• The Korean Journal of Physiology
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• v.29 no.2
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• pp.225-232
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• 1995

The present study was conducted to evaluate the effect of phorbol 12,13-dibutyrate (PDBu) on the contraction of rabbit jugular vein in vitro. PDBu concentrations of greater than 10 nM induced a periodic contraction which was composed of rapid contraction, plateau and slow relaxation. The frequency of periodic contraction increased as PDBu concentration increased. The PDBu-induced contraction was inhibited by staurosporine (100 nM), it was not changed by tetrodotoxin $(1\;{\mu}M).$ In $Ca^{2+}$-free medium, PDBu induced a sustaining contraction, but not periodic contraction. Addition of $Ca^{2+}$ to medium evoked periodic contraction which was inhibited by nifedipine, PDBu concentrations of greater than $0.1\;{\mu}M$ increased ^{45}Ca^{2+}$ uptake without changing $^{45}Ca^{2+}$ efflux. Charybdotoxin and apamin, $Ca^{2+}$-activated K^{+}$ channel blockers, did not affect the PDBu-induced periodic contraction, whereas tetraethylammonium (TEA) abolished the periodicity. Pinacidil $(10\;{\mu}M).$, a potassium channel activator, blocked PDBu induced periodic contraction, which was recovered by glybenclamide $(10\;{\mu}M).$. In high potassium solution, PDBu did not produce the periodic contraction. These results suggest that the PDBu-induced periodicity of contraction is modulated by voltage dependent $Ca^{2+}$ channel and ATP-sensitive $K^{+}$ channel.

• Journal of Life Science
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• v.29 no.12
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• pp.1393-1400
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• 2019

In the present study, we examined the effect of mitochondrial K⁺ channel opener diazoxide on the mitochondria-dependent apoptotic signaling in endothelial cells exposed to high glucose (HG) media. Endothelial cells derived from human umbilical veins were exposed to HG media containing 30 mM glucose, and the degree of apoptotic cell death associated with activation of the mitochondria-dependent apoptotic signaling pathway was determined. Exposure to HG media was seen to enhance apoptotic cell death in a time-dependent manner. In these cells, activation of caspases 3, 8, and 9 was observed, and while caspase-3 and -9 inhibitors suppressed the HG-induced apoptotic cell death, a caspase-8 inhibitor did not. The HG-treated cells exhibited disruption of mitochondrial membrane potential, formation of permeability transition pores, and cytosolic release of cytochrome c. Subsequently, diazoxide was seen to attenuate the HG-induced apoptotic cell death; caspase-9 activation was suppressed but caspase 8 was not. Diazoxide also suppressed the depolarization of mitochondrial membrane potential, the formation of mitochondrial permeability transition pores, and the release of cytochrome c. These effects were significantly inhibited by 5-hydroxydecanoate, a selective blocker of ATP-sensitive K⁺ channels (K_ATP). The present results demonstrate that diazoxide exhibits a beneficial effect to ameliorate HG-induced endothelial cell apoptosis. Opening the K_ATP could help preserve the functional integrity of mitochondria and provide an underlying mechanism to suppress HG-triggered apoptotic signaling.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.6
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• pp.463-469
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• 2000

It has been well known that 4-aminopyridine (4-AP) has an excitatory effect on vascular smooth muscle due to causing membrane depolarization by blocking $K^+-channel$. However, we observed that 4-AP had an inhibitory effect on the mesenteric artery of rat. Therefore, we investigated the mechanism of 4-AP-induced vasorelaxation. The mesenteric arcuate artery and its branches were isolated and cut into ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured. 4-AP $(0.1{\sim}10\;mM)$ induced a concentration-dependent relaxation, which was unaffected by NO synthase inhibitor, $N^G-nitro-L-arginine$ methylester $(100\;{\mu}M)$ or soluble guanylate cyclase inhibitor, methylene blue $(100\;{\mu}M).$ Glibenclamide $(100\;{\mu}M)$, ATP-sensitive $K^+$ channel blocker, did not exert any effect on the 4-AP-induced vasorelaxation. 4-AP relaxed the sustained contraction induced by 100 mM $K^+$ or $Ca^{2+}$ ionophore, A23187 $(100\;{\mu}M)$ in a dose-dependent manner. In addition, 4-AP significantly decreased the phasic contractile response to norepinephrine in the absence of extracellular $Ca^{2+}$. However, 4-AP did not block the $^{45}Ca$ influx of rat aorta. From the above results, we suggest that 4-AP may not block the $Ca^{2+}$ influx through $Ca^{2+}-channel,$ but act as a nonspecific vasorelaxant in arterial smooth muscle.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.207-213
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• 2007

