• Toxicological Research
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• 제18권4호
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• pp.355-362
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• 2002

Pathophysiological elevation of intracellular calcium concentration ($[Ca^{2+}]_1$) in the neuron has been considered as an important responsible factor in the neuronal cell damages. However the mechanism of increase of $[Ca^{2+}]_1$ and the relationship between $[Ca^{2+}]_1$ level and cytotocixity have not been fully demonstrated. In the present study, real-time alteration of $[Ca^{2+}]_1$and cellular response (cell damages) in the pheochromocytoma cells (PC12) stimulated by glutamate were investigated. Glutamate dose dependently decreased cell viability determined propidium iodide fluorescence method and morphology change. Conversely related with cell damages, glutamate dose dependently increased the level of［Ca$^{2+}$］$_{i}$ . To investigate the mechanism of glutamate-induced increase of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$, was first measured in the cell cultured in calcium free media and in the presence of dantrolene, an inhibitor of calcium release from ryanodine receptor located in endoplasmic reticulum (ER). Similar to the increase$[Ca^{2+}]_1$ in the calcium-containing media, glutamate dose dependently increased $[Ca^{2+}]_1$ in the cell cultured in free calcium media. However pretreatment (2 hr) with 20~50 $\mu\textrm{M}$ dantrolene substantial lowered glutamate-induced increase of $[Ca^{2+}]_1$, suggesting that release of calcium from ER may be major sourse of increase of $[Ca^{2+}]_1$ in PC12 cells. Dantrolene-induced inhibition of $[Ca^{2+}]_1$ resulted in recovery of cytotoxicity by glutamate. Relevance of N-methy-D-aspartate (NMDA) receptor, a type of glutamte receptor on glutamate-induced incense of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$ was also determined in the cells pretreated (2 hr) with NMDA receptor antagonist MK-80l. Glutamate-induced increase of $[Ca^{2+}]_1$ was reduced by MK-801 dose dependently. Furthermore, glutamate-induced cytotoxicity was also prevented by MK-80l. These results demonstrate that glutamte increase $[Ca^{2+}]_1$ dose dependently and thereby cause cytotoxicity. The increase of $[Ca^{2+}]_1$ may release from ER, especially through ryanodine receptor and/or through NMDA receptor Alteration of calcium homeostasis through disturbance of ER system and/or calcium influx through NMDA receptor could contribute glutamate-induced cell damages.s.

• The Korean Journal of Physiology and Pharmacology
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• 제20권6호
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• pp.629-639
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• 2016

The present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to $30{\mu}g/ml$), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine ($1{\mu}M$, an autonomic ganglionic bloker), pirenzepine ($2{\mu}M$, a muscarinic $M_1$ receptor antagonist), Ki14625 ($10{\mu}M$, an $LPA_{1/3}$ receptor antagonist), amiloride (1 mM, an inhibitor of $Na^+/Ca^{2+}$ exchanger), a nicardipine ($1{\mu}M$, a voltage-dependent $Ca^{2+}$ channel blocker), TMB-8 ($1{\mu}M$, an intracellular $Ca^{2+}$ antagonist), and perfusion of $Ca^{2+}$-free Krebs solution with 5mM EGTA (a $Ca^{2+}$chelater), while was not affected by sodium nitroprusside ($100{\mu}M$, a nitrosovasodialtor). Interestingly, LPA ($0.3{\sim}3{\mu}M$, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 ($10{\mu}M$). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin ($3{\mu}g/ml$). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic $Ca^{2+}$ increase by stimulation of the $Ca^{2+}$ influx as well as by the inhibition of $Ca^{2+}$ uptake into the cytoplasmic $Ca^{2+}$ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the $Ca^{2+}$ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.

• 대한한방종양학회지
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• 제4권1호
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• pp.177-197
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• 1998

