• Biomedical Science Letters
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• v.25 no.2
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• pp.123-130
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• 2019

Platelet aggregation is essential for hemostatic process in case of blood vessels damages. However, excessive platelet aggregation can cause cardiovascular disorders including atherosclerosis, thrombosis and myocardial infarction. Scopoletin is usually found in the roots of genus Scopolia or Artemisia, and is known to have anticoagulant and anti-malarial effects. This study investigated the effect of scopoletin on human platelet aggregation induced by U46619, an analogue of thromboxane $A_2(TXA_2)$. Scopoletin had anti-platelet effects by down-regulating $TXA_2$ and intracellular $Ca^{2+}$ mobilization ($[Ca^{2+}]_i$), the aggregation-inducing molecules generated in activated platelets. On the other hand, scopoletin increased the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are known to be intracellular $Ca^{2+}$ antagonists. This resulted in inhibition of fibrinogen binding to ${\alpha}IIb/{\beta}_3$ in U46619-induced human platelet aggregation. In addition, scopoletin inhibited the release of adenosine trisphosphate (ATP) in dose-dependent manner. This result means that the aggregation amplification activity through the granule secretion in platelets was suppressed by scopoletin. Therefore, we demonstrated that scopoletin has a potent antiplatelet effect and is highly likely to prevent platelet-derived vascular disease.

• Biomedical Science Letters
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• v.15 no.4
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• pp.273-279
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• 2009

Cordycepin (3'-deoxyadenosine) is an adenosine analogue isolated from Cordyceps militaris, and it has been used as an anti-cancer and anti-inflammation ingredient in traditional Chinese medicine. We investigated the effects of cordycepin on human platelet aggregation induced by thapsigargin, and determined the cytosolic free $Ca^{2+}$ levels ($[Ca^{2+}]_i$), an aggregation-stimulating factor. Cordycepin significantly inhibited thapsigargin-induced platelet aggregation. Its inhibitory effect was continually sustained at the maximal aggregation concentration of thapsigargin. The thapsigargin-induced $[Ca^{2+}]_i$ were clearly reduced by cordycepin in the presence of exogenous $CaCl_2$ or extracellular $Ca^{2+}$-chelator (EDTA). These results suggest that cordycepin inhibited thapsigargin-induced $Ca^{2+}$-influx from extracellular domain and thapsigargin-induced $Ca^{2+}$-mobilization from intracellular $Ca^{2+}$ storage. Accordingly, our data demonstrated that cordycepin may have a beneficial effect on platelet aggregation-mediated thrombotic diseases by inhibiting a $[Ca^{2+}]_i$-elevation.

• International Journal of Oral Biology
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• v.45 no.2
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• pp.64-69
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• 2020

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are secondary metabolites produced by anaerobic fermentation of dietary fibers in the intestine. Intestinal SCFAs exert various beneficial effects on intestinal homeostasis, including energy metabolism, autophagy, cell proliferation, immune reaction, and inflammation, whereas contradictory roles of SCFAs in the oral cavity have been reported. Herein, we found that low and high concentrations of SCFAs induce differential regulation of intracellular Ca²⁺ mobilization and expression of pro-inflammatory cytokines, such as interleukin (IL)-6 and IL-8, respectively, in gingival fibroblast cells. Additionally, cell viability was found to be differentially regulated in response to low and high concentrations of SCFAs. These findings demonstrate that the physiological functions of SCFAs in various cellular responses are more likely dependent on their local concentration.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.5
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• pp.427-433
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• 2013

Receptor activator of NF-${\kappa}B$ ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular $Ca^{2+}$ mobilization in a form of oscillations, which plays essential roles by activating sequentially $Ca^{2+}$/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether $Ca^{2+}$ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated $Ca^{2+}$ mobilization induced by aluminum fluoride ($AlF_4^-$), a G-protein activator, with or without RANKL and the effects of $AlF_4^-$ on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that $AlF_4^-$ induces intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) oscillations, which is dependent on extracellular $Ca^{2+}$ influx. Notably, co-stimulation of $AlF_4^-$ with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by $AlF_4^-$. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for $[Ca^{2+}]_i$ oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.419-425
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• 1998

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ($[Ca^{2+}]_i$) mobilization and cell proliferation were investigated. ATP-induced $[Ca^{2+}]_i$ increased in a dose-dependent manner $(10^{-7}\;M{\sim}10^{-3}\;M)$. ATP-induced $[Ca^{2+}]_i$ increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced $[Ca^{2+}]_i$ increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative $P_{2Y}$ receptor antagonist, dose-dependently weakened ATP-induced $[Ca^{2+}]_i$ mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.

• Biomedical Science Letters
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• v.10 no.1
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• pp.1-8
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• 2004

Cordycepin separated from Cordyceps militaris is a major physiologic active component in Cordyceps militaris. The platelet aggregation is stimulated by $Ca^{2+}$, which is either mobilized from intracellular endoplasmic reticulum or transported from extracellular space. cGMP antagonizes the actions of $Ca^{2+}$. Based on these facts, we have investigated the effects of cordycepin on the mobilization of $Ca^{2+}$ and the production of cGMP on collagen ($10\mu$g/ml)-induced human platelet aggregation. Cordycepin potently stimulated the human platelet aggregation induced by collagen ($10\mu$g/ml) in a dose-dependent manner. Cordycepin (500 $\mu$M) inhibited also the collagen-induced human platelet aggregation in the presence both 1 mM and 2 mM of $CaCl_2$. These are in accord with the results that cordycepin inhibited the $Ca^{2+}$- influx on collagen-induced human platelet aggregation. These results suggest that cordycepin decrease the intracellular $Ca^{2+}$ concentration to inhibit collagen-induced human platelet aggregation. Besides, cordycepin increased the level of cGMP on collagen-induced human platelet aggregation. This result is related with the decrease of intracellular $Ca^{2+}$ concentration, because cGMP inhibits the mobilization of $Ca^{2+}$. In addition, cordycepin inhibited the human platelet aggregation induced by LY -83583, inhibitor of guanylate cyclase. This result suggested that cordycepin inhibit the platelet aggregation by stimulating the activity of guanylate cyclase. In conclusion, we demonstrated that cordycepin might have the antiplatelet function by inhibiting $Ca^{2+}$-mobilization via the stimulation of the production of cGMP.

