_2O_2$ under pathological conditions may lead to cytosolic $Ca^{2+}$ accumulation and that the primary mechanism of $H_2O_2-induced$ $Ca^{2+}$ accumulation is likely to inhibit $Ca^{2+}$ efflux through PMCA rather than mobilize $Ca^{2+}$ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

• Natural Product Sciences
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• v.26 no.4
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• pp.295-302
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• 2020

Cudrania tricuspidata (C. tricuspidata) is a deciduous tree found in Japan, China and Korea. The root, stems, bark and fruit of C. tricuspidata has been used as traditional herbal remedies such as eczema, mumps, acute arthritis and tuberculosis. In this study, we investigated the potential efficacies of this natural compound by focusing on the inhibitory effect of cudraxanthone L (CXL) isolated from the roots of C. tricuspidata on human platelet aggregation. Our study focused on the action of CXL on collagen-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane A2 secretion. In addition, we investigated the inhibitory effect of CXL on thrombin-induced clot retraction. Our results showed that CXL inhibited collagen-induced human platelet aggregation, intracellular calcium mobilization, fibrinogen binding, fibronectin adhesion and clot retraction without cytotoxicity. Therefore, we confirmed that CXL has inhibitory effects on human platelet activities and has potential value as a natural substance for preventing thrombosis.

• Biomedical Science Letters
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• v.24 no.3
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• pp.253-262
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• 2018

Platelets are activated at sites of vascular injury via several molecules, such as adenosine diphosphate, collagen and thrombin. Full platelet aggregation is absolutely essential for normal hemostasis. Moreover, this physiological event can trigger circulatory disorders, such as thrombosis, atherosclerosis, and cardiovascular disease. Therefore, platelet function inhibition is a promising approach in preventing platelet-mediated circulatory disease. Many studies reported the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in platelet functions. However, these studies were limited. Thus, we examined MAPK signaling pathways in human platelets using specific MAPK inhibitors, such as PD98059, SB203580, and SP600125. We observed that these inhibitors were involved in calcium mobilization and influx in human platelets. They also suppressed thrombin-induced ${\alpha}$- and ${\delta}$-granule release. These results suggest that PD98059, SB203580, and SP600125 exhibit $Ca^{2+}$ antagonistic effects.