• 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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• 제17권3호
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• The Korean Journal of Physiology and Pharmacology
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• 제8권1호
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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.

• 대한약리학회지
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• 제25권1호
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• pp.31-42
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• 1989

Metoclopramide (MCP)는 역류성식도염, 위하수증, 항암제에 의한 구토 증상을 치료하는데 사용되고 있는 dopamine수용체 차단제로 알려져 있다. 본 연구에서는 흰쥐의 적출 관류부신에서 catecholamine (CA)의 분비작용에 미치는 영향을 검토하여 다음과 같은 결과를 얻었다. MCP는 부신정맥내로 주입하였을때 용량의존적으로 CA 분비작용을 나타냈다. 이러한 MCP의 CA 분비작용은 atropine 전처치로 차단되었으나 chlorisondamine 처치에 의해서는 완전 차단되지 못하였다. MCP 분비작용은 physostigmine, adenosine, ouabain의 전처치로 현저히 증강되었다. 그러나 5mM-EGTA 함유 $Ca^{++}-free\;Krebs$ 액으로 관류하였을때 MCP의 CA 분비작용은 현저히 억제되었다. MCP (200 ug/30 min)를 관류한 실험에서는 KCI이나 acetylcholine의 CA 분비작용이 유의있게 감소되었다. 이상의 실험결과로 보아 MCP는 칼슘의존적기전에 의해 CA를 유리 시키며, 이러한 분비작용은 부신에서 nicotine수용체 보다도 muscarine수용체의 활성화에 더 기인 되는 것으로 생각되며, chromaffin cell에 대한 일분 직접작용도 개재되어 나타나는 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.175-179
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• 2009

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1${\sim}$2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 ${\mu}M$ Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 ${\mu}M$ of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic $[Ca^{2+}]_i$ response; a transient $[Ca^{2+}]_i$ peak and sustained $[Ca^{2+}]_i$ increase secondary to ATP-stimulated $Ca^{2+}$ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

• The Korean Journal of Physiology and Pharmacology
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• 제3권3호
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• pp.339-350
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• 1999

The present study was attempted to examine the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on catecholamine (CA) secretion evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat adrenal gland. The perfusion of PACAP (10 nM) into an adrenal vein for 60 min produced a great 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 neuropeptide (NPY), which is known to be co-localized with norepinephrine in peripheral sympathetic nerves, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with PACAP (10 nM) under the presence of VIP antagonist $[(Lys^1,\;Pro^{2.5},\;Arg^{3.4},\;Tyr^6)-VIP\;(3\;{\mu}M)]$ for 20 min, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were not altered greatly in comparison to the case of PACAP-treatment only. Taken together, these results suggest that PACAP causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.95-95
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• 2001

Ciinidipine (FRC-8635) is a newly synthesized novel DHP type of organic Ca$\_$2＋/channel blockers that have been developed so far in Japan (Yoshimoto et al., 1991 : Hosono et at., 1992). It also has a blocking action on L-type voltage-dependent Ca$\^$2＋/channel (VDCCs) in the rabbit basilar artery (Oike et al., 1990) and a slow-onset and long-lasting hypotensive action in clinical and experimental studies (Ikeda et al., 1992 ; Tominaga et al., 1997). Recent electrophysiological data indicate that cilnidipine might be a dual-channel antagonist for peripheral neuronal N-type and vascular L-type Ca$\^$2＋/channels (Oike et al., 1990 ; Fujii et al., 1997; Uneyama et at., 1997). However, little is known about the involvement of N-type VDCCs in contributing to the muscarinic receptor-mediated CA secretion. Therefore, the present study was attempted to investigate the effect of cilinidipine on secretion of catecholamines (CA) evoked by ACh, high K$\^$＋/, DMPP and McN-A-343 from the isolated perfused rat adrenal gland. Cilnidipine (1-10 ${\mu}$M) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretory responses evoked by ACh (5.32${\times}$10$\^$-3/M), DMPP (10$\^$-4/ M for 2 min) and McN-A-343 (10$\^$-4/ M for 2 min). However, lower dose of lobeline did not affect CA secretion by high K$\^$＋/(5.6${\times}$10$\^$-2/ M), higher dose of it reduced greatly CA secretion of high K$\^$＋/. Cilnidipine itself did also fail to affect basal catecholamine output. Furthermore, in adrenal glands loaded with cilnidipine (10 ${\mu}$M), CA secretory response evoked by Bay-K-8644 (10 ${\mu}$M), an activator of L-type Ca$\^$2＋/channels was markedly inhibited while CA secretion by cyclopiazonic acid (10 ${\mu}$M), an inhibitor of cytoplasmic Ca$\^$2＋/-ATPase was no affected. Moreover, $\omega$-conotoxin GVIA (1 ${\mu}$M), given into the adrenal gland for 60 min, also inhibited time-dependently CA secretory responses evoked by ACh and high K$\^$＋/.