• BMB Reports
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• v.28 no.6
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• pp.499-503
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• 1995

In our previous studies (Chae et al., 1990; Chae et a1., 1993), we found that a phytochrome signal was clearly connected with the change in cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in oat cells. It was determined that the $[Ca^{2+}]_i$ change occured both by mobilization out of the intracellular $Ca^{2+}$ store and by influx from the medium. The specific aim of this work is to elucidate the processes connecting $Ca^{2+}$ mobilization and influx. The cells treated with thapsigargin (increasing $[Ca^{2+}]_i$ by inhibition of the $Ca^{2+}$-ATPase in the calcium pool) in the presence of external $Ca^{2+}$ showed the same increasing pattern (sustained increasing shape) of $[Ca^{2+}]_i$ as that measured in animal cells. Red light irradiation after thapsigargin treatment did not increase $[Ca^{2+}]_i$ These results suggest that thapsigargin also acts specifically in the processes of mobilization and influx of $Ca^{2+}$ in oat cells. When the cells were treated with TEA ($K^+$ channel blocker), changes in $[Ca^{2+}]_i$ were drastically reduced in comparison with that measured in the absence of TEA. The results suggest that the change in $[Ca^{2+}]_i$ due to red light irradiation is somehow related with $K^+$ channel opening to change membrane potential. The membrane potential change due to $K^+$ influx might be the critical factor in opening a voltage-dependent calcium channel for $Ca^{2+}$ influx.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.233-239
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• 2017

Intracellular calcium ($Ca^{2+}$) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide ($H_2O_2$) on intracellular $Ca^{2+}$ accumulation in mouse pancreatic acinar cells. Perfusion of $H_2O_2$ at $300{\mu}M$ resulted in additional elevation of intracellular $Ca^{2+}$ levels and termination of oscillatory $Ca^{2+}$ signals induced by carbamylcholine (CCh) in the presence of normal extracellular $Ca^{2+}$. Antioxidants, catalase or DTT, completely prevented $H_2O_2$-induced additional $Ca^{2+}$ increase and termination of $Ca^{2+}$ oscillation. In $Ca^{2+}$-free medium, $H_2O_2$ still enhanced CCh-induced intracellular $Ca^{2+}$ levels and thapsigargin (TG) mimicked $H_2O_2$-induced cytosolic $Ca^{2+}$ increase. Furthermore, $H_2O_2$-induced elevation of intracellular $Ca^{2+}$ levels was abolished under sarco/endoplasmic reticulum $Ca^{2+}$ ATPase-inactivated condition by TG pretreatment with CCh. $H_2O_2$ at $300{\mu}M$ failed to affect store-operated $Ca^{2+}$ entry or $Ca^{2+}$ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial $Ca^{2+}$ uniporter blocker, failed to attenuate $H_2O_2$-induced intracellular $Ca^{2+}$ elevation. These results provide evidence that excessive generation of $H_2O_2$ in pathological conditions could accumulate intracellular $Ca^{2+}$ by attenuating refilling of internal $Ca^{2+}$ stores rather than by inhibiting $Ca^{2+}$ extrusion to extracellular fluid or enhancing $Ca^{2+}$ mobilization from extracellular medium in mouse pancreatic acinar cells.

• Biomedical Science Letters
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• v.13 no.4
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• pp.333-339
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• 2007

Recently, much attention has been paid to human retinoblastoma since it provide a good model system for studying mechanisms underlying cell growth, differentiation, proliferation, and apoptosis, and for developing cancer therapy. However, until now it is unclear whether purinergic receptors are involved in the calcium mobilization in the retinoblastoma cells. In this regard, we measured possible purinergic signaling in WERI-Rb-1 cells using $Ca^{2+}$ imaging technique and RT-PCR method. ATP-induced $[Ca^{2+}]_i$ transients was maintained to about $90.7{\pm}1.0%$ of the control (n=48) even in the absence of extracellular calcium. The ATP-induced intracellular calcium response was only attained to $10.4{\pm}1.8%$ (n=55) of peak amplitude of the control after preincubation of 1 ${\mu}MU-73122$, a PLC inhibitor, but it was not affected by 1 ${\mu}MU-73343$, a inactive form of U-73122. And also ATP-induced $[Ca^{2+}]_i$ rise was almost attenuated by 20 ${\mu}M$ 2-APB, a putative $IP_3$ receptor inhibitor. Two subtypes of $IP_3$ receptor $(IP_{3-1}R,\;IP_{3-2}R)$ were identified by a RT-PCR method. These findings suggest that purinergic stimuli can cause calcium mobilization via $PLC-IP_3$ pathway after the activation of P2Y receptors in the retinoblastoma cells, which may play important roles in cell proliferation, differentiation, growth, and cell death.

• BMB Reports
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• v.40 no.5
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• pp.678-683
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• 2007

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 ${\mu}g/ml$)-stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metallo-proteinase-9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular $Ca^{2+}$ mobilization, and thromboxane $A_2$ ($TXA_2$) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or $TXA_2$ synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular $Ca^{2+}$ mobilization and $TXA_2$ production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.