This experimental study was carried out to evaluate the effect of Yipahnsan on angiogenic inhibition mechanism. This study investigates the effects of Yipahnsan on angiogenic inhibition mechanism evaluate cell adhesive inhibition effect, DNA fragmantaion analysis, nuclear condensation assay, FACScan analysis, angiogenic lumen formation assay, immunocytochemistry analysis, RT-PCR for mRNA expression, western blot analysis, confocal analysis for $Ca^{2+}influx$. The results were summarized as follows : 1. The cell adhesive inhibition ability was strongly increased from $5{\mu}g/ml$ on ECV304 cell line and ECVPAR cell line. 2. YY water extract caused $G_0/G_1$ arrest peak to existed on the ECV304 cell line. 3. YY water extract caused inhibition of proliferation and inducement of apoptosis on the collagen coated plate in ECV304 cell line. 4. YY water extract inhibited the lumen formation on the matrigel coated plate in ECV304 cell line. 5. YY water extract inhibited the expressions of LFA-1 and ELAM-1 on ECV304 cell line and ECVPAR cell line. 6. YY water extract inhibited the expressions of MMP-9 and uPA on ECV304 cell line and ECVPAR cell line. 7. YY water extract inhibited the expression of integrin ${\alpha}_v{\beta}_3$ on ECV304 cell line and ECVPAR cell line. 8. YY water extract decreased the change of $Ca^{2+}$ in intracellular on ECV304 cell line and ECVPAR cell line. According to the results, Yipahnsan showed to be a key antagonist of integrin ${\alpha}_v{\beta}_3$, and to be induction of apoptosis by p53 through flow cytometry. This report also demonstrated that expressions of MMP-9 and uPA were blocked under the angiogenesis model. Thus, we suggested that Yipahnsan blocks angiogenesis by inducing apoptosis in ECV304 and ECVPAR cell lines, and another oriental herbal medicine that treats qi-stagnation and blood-stasis type also has angiogenic inhibition effects.

• Toxicological Research
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• 제14권3호
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• pp.349-356
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• 1998

We identified S9940, a novel microbial metabolite from Streptomyces spp., to inhibit the release of neurotransmitter from PC12 cells. S9940 is an inhibitor of trifiated norepinephrine ([$^{3}H$]-NE) release in high $K^+$ buffer solution containing ionomycin, indicating that S9940 inhibits neurotransmitter release after the influx of $Ca^{2+}$ ions. We also examined the effect of S9940 on $\beta-glucuronidase$ release from guinea pig neurophils and the effect on the neurite extension of PC12 cells and rat hippocampal neurons. As a result, S9940 inhibited $\beta-glucuronidase$ release: when treated with $5{\mu}g/ml$ of S9940, which prevented [$^{3}H$]-NE release, the inhibition of neurite extension for both PC12 cells and rat hippocampal neurons was observed.

• Preventive Nutrition and Food Science
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• 제21권4호
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• pp.323-329
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• 2016

Achyranthes japonica Nakai (AJN) water extract has a variety of physiological properties, including anti-diabetic, anti-cancer, anti-inflammatory, anti-microbial, and anti-oxidative activities. In the present study, the inhibitory effects of AJN extract were investigated in high affinity immunoglobulin E receptor ($Fc{\varepsilon}RI$)-mediated KU812F cells activation. AJN extract showed suppressive effects on histamine release and intracellular calcium [$Ca^{2+}$]i elevation from anti$Fc{\varepsilon}RI$ antibody (CRA-1)-stimulated cells in a dose-dependent manner. Flow cytometric analysis showed that AJN extract treatment caused a dose-dependent decrease in the cell surface $Fc{\varepsilon}RI$ expression and the binding between the cell surface $Fc{\varepsilon}RI$ and the IgE antibody. Moreover, reverse transcription-polymerase chain reaction analysis showed that levels of the mRNA for the $Fc{\varepsilon}RI$ ${\alpha}$ chain was decreased by treatment with AJN extract. These results indicate that AJN extract may exert anti-allergic effects via the inhibition of calcium influx and histamine release, which occurs as a result from the downregulation of the binding of IgE antibody to cell surface $Fc{\varepsilon}RI$. This mechanism may occur through $Fc{\varepsilon}RI$ expression inhibition.

• The Korean Journal of Physiology and Pharmacology
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• 제4권2호
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• pp.149-158
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• 2000

The present study was attempted to examine the effect of staurosporine (STS) on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused rat adrenal gland and to establish its mechanism of action. The perfusion of STS $(3{\times}10^{-7}{\sim}3{\times}10^{-8}\;M)$ into an adrenal vein for 20 min produced a dose-dependent inhibition in CA secretion evoked by ACh $(5.32{\times}10^{-3}\;M),$ high $K^+\;(5.6{\times}10^{-2}\;M),$ DMPP $(10^{-4}\;M\;for\;2\;min),$ McN-A-343 $(10^{-4}\;M\;for\;2\;min),$ cyclopiazonic acid $(10^{-5}\;M\;for\;4\;min)$ and Bay-K-8644 $(10^{-5}\;M\;for\;4\;min).$ Also, in the presence of tamoxifen $(2{\times}10^{-6}\;M),$ which is known to be a protein kinase inhibitor, CA secretory responses evoked by ACh, high $K^+,$ DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with STS $(10^{-7}\;M)$ under the presence of phorbol-12, 13-dibutyrate $(10^{-7}\;M),$ a specific activator of protein kinases (for 20 min), the inhibitory effect of STS on CA secretory responses evoked by ACh, high $K^+,$ DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid was greatly recovered to the extent of the control release as compared to those in the presence of STS only. These results demonstrate that STS causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating strongly that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells through preventing activation of protein kinases. Furthermore, these findings also suggest that these STS-sensitive protein kinases play a modulatory role partly in regulating the rat adrenomedullary CA secretion.

