• Biomedical Science Letters
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• v.25 no.4
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• pp.302-312
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• 2019

The aim of this work was to investigate the effect of black tea extract (BTE) on collagen -induced platelet aggregation. In this study, BTE (10~500 ㎍/mL) was shown to inhibit platelet aggregation via thromboxane A₂ (TXA₂) down-regulation by blocking cyclooxygenase-1 (COX-1) activity. Also, BTE decreased intracellular Ca²⁺ mobilization ([Ca²⁺]_i). Additionally, BTE enhanced the levels of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are aggregation-inhibiting molecules. BTE inhibited the phosphorylation of phospholipase C (PLC) γ2 and syk activated by collagen. BTE regulated platelet aggregation via cAMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) Ser¹⁵⁷. The anti-platelet effects of BTE in high fat diet (HFD)-induced obese rats were evaluated. After eight weeks of BTE treatment (300 and 600 mg/kg), the platelet aggregation rate in the treated groups was significantly less than that in the HFD-fed control group. Also, BTE exhibited a hepatoprotective effect and did not exert hepatotoxicity. Therefore, these data suggest that BTE has anti-platelet effects on collagen-stimulated platelet aggregation and may have therapeutic potential for the prevention of platelet-mediated thrombotic diseases.

• Biomedical Science Letters
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• v.15 no.4
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• pp.319-326
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• 2009

Extracellular ATP elicits diverse physiological effects by binding to the G-protein-coupled P2Y receptors on the plasma membrane. In addition to the short-term effects of extracellular nucleotides on cell functions, there is evidence that such purinergic signalling can have long-term effects on cell proliferation, differentiation and death. The 3T3-L1 cell line derived from mouse embryo is a well-established and commonly utilized in vitro model for adipocytes differentiation and function. However, the distributions and roles of P2Y subtypes are still unknown in the preadipocyte. In this study, we identified the distributions and roles of P2Y subtypes in preadipocyte using $Ca^{2+}$ imaging and realtime PCR. ATP increased the $[Ca^{2+}]_i$ in a concentration-dependent manner. ATP increased $Ca^{2+}$ in absence and/or presence of extracellular $Ca^{2+}$. Suramin, non-selective P2Y blocker, largely blocked the ATP-induced $Ca^{2+}$ response. U73122, a PLC inhibitor, completely inhibited $Ca^{2+}$ mobilization in 3T3-L1 cells. The mRNA expression by realtime PCR of P2Y subtypes was $P2Y_2:P2Y_5:P2Y_6=1.0:12.5:0.3$. In conclusion, we showed that $P2Y_5$ receptor is a dominant purinergic receptor in preadipocytes, and multiple P2Y receptors could involve in differentiation and migration via regulating of intracellular calcium concentration.

• 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 Physiology and Pharmacology
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• v.2 no.1
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• pp.63-68
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• 1998

Nitric oxide (NO)-mediated relaxation in vascular smooth muscle involves not only activation of guanylate cyclase but also hyperpolarization of the membrane. It has been shown that depolarization decreases the [$Ca^{2+}$] sensitivity of myosin light chain kinase in arterial smooth muscle, and nitric oxide (NO)-mediated relaxation was attenuated in this situation. However, why potassium inhibits or attenuates the action of EDRF/NO is not clear. Therefore, we investigated the magnitude of relaxation and cGMP contents using measures known to release NO, such as photorelaxation, photo activated NO-mediated relaxation, and NO-donor (SNP)-mediated relaxation in porcine coronary arterial rings in which contractile conditions were made by different degree of depolarization, i.e., contraction in response to U46619 or U46619 plus KCl. In all cases, the magnitude of relaxation was significantly greater (P＜0.05) in U46619-contracted rings than in U46619+KCl-contracted ones. Although accumulation of cGMP was evident with three measures employed in the present study, no difference was found in cGMP contents between U46619 and U46619+KCl conditions, indicating that the diminished relaxation in KCl containing solution is cGMP-independent mechanism(s). To understand this further, cytosolic $Ca^{2+}$ changes due to NO were compared in rat thoracic aorta by exploiting photoactivated NO using streptozotocin (STZ) that was contracted with either NE or KCl. Fura-3 $[Ca]_{cyt}$ signal caused by NO was small and transient in high $K^+$-, but large and sustained in NE-contracted aorta. The inhibitory potency of STZ expressed in terms of $IC_{50}$ was 5.14 and 3.88 ${\mu}M$ in NE and in high $K^+$, respectively. These results suggest that modification of the cellular mobilization of $Ca^{2+}$ rather than cGMP levels may be an important mechanism for the NO-mediated relaxation when vascular membrane is depolarized, such as atherosclerosis and hypertension.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.377-384
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• 1998