This study was designed to characterize ureteral smooth muscle motility and also to study the effect of forskolin(FSK) and isoproterenol(ISO) on smooth muscle contractility in murine ureter. High $K^+$(50 mM) produced tonic contraction by $0.17{\pm}0.06mN$(n=19). Neuropeptide and neurotransmitters such as serotonin($5{\mu}M$), histamine($20{\mu}M$), and carbarchol(CCh, $10{\sim}50{\mu}M$) did not produce significant contraction. However, CCh($50{\mu}M$) produced slow phasic contraction in the presence of 25 mM $K^+$. Cyclopiazonic acid(CPA, $10{\mu}M$), SR $Ca^{2+}$-ATPase blocker, produced tonic contraction(0.07 mN). Meanwhile, inhibition of mitochondria by protonophore carbnylcyanide m-chlorophenylhydrazone(CCCP) also produced weak tonic contraction(0.01 mN). The possible involvement of $K^+$ channels was also pursued. Tetraethyl ammonium chloride(TEA, 10 mM), glibenclamide($10{\mu}M$) and quinidine($20{\mu}M$) which are known to block $Ca^{2+}$-activated $K^+$ channels($K_{Ca}$ channel), ATP-sensitive $K^+$ channels($K_{ATP}$) and nonselective $K^+$ channel, respectively, did not elicit any significant effect. However, $Ba^{2+}$($1{\sim}2mM$), blocker of inward rectifier $K^+$ channels($K_{IR}$ channel), produced phasic contraction in a reversible manner, which was blocked by $1{\mu}M$ nicardipine, a blocker of dehydropyridine-sensitive voltage-dependent L-type $Ca^{2+}$ channels($VDCC_L$) in smooth muscle membrane. This $Ba^{2+}$-induced phasic contraction was significantly enhanced by $10{\mu}M$ cyclopiazonic acid(CPA) in the frequency and amplitude. Finally, regulation of $Ba^{2+}$-induced contraction was studied by FSK and ISO which are known as adenylyl cyclase activator and $\beta$-adrenergic receptor agonist, respectively. These drugs significantly suppressed the frequency and amplitude of $Ba^{2+}$-induced contraction(p<0.05). These results suggest that $Ba^{2+}$ produces phasic contraction in murine ureteral smooth muscle which can be regulated by FSK and $\beta$-adrenergic stimulation.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.314-321
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• 2015

Background: Ginseng belongs to the genus Panax. Its main active ingredients are the ginsenosides. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal (GI) tract. To understand the effects of ginsenoside Re (GRe) on GI motility, the authors investigated its effects on the pacemaker activity of ICCs of the murine small intestine. Methods: Interstitial cells of Cajal were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICCs. Changes in cyclic guanosine monophosphate (cGMP) content induced by GRe were investigated. Results: Ginsenoside Re ($20-40{\mu}M$) decreased the amplitude and frequency of ICC pacemaker activity in a concentration-dependent manner. This action was blocked by guanosine 50-[${\beta}-thio$]diphosphate [a guanosine-5'-triphosphate (GTP)-binding protein inhibitor] and by glibenclamide [an adenosine triphosphate (ATP)-sensitive $K^{+}$ channel blocker]. To study the GRe-induced signaling pathway in ICCs, the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) and RP-8-CPT-cGMPS (a protein kinase G inhibitor) were examined. Both inhibitors blocked the inhibitory effect of GRe on ICC pacemaker activity. L-NG-nitroarginine methyl ester ($100{\mu}M$), which is a nonselective nitric oxide synthase (NOS) inhibitor, blocked the effects of GRe on ICC pacemaker activity and GRe-stimulated cGMP production in ICCs. Conclusion: In cultured murine ICCs, GRe inhibits the pacemaker activity of ICCs via the ATP-sensitive potassium ($K^{+}$) channel and the cGMP/NO-dependent pathway. Ginsenoside Re may be a basis for developing novel spasmolytic agents to prevent or alleviate GI motility dysfunction.

• Toxicological Research
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• v.14 no.4
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• pp.507-513
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• 1998

The purpose of this study was to clarify the effect of silica on cytosolic free calcium mobilization and cell injury in primary cultured rat hepatocytes. Cytosolic free calcium concentration ([Ca$^{2+}$]) was measured employing calcium sensitive fluorescent dye, Fura-2 / AM, and cell injury was evaluated by determination of cellular ATP contents. Silica increased [Ca$^{2+}$], in a concentration-dependent manner in hepatocytes (10$^{-5}$ ~10$^{-2}$ M). Silica caused a biphasic increase in [Ca$^{2+}$], which was composed of an initial rapid rise and following sustained phase. $Ca^{2+}$ removal from the medium resulted in abolishment of initial and sustained phase of silica (10$^{-2}$ M)-induced [Ca$^{2+}$], in hepatocytes. The pretreatment with nifedipine (1 $\mu$M) attenuated silica-induced [Ca$^{2+}$], increases. Silica decreased cellular ATP contents in a dose-dependent manner. This silica-induced cell injury was attenuated by the pretreatment with EGTA (100 $\mu$M) and nifedipine (1 $\mu$M). This study suggests that the elevation of [Ca$^{2+}$], caused by silica may be due mainly to influx through a plasma membrane $Ca^{2+}$ channel and hepatotoxicity by silica relate with alteration of calcium homeostasis.ium homeostasis.

