• 대한수의학회지
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• 제31권3호
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• pp.253-258
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• 1991

The aims of this study were to investigate the effect of adenosine triphosphate (ATP), which has been known as the neurotransmitter of nonadrenergic, noncholinergic nerves, and the source of $Ca^{\sharp}$ in the effect of ATP on the isolated renal artery of pig. The results of this study were summarized as follows: 1. ATP caused the contraction and the contractile responses were increased in a dose-dependent manner between the concentration of ATP $2{\times}10^{-3}M$ and $10^{-2}M$ on the isolated renal artery of pig. 2. The contractile responses induced by ATP $(5{\times}10^{-3}M)$ were not blocked by pretreatment with cholinergic receptor blocker (atropine, $10^{-6}M$), $\alpha$-adrenergic recptor blocker(phentolamine, $10^{-6}M$) or $\beta$-adrenergic receptor blocker (propranolol, $10^{-6}M$), and $H_1$-receptor blocker (pyrilamine, $10^{-6}M$) or $H_2$-receptor blocker (cimetidine, $10^{-6}M$) on the isolated renal artery of pig. 3. The contractile responses induced by ATP $(5{\times}10^{-3}M)$ were not appeared in $Ca^{\sharp}$-free medium. As the concentration of $Ca^{\sharp}$ in $Ca^{\sharp}$-free medium was increased, the contractile responses induced by ATP $(5{\times}10^{-3}M)$ were enhanced but were completely inhibited by pretreatment with $Ca^{\sharp}$-channel blocker, papaverine $(5{\times}10^{-5}M)$ or verapamil $(5{\times}10^{-5}M)$ on the isolated renal artery of pig.

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.741-748
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• 1997

In the present study, we have investigated the effect of metabolic inhibition on the inward rectifier K current ($I_{K1}$). Using whole cell patch clamp technique we applied voltage ramp from +80 mV to -140 mV at a holding potential of -30 mV and recorded the whole cell current in single ventricular myocytes isolated from the rabbit heart. The current-voltage relationship showed N-shape (a large inward current and little outward current with a negative slope) which is a characteristic of $I_{K1}$. Application of 0.2 mM dinitrophenol (DNP, an uncoupler of oxidative phosphorylation as a tool for chemical hypoxia) to the bathing solution with the pipette solution containing 5 mM ATP, produced a gradual increase of outward current followed by a gradual decrease of inward current with little change in the reversal potential (-80 mV). The increase of outward current was reversed by glibenclamide ($10\;{\mu}M$), suggesting that it is caused by the activation of $K_{ATP}$. When DNP and glibenclamide were applied at the same time or glibenclamide was pretreated, DNP produced same degree of reduction in the magnitude of the inward current. These results show that metabolic inhibition induces not only the increase of $K_{ATP}$ channel but also the decrease of $I_{K1}$. Perfusing the cell with ATP-free pipette solution induced the changes very similar to those observed using DNP. Long exposure of DNP (30 min) or ATP-free pipette solution produced a marked decrease of both inward and outward current with a significant change in the reversal potential. Above results suggest that the decrease of $I_{K1}$ may contribute to the depolarisation of membrane potential during metabolic inhibition.

• 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.

• 생명과학회지
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• 제20권3호
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• pp.457-461
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• 2010

가족성 저칼륨성 주기성마비는 간헐적으로 발생하는 저칼륨혈증을 동반한 가역적 이완성 근육마비를 특정으로 하는 상염색체 우성 유전질환이다. 골격근 세포막에 위치한 $K_{ATP}$ 채널의 활성도 감소가 저칼륨성 주기성 마비의 발병과 관련 있는 것으로 보고되고 있으나 아직까지 명확한 기전이 밝혀져 있지 않다. 본 연구에서는 $K_{ATP}$ 채널을 구성하는 단위체인 Kir6.2를 대상으로 가족성 저칼륨성 주기성마비 환자의 골격근 세포에서 $K_{ATP}$ 채널의 활성도 감소가 발생하는 분자생물학적 기전을 알아보고자 하였다. 환자와 정상인의 골격근 세포내 Kir6.2 단위체의 유전자인 KCNJ11 의 mRNA발현 수준과 단백질 발현양상을 확인한 결과, 정상 세포외 칼륨 농도인 4mM 칼륨 완충용액에 노출된 경우 KCNJ11 mRNA와 단백질 수준의 정량적 차이는 관찰되지 않았다. 그러나 환자에서 마비를 유발할 수 있는 저칼륨 농도인 1mM의 칼륨 완충용액에 노출시킨 경우 정상세포는 KCNJ11 mRNA의 발현이 감소하였고, 그 산물인 Kir6.2 단백질의 정량적 차이를 확인한 결과 세포막에 존재하는 단백질의 양 또한 유의하게 감소하였다. 그러나 환자의 경우 1mM의 칼륨 완충용액에 노출시 KCNJ11 mRNA 발현수준에 차이가 없었고, 더불어 세포막과 세포질 상의 Kir6.2 단백질 분포에도 변화가 나타나지 않았다. 이는 환자 세포의 경우 세포막 단백질이 세포질로 회수되지 못하여 $K_{ATP}$ 채널의 폐쇄가 유지되어 탈분극이 지속되며 이로 인해 환자에서 마비 증상을 유발할 수 있음을 시사하는 결과로 본 질환의 새로운 발병 기전을 설명할 수 있는 근거로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제14권1호
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• pp.21-28
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• 2010