• The Korean Journal of Physiology and Pharmacology
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• 제10권5호
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• pp.273-282
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• 2006

The aim of the present study was to investigate the effects of R-(-)-2,10,11-trihydroxy-N-propylnoraporphine [R-(-)-TNPA], a selective agonist of dopaminergic $D_2$ receptor and S(-)-raclopride, a selective antagonist of dopaminergic $D_2$ receptor, on the secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused model of the rat adrenal gland, and also to establish its mechanism of action. R-(-)-TNPA $(10{\sim}100\;{\mu}M)$ perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretory responses evoked by ACh (5.32 mM), high $K^+$ (56 mM), DMPP $(100\;{\mu}M)$ and McN-A-343 $(100\;{\mu}M)$. R-(-)-TNPA itself did also fail to affect basal CA output. Also, in adrenal glands loaded with R-(-)-TNPA $(30\;{\mu}M)$, the CA secretory responses evoked by Bay-K-8644 $(10\;{\mu}M)$, an activator of L-type $Ca^2+$ channels and cyclopiazonic acid $(10\;{\mu}M)$, an inhibitor of cytoplasmic $Ca^{2+}-ATPase$ were also inhibited. However, S(-)-raclopride $(1{\sim}10\;{\mu}M)$, given into an adrenal vein for 60 min, enhanced the CA secretory responses evoked by ACh, high $K^+$, DMPP and McN-A-343 only for the first period (4 min), although it alone has weak effect on CA secretion. Moreover, S(-)-raclopride $(3.0\;{\mu}M)$ in to an adrenal vein for 60 min also augmented the CA release evoked by BAY-K-8644 and cyclopiazonic acid only for the first period (4 min). However, after simultaneous perfusion of R-(-)-TNP A $(30\;{\mu}M)$ and S(-)-raclopride $(3.0\;{\mu}M)$, the inhibitory responses of R(-)-TNPA $(30\;{\mu}M)$ on the CA secretion evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644, and cyclopiazonic acid were significantly reduced. Taken together, these experimental results suggest that R-(-)-TNPA greatly inhibits the CA secretion from the perfused rat adrenal medulla evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) and membrane depolarization, but S(-)-raclopride rather enhances the CA release by them. It seems that this inhibitory of R-(-)-TNPA may be mediated by stimulation of inhibitory dopaminergic $D_2$ receptors located on the rat adrenomedullary chromaffin cells, while the facilitatory effect of S(-)-raclopride is due to the blockade of dopaminergic $D_2$ receptors, which are relevant to extra- and intracellular calcium mobilization. Therefore, it is thought that dopaminergic $D_2$ receptors may be involved in regulation of CA release in the rat adrenal medulla.

• 대한핵의학회지
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• 제29권3호
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• pp.294-306
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• 1995

국소뇌허혈에서 MK-801의 신경세포방어효과를 검토하기 위하여 흰쥐의 중뇌동맥을 폐색시켜 뇌허혈 및 경색을 유발하기 전과 후에 MK-801을 투여한 후 국소뇌포도당이용률의 변화를 2-deoxy-D-(14C)glu-cose 자가방사기록법을 이용하여 측정하고 이를 대조군과 비교하였다. 중대뇌동맥폐색 시술 전과 후에 평균동맥압, 직장체온, 동맥혈 PH, $pCO_2,\;pO_2$의 유의한 차이는 없었다. [$^{14}C$]DG 실험 중 대조군, MK-801 전처처군과 MK-801 후처치군 사이에서 평균동맥압, 직장 체온, 동맥혈장 당농도, 동맥혈 pH, $pCO_2,\;pO_2$의 유의한 차이는 없었다. 대조군에서 중대뇌동맥폐색 24시간 후에 폐색된 대뇌반구의 전두엽피질, 두정엽피질, 담창구, 편도, 해마 CA-2 지역, CA-3 지역, 내비회, 후두엽피질, 측두엽피질의 국소뇌포도당이용률은 반대쪽정상 대뇌반구 상동부위의 국소뇌포도당이용률보다 유의하게 낮았다. MK-801 전처치군에서 전두엽피질, 두정엽피질, 편도, 해마 CA-2 지역, 내비회, 후두엽피질, 측두엽피질의 병변/정상 국소뇌포도당이용률 비는 대조군의 그것보다 유의하게 높았다. 한편 MK-801 후처치군에서는 전두엽피질, 두정엽피질, 해마 CA-2 지역, 후두엽피질, 측두엽피질의 병변/정상 국소뇌포도 당이용률 비가 대조관의 그것보다 유의하게 높았다. MK-801 전처치군 정상 대뇌반구의 미상핵-피각, 전두엽피질, 두정엽피질, 대상회전, 담창구, 시상, 뇌량, 해마 CA-1, CA-2, CA-3 지역, 후두엽피질, 측두엽피질의 국소뇌포도당이용률은 대조군 정상 대뇌반구의 그것보다 유의하게 높았다. 반면 MK-801 후처치군 정상 대뇌반구의 국소뇌포도당이용률은 대조군 정상대뇌반구의 국소뇌포도당이용률과 유의한 차이가 없었다. 뿐만 아니라 폐색된 대뇌반구에서도 정상 대뇌반구와 매우 유사한 결과를 보였다. 즉, MK-801 전처치군에서는 미상핵-피각, 전두엽피질, 두정엽피질, 대상회전, 담창구, 시상, 뇌량, 해마 CA-1, CA-2, CA-3 지역, 내비회, 후두엽피질, 측두엽피질의 국소뇌포도당이용률이 대조군보다 유의하게 높은 반면, MK-801 후처치군에서는 대조군과 유의한 국소뇌포도당이용률의 차이를 나타내지 않았다. 이상의 결과로부터 국소뇌허혈에서 MK-801은 신경세포방어효과를 나타낼 것으로 생각된다. MK-801은 뇌포도당이용률을 국소적으로 증가시키며, 이는 NMDA 수용체 길항제 투여와 뇌기능 변화의 유관성을 시사하는 것이다.

• 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.