• Applied Biological Chemistry
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• 제40권3호
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• pp.202-208
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• 1997

콩 뿌리조직의 이온 흡수와 관련된 생리활성을 조사하기 위하여 뿌리조직으로부터 마이크로솜을 분리하였고, 마이크로솜 ATPase (이온점프) 활성을 분광학적 방법인 enzyme-coupled 분석방법에 따라 측정하였다. 마이크로솜 ATPase의 활성에 미치는 여러 가지 이온의 효과 또는 ATPase의 이온선택성을 조사하기 위하여 $10mM\;Na^+$과 $120mM\;K^+$을 포함하는 대조용액에서의 평균활성을 측정한 결과 190 nmol/min/mg protein으로 나타났다. 대조활성에 비하여 $Na^+$을 포함하지 않은 $130mM\;K^+$ 용액에서는 활성이 150%로 증가하였고, $K^+$을 포함하지 않은 $130mM\;Na^+$ 용액에서는 활성이 63%로 감소되었다. 반응용액의 $K^+$ 농도에 따른 활성변화를 측정한 결과, ATPase의 활성은 외부용액의 $K^+$ 농도 증가에 따라 활성이 증가됨을 알 수 있었다. 또한 마이크로솜 ATPase 활성은 반응용액의 pH 감소에 따라 증가되어 $pH\;6{\sim}7$에서는 비교적 높은 활성을 보였으나, pH 8 이상에서는 급격히 활성이 감소되었고, pH 9에서는 80%이상의 활성이 저해되었다. $Ca^{2+}$에 의한 이온펌프의 활성조절 여부를 평가하기 위해서 마이크로솜 내부 및 외부의 $Ca^{2+}$에 의한 ATPase 활성변화를 측정하였다. 마이크로솜 ATPase의 활성은 반응액의 $Ca^{2+}$ 농도가 낮아질수록 증가하여 $10^{-9}M$ 이하에서 최대활성이 관측되었고, $Ca^{2+}$ 농도가 증가할수록 활성은 감소하여 $500\;{\mu}M$ 전후에서 50%의 활성이 감소하였다. 또한 ATPase의 활성은 마이크로솜 내부의 $Ca^{2+}$ 농도증가에 의해서 저해되어, $Ca^{2+}\;ionophore\;A23187$처리에 의한 외부의 $Ca^{2+}$ 유입에 의해서 약30%의 활성감소를 보였으며, EGTA 처리에 의한 $Ca^{2+}\;chelation$에 의해서 마이크로솜 내부의 $Ca^{2+}$ 농도가 감소되었을 때, ATPase 활성은 증가하였다. 위의 조건에서 실제 마이크로솜 내부로의 $Ca^{2+}$ 유입 여부는 $‘Ca^{2+}’$를 이용하여 확인하였다. 이상의 결과는 마이크로솜 막에 위치한 ATPase의 내부 및 외부에 $Ca^{2+}$에 의한 효소활성 조절부위가 각각존재함을 시사한다.

• The Korean Journal of Physiology and Pharmacology
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• 제14권4호
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• pp.241-248
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• 2010