Phenolic compounds affect intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced $Ca^{2+}$ signaling in PC12 cells using fura-2-based digital $Ca^{2+}$ imaging and whole-cell patch clamping. Treatment with ATP ($100\;{\mu}M$) for 90 s induced increases in $[Ca^{2+}]_i$ in PC12 cells. Pretreatment with octyl gallate (100 nM to $20\;{\mu}M$) for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ response in a concentration-dependent manner ($IC_{50}=2.84\;{\mu}M$). Treatment with octyl gallate ($3\;{\mu}M$) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular $Ca^{2+}$ with nominally $Ca^{2+}$-free HEPES HBSS or depletion of intracellular $Ca^{2+}$ stores with thapsigargin ($1\;{\mu}M$). Treatment for 10 min with the L-type $Ca^{2+}$ channel antagonist nimodipine ($1\;{\mu}M$) significantly inhibited the ATP-induced $[Ca^{2+}]_i$ increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the $[Ca^{2+}]_i$ increase induced by 50 mM KCI. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein ($50\;{\mu}M$) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced $[Ca^{2+}]_i$ increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of $Ca^{2+}$ from extracellular space and P2Y receptor-induced release of $Ca^{2+}$ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced $Ca^{2+}$ responses by inhibiting the secondary activation of voltage-gated $Ca^{2+}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• 제5권3호
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• pp.231-241
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• 2001

The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in $Na^+$ and $Cl^-$ homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current $(I_{sc})$ was measured. M-1 cells developed amiloride-sensitive current $5{\sim}7$ days after seeding. Apical and basolateral addition of ATP induced increase in $I_{sc}$ in M-1 cells, which was partly retained in $Na^+-free$ or $Cl^--free$ solution, indicating that ATP increased $Na^+$ absorption and $Cl^-$ secretion in M-1 cells. $Cl^-$ secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and $Ca^{2+}-activated$ chloride channels, but $Na^+$ absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express $P2Y_2,\;P2X_3\;and\;P2Y_4$ receptors. These results showed that ATP regulates $Na^+$ and $Cl^-$ transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

• The Korean Journal of Physiology and Pharmacology
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• 제8권2호
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• pp.95-100
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• 2004

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium $(K_{ATP})$ channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that $K_{ATP}$ channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide $(100\;{\mu}M)$, SNAP $(300\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) $(100\;{\mu}M)$ followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide $(50\;{\mu}M)$, 5-hydroxydecanoate (5-HD) $(100\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) $(100\;{\mu}M)$. These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of $K_{ATP}$ channels in rat ventricular myocytes.

• Biomolecules & Therapeutics
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• 제4권4호
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• pp.373-377
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• 1996

The effect of potassium channel openers, KR-30450, KR-30818 and lemakalim have been compared against several spasmogens in guinea pig bronchi. In guinea pig bronchi, KR-30450 had a greater relaxant effect than lemakalim and KR-30818 against tone induced by histamine $10^{-5}M$ ($EC_{50}$ $\mu$M: KR-30450, 0.108$\pm$0.077; KR-30818, 0.403$\pm$0.023; lemakalim, 0.968$\pm$0.036) and prostaglandin $F_{2\alpha} 3\times10^{-6} M$ ($EC_{50}$ $\mu$M: KR-30450, 0.018$\pm$0.001; KR-30818, 0.028$\pm$0.003; lemakalim, 0.138$\pm$0.019). Relaxant effect of KR-30450 and KR-30818 were significantly reduced by 20 min pretreatment of tissues with $10^[-6}$ M glibenclamide, a selective blocker of ATP-sensitive potassium channel. Against acetylcholine-induced tone in guinea pig bronchi, however, these compounds had little effect. In summary, KR-30450 and KR-30818 showed greater relaxant effect than lemakalim in guinea pig bronchi (KR-30450>KR-30818>lemakalim). These relaxant actions are suggested to be mediated at least in part by a mechanism which involves the opening of ATP-sensitive potassium channel.

• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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• pp.201-206
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• 2004

Mitochondrial ATP-sensitive potassium $(mitoK_{ATP})$ channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of $mitoK_{ATP}$ channels remains unclear. In this study, we investigated the subunit composition of $mitoK_{ATP}$ channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while $mitoK_{ATP}$ channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of $mitoK_{ATP}$ channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands $({\sim}44,\;{\sim}46,\;and\;{\sim}30\;kDa)$ were detected, and a specific ${\sim}46-kDa$ immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of $mitoK_{ATP}$ channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

• Journal of Korean Neurosurgical Society
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• 제29권6호
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• pp.719-724
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• 2000

Objectives : It has been reported that the presence of a pharmacologically inactive foreign substance, polystyrene latex bead, in subarachnoid space activates a non-specific immunological response and elicits arterial narrowing. Recently the activation of potassium($K^+$) channels may be of benefit in relieving cerebral vasospasm. The present study examined the effects of systemic administration of a ATP-sensitive $K^+$ channel activator, cromakalim, on the polystyrene latex bead-induced cerebral vasospasm. Methods : The spasm models similar to that caused by subarachnoid blood injection were created by injection of bead into rabbit cisterna magna. Intravenous injections of cromakalim were administered twice daily(bid) 30 minutes after induction of vasospasm. Animals were killed by perfusion-fixation 2 days after vasospasm. Basilar arteries were removed and sectioned, and the luminal cross-sectional areas were measured. Results : Injection of bead elicited an arterial constriction, reducing arterial diameter to 33.3% of resting tone. Cromakalim inhibited bead-induced constriction at a dose of 0.3mg/kg(Mann-Whitney test, p<0.01). Conclusion : These results support the concept that the cellular events triggered by inactivation of ATP-sensitive $K^+$ channels are responsible for the pathogenesis of vasospasm. The findings also indicate that cromakalim represents a potential therapeutic agents for the treatment of cerebral vasospasm.