• Molecules and Cells
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• v.27 no.5
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• pp.591-599
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• 2009

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not ${\alpha}7$, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of ${\alpha}7$ nAChR induces alterations in channel gating properties and converts ${\alpha}7$ nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside $Rg_3$ ($Rg_3$) activity against the ${\alpha}7$ nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to $Rg_3$. We further characterized $Rg_3$ regulation of L247T receptors. We found that $Rg_3$ inhibition of mutant ${\alpha}7$ nAChR channel currents was reversible and concentration-dependent. $Rg_3$ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between $Rg_3$ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in $Rg_3$ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that $Rg_3$ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas $Rg_3$ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for $Rg_3$ at the channel pore.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.13-21
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• 1996

Intracellular calcium concentration ($[Ca^{2+}]_i$) may play a crucial role in a variety of neuronal functions. Here we report that in primary culture of mouse cerebellar granule cells nicotinic acetylcholine receptors (nAChRs) are expressed in a specific developmental stage and involved in the regulation of intracellular calcium homeostasis. Nicotine-mediated calcium responses were measured using $^{45}Ca^{2+}$ or fluorometrically using the calcium-sensitive fluorescent dye fura-2. Maximal uptake of $^{45}Ca^{2+}$ evoked by nicotine in mouse cerebellar granule cells were revealed $8{\sim}12$ days in culture. In contrast, nicotine did not alter the basal $^{45}Ca^{2+}$ uptake in cultured glial cells. In cerebellar granule cells nicotine-evoked $^{45}Ca^{2+}$ uptake was largely blocked by the NMDA receptor antagonists. Glutamate pyruvate transaminase (GPT). which removes endogenous glutamate, also prevented nicotine effects, implying the indirect involvement of glutamate in nicotine-mediated calcium responses. Fluorometric studies using fura-2 showed two phases of nicotine-evoked $[Ca^{2+}]_i$ rises: the initial rising phase and the later plateau phase. Interestingly, the NMDA receptor antagonists and GPT appeared to inhibit only the later plateau phase of nicotine-evoked $[Ca^{2+}]_i$ rises. The present results imply that nicotine mediated $^{45}Ca^{2+}$ uptake and $[Ca^{2+}]_i$ rises are attributed to the calcium fluxes through both nAchRs and NMDA receptors in a time-dependent manner. Consequently, nAChRs may play an important role in neuronal development by being expressed in a specific developmental stage and regulating the intracellular calcium homeostasis.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.191-200
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• 2005

Many studies have shown that the development of mouse early 2-cell embryos in vitro is related with the intracellular $Ca^{2+}$ changes. In ICR strain mouse, the development of embryos arrests at early 2-cell stage, but the arrested early 2-cell embryos can be rescued by the addition of $Ca^{2+}$-related materials. Acetylcholine (ACh) increases intracellular $Ca^{2+}$ concentration ([$Ca^{2+}$]i) via the mAChR-PLC-IP3 pathway in mouse oocytes. We examined whether ACh rescues 2-cell block in mouse. In early 2-cell embryos, ACh increased [$Ca^{2+}$]i in a dose-dependent manner (p<0.001), and had an effect on rescue of 2-cell block and embryonic development. To identify the signal pathway involved in ACh-induced rescue of 2-cell block, we first applied an agonist of ACh receptor (AChR). Like ACh, carbachol increased intracellular $Ca^{2+}$ concentration ([$Ca^{2+}$]i) and atropine, an antagonist of ACh receptor, blocked the ACh-induced $Ca^{2+}$ increase. In $Ca^{2+}$-free medium, ACh also increased [$Ca^{2+}$]i, indicating that $Ca^{2+}$ increased by ACh is mainly released from the intracellular $Ca^{2+}$ store. The ACh-induced $Ca^{2+}$ increase was blocked by PLC inhibitor (U73122), ryanodine receptor (RyR) antagonist (dantrolene), and CaM KII inhibitor (KN-93), but not by IP3R antagonists (xestospongin C). These results show that ACh increases intracellular $Ca^{2+}$ concentration via mAChR/PLC/RyR, and this contributes to the rescue of 2-cell block.