The present sutdy aimed to determine whether olmesartan, an angiotensin II (Ang II) type 1 ($AT_1$) receptor blocker, can influence the CA release from the isolated perfused model of the rat adrenal medulla. Olmesartan ($5{\sim}50{\mu}M$) perfused into an adrenal vein for 90 min produced dose- and time-dependent inhibition of the CA secretory responses evoked by ACh (5.32 mM), high $K^+$ (56 mM, a direct membrane-depolarizer), DMPP (100 ${\mu}M$) and McN-A-343 (100 ${\mu}M$). Olmesartan did not affect basal CA secretion. Also, in adrenal glands loaded with olmesartan (15 ${\mu}M$), the CA secretory responses evoked by Bay-K-8644 (10 ${\mu}M$, an activator of voltage-dependent L-type $Ca^{2+}$ channels), cyclopiazonic acid (10 ${\mu}M$, an inhibitor of cytoplasmic $Ca^{2+}$-ATPase), veratridine (100 ${\mu}M$, an activator of voltage-dependent $Na^+$ channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations ($150{\sim}300{\mu}M$), olmesartan rather enhanced the ACh-evoked CA secretion. Taken together, these results show that olmesartan at low concentrations inhibits the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by direct membrane depolarization from the rat adrenal medulla, but at high concentrations it rather potentiates the ACh-evoked CA secretion. It seems that olmesartan has a dual action, acting as both agonist and antagonist at nicotinic receptors of the isolated perfused rat adrenal medulla, which might be dependent on the concentration. It is also thought that this inhibitory effect of olmesartan may be mediated by blocking the influx of both $Na^+$ and $Ca^{2+}$ into the rat adrenomedullary chromaffin cells as well as by inhibiting the $Ca^{2+}$ release from the cytoplasmic calcium store, which is thought to be relevant to the $AT_1$ receptor blockade, in addition to its enhancement on the CA secreton.

• Biomolecules & Therapeutics
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• 제16권2호
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• pp.147-160
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• 2008

The present study was designed to examine effects of polyphenolic compounds isolated from red wine (PCRW) on the release of catecholamines (CA) from the isolated perfused model of the rat adrenal medulla, and to clarify its mechanism of action. PCRW (20${\sim}$180 ${\mu}$g/mL), given into an adrenal vein for 90 min, caused inhibition of the CA secretory responses evoked by ACh (5.32 mM), high $K^+$ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic $N_N$ receptor agonist, 100 ${\mu}$M) and McN-A-343 (a selective muscarinic $M_1$ receptor agonist, 100 ${\mu}$M) in dose- and time-dependent fashion. PCRW itself did not affect basal CA secretion (data not shown). Following the perfusion of PCRW (60 ${\mu}$g/mL), the secretory responses of CA evoked by Bay-K-8644 (a L-type dihydropyridine $Ca^{2+}$ channel activator, 10 ${\mu}$M), cyclopiazonic acid (a cytoplasmic $Ca^{2+}$-ATPase inhibitor, 10 ${\mu}$M) and veratridine (an activator of voltage-dependent $Na^+$ channels, 10 ${\mu}$M) were also markedly blocked, respectively. Interestingly, in the simultaneous presence of PCRW (60 ${\mu}$g/mL) and L-NAME (a selective inhibitor of NO synthase, 30 ${\mu}$M), the inhibitory responses of PCRW on the CA secretion evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyclpiazonic acid were recovered to considerable level of the corresponding control release compared with those effects of PCRW-treatment alone. Practically, the amount of NO released from adrenal medulla after loading of PCRW (180 ${\mu}$g/mL) was significantly increased in comparison to the corresponding basal released level. Collectively, these results obtained here demonstrate that PCRW inhibits the CA secretory responses evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization from the isolated perfused adrenal gland of the normotensive rats. It seems that this inhibitory effect of PCRW is mediated by blocking the influx of both ions through $Na^+$ and $Ca^+{2$} channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the release of $Ca^{2+}$ from the cytoplasmic calcium store, which are due at least partly to the increased NO production through the activation of nitric oxide synthase. Based on these data, it is also thought that PCRW may be beneficial to prevent or alleviate the cardiovascular diseases, such as hypertension and angina pectoris.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.246-254
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• 2018

Enzyme fermentation is a type of food processing technique generally used to improve the biological activities of food and herbal medicines. In this study, a Syzygii Flos (clove) extract was fermented using laccase derived from Trametes versicolor (LTV). The fermented clove extract showed greater neuroprotective effects against glutamate toxicity on HT22 than the non-fermented extract did. HPLC analysis revealed that the eugenol (1) and dehydrodieugenol (2) contents had decreased and increased, respectively, after fermentation. The content of 2 peaked at 1 h after fermentation to $103.50{\pm}8.20mg/g_{ex}$ (not detected at zero time), while that of 1 decreased to $79.54{\pm}4.77mg/g_{ex}$ ($185.41{\pm}10.16mg/g_{ex}$ at zero time). Compound 2 demonstrated promising HT22 neuroprotective properties with inhibition of $Ca^{2+}$ influx, the overproduction of intracellular reactive oxygen species, and lipid peroxidation. In addition, LTV showed the best fermentation efficacy compared with laccases derived from Pleurotus ostreatus and Rhus vernicifera.