• Korean Journal of Veterinary Research
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• v.39 no.5
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• pp.878-895
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• 1999

Ischemic preconditioing (IPC), i.e., a preliminary brief episode of ischemia and reperfusion, has been shown to reduce the cell damage induced by long ischemia and reperfusion. Superoxide radical which is produced during reperfusion after ischemia was recognized as a factor of the ischemic injury and it is dismutated into $H_2O_2$ and $O_2$ by two types of intracellular superoxide dismutase (SOD), Cu,Zn-SOD in cytoplasm and Mn-SOD in mitochondria. Recently oxygen free radicals are suggested to induce the apoptosis, however mechanism of the reduced apoptosis by ischemic preconditioing was unknown, while many studies performed in mammalian heart indicated that ATP-sensitive $K^+$ ($K_{APT}$) channel activation related with the protective effects. The aim of present study is to investigate 1) whether IP upregulate the Cu,Zn-SOD and Mn-SOD activities, and 2) whether ischemic preconditioning decreases apoptosis via $K_{APT}$ channel activation in timely reperfused skeletal muscle after long ishemia. The experimental animals, Sprague-Dawley rats weighing 250~300g, were divided into 8 groups; 1) control group, 2) ischemic preconditioning only groups, 3) pinacidil, a $K_{APT}$ channel opener, treatment only groups, 4) glibenclamide, a $K_{APT}$ channel blocker, treatment only groups, 5) ischemia groups, 6) ischemia after IPC groups, 7) ischemia and pinacidil treatment groups, and 8) IP and ischemia after glibenclamide pretreatment groups. Animals of the control group were administered with the vehicle (DMSO) alone. Pinacidil (1mg/kg) was administered intravenously 5 minutes after initiation of ischemia, and glibenclamide (0.5mg/kg) was injected intravenously 20 minutes before IPC. In rats that were ischemic preconditioned, the left common iliac artery was occluded for 5 minutes followed by 5 minutes of reperfusion by three times using vascular clamp. Ischemia was done by occlusion of the same artery for 4 hours. The specimens of left rectus femoris muscle were obtained immediately (0 hour), 12 hours, 24 hours after drug administrations, IP or ischemia and reperfusion. The immunoreactivities of SOD and its alterations were observed by use of sheep antihuman Cu,Zn-SOD and Mn-SOD antibodies on the $10{\mu}m$ cryosections. The incidencies of apoptosis were observed by TUNEL methods with in situ apoptosis detection kit on $6{\mu}m$ paraffine section. The results obtained were as follows : 1. After IPC, immunoreactivities of Cu,Zn-SOD mainly in the small-sized fibers were increased by 24 hours, that of Mn-SOD at 0 hour and 24 hours. 2. No significant changes in immunoreactivities of SOD was observed in the pinacidil and in the glibenclamide treatment only groups, and in the ischemia only groups. 3. The immunoreactivities of the Cu,Zn-SOD were increased in the ischemia after IPC groups and the ischemia and pinacidil treatment groups. 4. The immunoreactivities of the Cu,Zn-SOD in the IPC and ischemia after glibenclamide pretreatment groups were not increased except for the 12 hours reperfusion group. But, Mn-SOD immunoreactivities were increased in the 0 hours, 12 hours and 24 hours after reperfusion. 5. In the control group, the IPC only groups, and the pinacidil treatment only groups, negative or trace apoptotic reactions were observed, but the positive apoptotic reaction occured in the glibenclamide treatment groups. 6. Moderate or many number of apoptosis were revealed in the ischemia groups, and also the IPC and ischemia after glibenclamide pretreatment group except for 12 hours and 24 hours after reperfusion. However, the incidence of apoptosis was decreased in the ischemia after IPC groups and in the ischemia and pinacidil treatment groups. 7. There is a coincidence between the increase of Cu,Zn-SOD immunoreactivities and the decrease of apoptosis in the presence of ischemia and reperfusion. These results suggest that the protective effects of ishemic preconditioing may related to the SOD activation, and the ischemic preconditioning decreases the apoptosis partially via $K_{APT}$ channel activation in timely reperfused rat skeletal muscle. It is also suggested that inhibition of apoptosis by IPC may related with the SOD activation.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.647-655
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• 1997