• The Korean Journal of Pharmacology
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• v.27 no.2
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• pp.167-181
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• 1991

The present study was an attempt to investigate the effect of forskolin on secretion of catecholamines (CA) evoked by Ach, excess $K^+$, DMPP, McN-A-343 and caffeine from the isolated perfused rat adrenal glands and to elucidate its mechanism of action. The perfusion with forskolin (1.0 uM) for 1 min into the adrenal vein enhanced markedly the secreation of CA evoked by Ach (50 ug), excess $K^+$ (56 mM) DMPP (100 uM) and by caffeine (0.3 mM) but did not that by McN-A-343. Forskolin alone did not potentiate the CA secretion. Moreover, forskolin augmented the CA release evoked by the above same stimulation even in the absence of extracellular calcium. The 1 min perfusion of 300 uM-dibutyryl cyclic AMP (DBcAMP), which is known to increase cyclic AMP levels, led to enhancement of Ca secretion evoked by Ach, excess $K^+$ and DMPP but did not that by McN-A-343 and caffeine. DBcAMP by itself also did not augment the CA secretion. In the calcium-free medium DBcAMP significantly enhanced the CA secretion by the same stimulation, except for the case of McN-A-343. These experimental results suggest that forskolin activates adenylate cyclase, resulting the elevation of cyclic AMP which may potentiate cholinergic nicotinic receptor-mediated and also depolarization-dependent CA secretion and that it may alter the intracellular calcium homeostasis in the rat adrenal glands.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.31-42
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• 1988

The effect of Panaxadiol(PD), which is an active component of Korean Ginseng Saponins, on the secretion of catecholamines (CA) from the rabbit adrenal gland and its mode of action were investigated in the present study. $PD(400{\mu}g)$ increased significantly the secretion of CA from the isolated perfused rabbit adrenal gland. PD-induced secretion of CA was reduced markedly by treatment of atropine, CA secretion induced by Ach or PD was potentiated significantly by physostigmine-treatment. Chlorisondamine did inhibit CA secretion of PD or Ach. Perfusion of $PD(400{\mu}g)$ for 30 min enhanced the secretory activity of CA by Ach. Ouabain weakened the secretory response induced by PD but rather enhanced the response by Ach. Adenosine-treatment resulted in marked enhancement of CA secretion by PD or Ach, Pefusion with $Ca^{2+}-free$ Krebs containing EGTA (5 mM) for about 30 min totally blocked secretory effect induced by Ach and also weakened that by PD. From the above experimental results, it is suggested that PD causes secretion of catecholamines from the rabbit adrenal gland by a calcium-dependent exocytotic mechanism. The secretory effect of PD is due to the stimulation of cholinergic muscarinic and nicotinic receptors present in the adrenal gland and partly to a direct action on the chromaffin cell itself.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.219-225
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• 2004

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons $(EC_{50};\;41.4\;{\mu}M\;and\;64.0\;{\mu}M,\;respectively)$. ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for ${\alpha}3{\beta}4$ nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons $(IC_{50};\;0.53\;{\mu}M\;and\;0.22\;{\mu}M,\;respectively)$. Conversely, ${\alpha}-bungarotoxin$, ${\alpha}-methyllycaconitine$, and $dihydro-{\beta}-erythroidine$, which are known as potent and sensitive blockers for ${\alpha}7$ or ${\alpha}4{\beta}2$ nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that ${\alpha}3$ and ${\beta}4$ subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing ${\alpha}3$ and ${\beta}4$ subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

• Proceedings of the Ginseng society Conference
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• 1988.08a
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• pp.55-62
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• 1988

The effect of panaxadiol (PD). an active component of Korean ginseng saponins on the secretion of catecholamines (CA) from the rabbit adrenal gland and its mode of action were investigated. PD ($400\;{\mu}g$) increased significantly the secretion of CA from the isolated perfused rabbit adrenal gland. PD-induced secretion of CA was reduced markedly by treatment with atropine. CA secretion induced by Ach or PD was potentiated by physostigmine treatment. Chlorisondamine inhibited CA secretion of PD or Ach. Perfusion of PD ($400\;{\mu}g$) for 30 minutes enhanced the secretory activity of CA by Ach. Ouabain weakened the secretory rsponse caused by PD but enhanced the response by Ach. Adenosine treatment resulted in marked increase in CA secretion by PD or Ach. Perfusion with calcium free Krebs solution containing 5 mM EDTA for 30 minutes completely blocked the secretory effect induced by Ach and also weakened that evoked by PD. It is suggested that PD causes the secretion of CA from the rabbit adrenal gland by a calcium dependent exocytotic process. The secretory effect of PD is due to the stimulation of cholinergic muscarinic and nicotinic-receptors present in the adrenal gland partly by direct action on the chromaffin cell.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.4
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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.

• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.40-46
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• 1998

We investigated the influence of the root of Panax ginseng C. A. Meyer on the secretion of catecholamines from bovine adrenal chromaffin cells, which are used as a model of nervous systems. In two major parts extracted from the ginseng root, the crude saponin fraction, but not the non-saponin fraction, reduced the secretion from the cells, stimulated by acetylcholine (ACh). Ginseng saponins (ginsenosides) are classified into three groups, the panaxadiol, the panaxatriol and the oleanolic acid groups, on the basis of the chemical structures of their saponins. Both the panaxadiol and the panaxatriol saponins, excluding only one oleanolic acid saponin ginsenoside-Ro, generally reduced the ACh-evoked secretion. The inhibitory effects of the panaxatriol were much stronger than those of the panaxadiol. However, ginsenoside-Rg, and -Rh3 in the panaxadiol saponins were the potent inhibitors comparable to the panaxatriol saponins. Ginsenoside-Rg2 in the panaxatriol was the most effective. It is probable that the ginsenoside inhibition of the catecholamine secretion is due to the suppression of the function of the nicotinic ACh receptor-cation channels. On the other hand, ginsenoside-Rg2 did not affect the angiotensin II-, the bradykinin-, the histamine- and the neurotensin- induced catecholamine secretions from the chromaffin cells and the muscarine- and the histamine- induced contraction of the ileum in guinea-pigs. Ginsenoside-Rbl, a panaxadiol saponin, and ginsenoside-Ro had no or only a slight effect on them. On the contrary, ginsenoside-Rg3 not only competitively inhibited the muscarine-induced ileum contraction but also reduced the angiotensin R -, the bradykinin-, the histamine- and the neurotensin-induced catecholamine secretions. Thus, the ginseng root contains active ingredients, namely some ginsensides, which suppress the responses induced by receptor stimulation. The inhibitory effects of ginseng saponins may be one of the action mechanisms for the pharmacological effects of the Panax ginseng root.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.1
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• pp.13-23
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• 2008

The aim of the present study was designed to establish comparatively the inhibitory effects of $D_1$-like and $D_2$-like dopaminergic receptor agonists, SKF81297 and R(-)-TNPA on the release of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. SKF81297 $(30{\mu}M)$ and R-(-)-TNPA $(30{\mu}M)$ perfused into an adrenal vein for 60 min, produced great inhibition in the CA secretory responses evoked by ACh $(5.32{\times}10^{-3}\;M)$, DMPP $(10^{-4}\;M)$, McN-A-343 $(10^{-4}\;M)$, high $K^+$ $(5.6{\times}10^{-2}\;M)$, Bay-K-8644 $(10{\mu}M)$, and cyclopiazonic acid $(10{\mu}M)$, respectively. For the release of CA evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: SKF81297>R-(-)-TNPA. However, R(+)-SCH23390, a selectve $D_1$-like dopaminergic receptor antagonist, and S(-)-raclopride, a selectve $D_2$-like dopaminergic receptor antagonist, enhanced the CA secretory responses evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid only for $0{\sim}4$ min. The rank order for the enhancement of CA release evoked by high $K^+$, McN-A-343 and cyclopiazonic acid was R(+)-SCH23390>S(-)-raclopride. Also, the rank order for ACh, DMPP and Bay-K-8644 was S(-)-raclopride > R(+)-SCH23390. Taken together, these results demonstrate that both SKF81297 and R-(-)-TNPA inhibit the CA release evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization from the isolated perfused rat adrenal gland without affecting the basal release, respectively, but both R(+)-SCH23390 and S(-)-raclopride facilitate the CA release evoked by them. It seems likely that the inhibitory effects of SKF81297 and R-(-)-TNPA are mediated by the activation of $D_1$-like and $D_2$-like dopaminergic receptors located on the rat adrenomedullary chromaffin cells, respectively, whereas the facilitatory effects of R(+)-SCH23390 and S(-)-raclopride are mediated by the blockade of $D_1$-like and $D_2$-like dopaminergic receptors, respectively: this action is possibly associated with extra- and intracellular calcium mobilization. Based on these results, it is thought that the presence of dopaminergic $D_1$ receptors may play an important role in regulation of the rat adrenomedullary CA secretion, in addition to well-known dopaminergic $D_2$ receptors.