Gold compounds depress phagocytic cell responses, including chemotaxis, and respiratory burst. However, the effects of gold compounds on the function of phagocytic cells are variable according to the preparation of medicine. In this study, effect of tetrachloroauric acid on activated neutrophil responses, including respiratory burst, lysosomal enzyme release and change of intracellular $Ca^{2+}$ level and on the synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by macrophages was studied. This study further examines how gold compounds affect the activation processes. The respiratory burst stimulated by complement C5a, degraded IgG and PMA in neutrophils was inhibited by tetrachloroauric acid. In contrast to C5a and degraded IgG, PMA-stimulated superoxide production was weakly inhibited by tetrachloroauric acid. Staurosporine, genistein, EGTA and verapamil inhibited superoxide and $H_2O_2$ production caused by C5a and degraded IgG. PMA-stimulated superoxide production was inhibited by staurosporine but was not affected by genistein. Tetrachloroauric acid, genistein, EGTA and verapamil inhibited the release of acid phosphatase and myeloperoxidase, while the effect of staurosporine was not detected. The synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by $interleukin-1{\beta}$ in macrophages was inhibited by tetrachloroauric acid. Preincubation with tetrachloroauric acid, genistein, EGTA and verapamil, the elevation of [$Ca^{2+}_i$] evoked by C5a was inhibited. Store-regulated $Ca^{2+}$ entry in thapsigargin-pretreated neutrophils was decreased by the addition of tetrachloroauric acid and genistein. The effect of staurosporine on intracellular $Ca^{2+}$ mobilization was not observed. In conclusion, tetrachloroauric acid may suppress neutrophil responses through its inhibitory action on elevation of intracellular $Ca^{2+}$ level and protein kinase C. It might exhibit an inhibitory effect on the action of protein tyrosine kinase. Tetrachloroauric acid depresses cytokine production by macrophages.

• Journal of Ginseng Research
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• v.39 no.3
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• pp.279-285
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• 2015

Background: Korean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng's therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. Methods: The platelet aggregation was induced by collagen, the ligand of integrin ${\alpha}_{II}{\beta}_I$ and glycoprotein VI. The crude saponin's effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin ${\alpha}_{II}b{\beta}_{III}$ was examined by fluorocytometry. Results: CSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed $[Ca^{2+}]_i$ mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin ${\alpha}_{IIb}{\beta}_3$. Conclusion: Our present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.129-135
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• 2011

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-$HT_{2B,\;3,\;4,\;and\;7}$ receptors in ICC. However, SDZ 205557 (a 5-$HT_4$ receptor antagonist), SB 269970 (a 5-$HT_7$ receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-$HT_3$ antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-$HT_{2B}$ receptor antagonist). Based on $[Ca^{2+}]_i$ analysis, we found that 5-HT increased the intensity of $[Ca^{2+}]_i$. The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-$HT_{3,\;4,\;and\;7}$ receptors via $[Ca^{2+}]_i$ mobilization and regulation of mitogen-activated protein kinases.

• Journal of Ginseng Research
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• v.37 no.2
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• pp.176-186
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• 2013

In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with $IC_{50}$ value of about 81.1 ${\mu}g/mL$. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free $Ca^{2+}$ ($[Ca^{2+}]_i$), one of aggregation-inducing molecules. Of two $Ca^{2+}$-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of $[Ca^{2+}]_i$ elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-$Ser^{157}$, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-$Ser^{239}$, a cGMP-dependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-$Ser^{157}$ phosphorylation through A-kinase activation to inhibit $[Ca^{2+}]_i$ mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.

• Korean Journal of Pharmacognosy
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• v.51 no.4
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• pp.231-237
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• 2020

Euchrestaflavanone B (EFB) is a flavonoid that can be found in root bark, particularly in Cudrania tricuspidata (C. tricuspidata). The extract of C. tricuspidata is widespread throughout Asia and used in traditional medicine. In a previous study, we found anti-platelet effects of substances isolated from C. tricuspidata on collagen-induced human platelets. However, the C. tricuspidata still contains numerous substances, thus, we have searched new candidate, EFB isolated from C. tricuspidata for anti-platelet effect. Our results showed that EFA inhibited collagen-induced platelet aggregation and glycoprotein IIb/IIIa (αIIb/β3)-mediated signaling events, including platelet adhesion, granule secretion, thromboxane A2 production and clot retraction. These results suggest that EFA has inhibitory effects on human platelet activities and thrombus formation and has potential value as a natural substance for preventing platelet-induced thrombosis.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.