The purpose of present study is to investigate the influence of a spinal gamma-aminobutyric acid B($GABA_B$) receptor on a central regulation of blood pressure(BP) and heart rate(HR), and to define its mechanism in the spinal cord. In urethane-anesthetized, d-tubocurarine-paralyzed and artificially ventilated male Sprague-Dawley rats, intrathecal administration of drugs were carried out using injection cannula(33-gauge stainless steel) through the guide cannula(PE 10) which was inserted intrathecally at lower thoracic level through the puncture of a atlantooccipital membrane. Intrathecal injection of an $GABA_B$ receptor agonist, baclofen(30, 60, 100 nmol) decreased both BP and HR dose-dependently. Pretreatment with 8-bromo-cAMP(50 nmol), a cAMP analog, or glipizide(50 nmol), a ATP-sensitive $K^+$ channel blocker, attenuated the depressor and bradycardic effects of baclofen(100 nmol), but not with 8-bromo-cGMP(50 nmol), a cGMP analog. These results suggest that the $GABA_B$ receptor in the spinal cord plays an inhibitory role in central cardiovascular regulation and that this depressor and bradycardic actions are mediated by the decrease of cAMP via the inhibition of adenylate cyclase and the opening of $K^+$ channel.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.148-149
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• 1998

It has been known that potassium channel openers are a new class of molecules that have attracted general interest because of their potent antihypertensive activity in vivo and vasorelaxant activity in vitro (Hamilton and Weston, 1989). In the present study, it was attempted to examine the effect of the potassium channel opener on catecholamine (CA) secretion evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat adrenal gland. The perfusion of pinacidil (30-300 uM) into an adrenal vein for 20 min produced relatively dose-dependent inhibition in CA secretion evoked by ACh (5.32 mM), high $K^{+}$ (56 mM), DMPP (100 uM for 2 min), McN-A-343 (100 uM for 2 min), cyclopiazonic acid (10 uM for 4 min) and Bay-K-8644 (10 uM for 4 min). Also, under the presence of minoxidil (100 uM), which is also known to be a potassium channel activator, 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 pinacidil (100 uM) under the presence of glibenclamide (1 uM), an antidiabetic sulfonylurea that has been shown to be a specific blocker of ATP-regulated potassium channels (for 20 min), CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were considerably recovered to a considerable extent of the normal release as compared to that of pinacidil only. These results, taken together, suggest that pinacidil cause 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. Furthermore, these findings suggest strongly that these potassium channel openers-sensitive membrane potassium channels also play an important role in regulating CA secretion.

• The Korean Journal of Pharmacology
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• v.30 no.2
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• pp.191-203
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• 1994

$K^+$ channel openers (KCOs) are known to have a wide range of effects by opening the $K^+$ channel in plasma membranes of various smooth muscles, cardiac muscle and pancreatic ${\beta}-cell$. In the present study, we investigated the effects of 5 types of KCOs, cromakalim, RP49356, pinacidil, nicorandil and diazoxide on the contractility of isolated rat uterus. All KCOs tested inhibited the uterine contraction induced by 0.2 nM oxytocin in a dose-dependent manner. Individual KCO and its $pD_2$ values were cromakalim 6.5, RP49356 6.3, pinacidil 5.92, nicorandil 4.43 and diazoxide 4.18. The relaxant effects of KCO were inhibited by glibenclamide (0.3, 1 and $10\;{\mu}M$) with $pA_2$ values of cromakalim 6.91, RP49356 6.59, pinacidil 6.55, nicorandil 5.97 and diazoxide 6.37. In addition, the relaxant effect of cromakalim or pinacidil was antagonised by TEA, a non-selective $K^+$ channel blocker, but not by apamin. Contractions induced by low concentration of KCI (< 40 mM) were inhibited by cromakalim $(100{\mu}M)$ and nicorandil $(300{\mu}M)$, but those evoked by higher concentration (> 40 mM) of KCI were little affected. In ovariectomized rat uterus, cromakalim dose-dependently inhibited oxytocin-induced contraction and glibenclamide $(10{\mu}M)$ inhibited the relaxant effect of cromakalim with $pD_2$ and $K_B$ values of 7.48 and $1.26{\times}10^{-7}M$, respectively. In estrogen-primed rat uterus, these values were 6.51 and $1.57{\times}10^{-7}M$, respectively, indicating that the cromakalim is less effective on the estrogen-treated uterine smooth muscle. Our results suggest that the KCO-sensitive $K^+$ channels participate in the motility of uterine smooth muscle and such channels are, at least in part, under the control of estrogen. In addition, our data Indicate that the type of $K^+$ channels activated by KCO is ATP-sensitive $K^+$ channels which is blocked by glibenclamide.