• Animal cells and systems
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• v.3 no.2
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• pp.181-185
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• 1999

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetylcholine receptors (nAChRs) $\alpha$$_{6}$ subtype in oocytes was systematically increased by more than 100-fold, which was confirmed both by the $\alpha$-Bungarotoxin ($\alpha$,/TEX>Bgt) binding assay and the current measurement.t.

• Journal of Ginseng Research
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• v.29 no.1
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• pp.19-26
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• 2005

The last two decades have shown a marked expansion in publications of diverse effects of Panax ginseng. Ginsenosides, as active ingredients of Panax ginseng, are saponins found in only ginseng. Recently, a line of evidences shows that ginsenosides regulate various types of ion channel activity such as $Ca^{2+},\;K^+,\;Na^+,\;Cl^-$, or ligand gated ion channels (i.e. $5-HT_3$, nicotinic acetylcholine, or NMDA receptor) in neuronal, non-neuronal cells, and heterologously expressed cells. Ginsenosides inhibit voltage-dependent $Ca^{2+},\;K^+,\;and\;Na^+$ channels, whereas ginsenosides activate $Ca^{2+}-activated\;Cl^-\;and\;Ca^{2+}-activated\;K^+$ channels. Ginsenosides also inhibit excitatory ligand-gated ion channels such as $5-HT_3$, nicotinic acetylcholine, and NMDA receptors. This review will introduce recent findings on the ginsenoside-induced differential regulations of ion channel activities and will further expand the possibilities how these ginsenoside-induced ion channel regulations are coupled to biological effects of Panax ginseng.

• Archives of Pharmacal Research
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• v.26 no.1
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• pp.28-33
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• 2003

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate excitatory ligand-gated ion channel activity such as nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides affect inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human recombinant $GABA_A$ receptor (${\alpha}_1{\beta}_1{\gamma}_{2s}$) channel activity expressed in Xenopus oocytes using a two-electrode voltage-clamp technique. Among the eight individual ginsenosides examined, namely, $Rb_1$, $Rb_2$, Rc, Rd, Re, Rf, $Rg_1$ and $Rg_2$, we found that Rc most potently enhanced the GABA-induced inward peak current ($I_{GABA}$). Ginsenoside Rc alone induced an inward membrane current in certain batches of oocytes expressing the $GABA_A$ receptor. The effect of ginsenoside Rc on $I_{GABA}$ was both dose-dependent and reversible. The half-stimulatory concentration ($EC_{50}$) of ginsenoside Rc was 53.2$\pm$12.3 $\mu$M. Both bicuculline, a $GABA_A$ receptor antagonist, and picrotoxin, a $GABA_A$ channel blocker, blocked the stimulatory effect of ginsenoside Rc on $I_{GABA}$. Niflumic acid (NFA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), both $CI^{-1}$ channel blockers, attenuated the effect of ginsenoside Rc on I$I_{GABA}$. This study suggests that ginsenosides regulated $GABA_A$ receptor expressed in Xenopus oocytes and implies that this regulation might be one of the pharmacological actions of Panax ginseng.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.155-164
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• 2008

Resveratrol has been known to possess various potent cardiovascular effects in animal, but there is little information on its functional effect on the secretion of catecholamines (CA) from the perfused model of the adrenal medulla. Therefore, the aim of the present study was to determine the effect of resveratrol on the CA secretion from the isolated perfused model of the normotensive rat adrenal gland, and to elucidate its mechanism of action. Resveratrol (10${\sim}100{\mu}$M) during perfusion into an adrenal vein for 90 min inhibited 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 both a time- and dose- dependent fashion. Also, in the presence of resveratrol (30${\mu}$M), the secretory responses of CA evoked by veratridine 8644 (an activator of voltage-dependent$Na^+$ channels, 100${\mu}$M), Bay-K-8644 (a L-type dihydropyridine $Ca^{2+}$ channel activator, 10${\mu}$M), and cyc1opiazonic acid (a cytoplasmic $Ca^{2+}$-ATPase inhibitor, 10${\mu}$M) were significantly reduced. In the simultaneous presence of resveratrol (30${\mu}$M) and L-NAME (an inhibitor of NO synthase, 30${\mu}$M), the CA secretory evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyc1opiazonic acid were recovered to a considerable extent of the corresponding control secretion compared with the inhibitory effect of resveratrol alone. Interestingly, the amount of nitric oxide (NO) released from the adrenal medulla was greatly increased in comparison to its basal release. Taken together, these experimental results demonstrate that resveratrol can inhibit the CA secretory responses evoked by stimulation of cholinergic nicotinic receptors, as well as by direct membrane-depolarization in the isolated perfused model of the rat adrenal gland. It seems that this inhibitory effect of resveratrol is exerted by inhibiting an influx of both ions through $Na^+$ and $Ca^{2+}$ channels into the adrenomedullary cells as well as by blocking the release of $Ca^{2+}$ from the cytoplasmic calcium store, which are mediated at least partly by the increased NO production due to the activation of NO synthase.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.4
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• pp.407-414
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• 2016

This study was performed to investigate whether the spinal cholinergic and serotonergic analgesic systems mediate the relieving effect of electroacupuncture (EA) on oxaliplatin-induced neuropathic cold allodynia in rats. The cold allodynia induced by an oxaliplatin injection (6 mg/kg, i.p.) was evaluated by immersing the rat's tail into cold water ($4^{\circ}C$) and measuring the withdrawal latency. EA stimulation (2 Hz, 0.3-ms pulse duration, 0.2~0.3 mA) at the acupoint ST36, GV3, or LI11 all showed a significant anti-allodynic effect, which was stronger at ST36. The analgesic effect of EA at ST36 was blocked by intraperitoneal injection of muscarinic acetylcholine receptor antagonist (atropine, 1 mg/kg), but not by nicotinic (mecamylamine, 2 mg/kg) receptor antagonist. Furthermore, intrathecal administration of $M_2$ (methoctramine, $10{\mu}g$) and $M_3$ (4-DAMP, $10{\mu}g$) receptor antagonist, but not $M_1$ (pirenzepine, $10{\mu}g$) receptor antagonist, blocked the effect. Also, spinal administration of $5-HT_3$ (MDL-72222, $12{\mu}g$) receptor antagonist, but not $5-HT_{1A}$ (NAN-190, $15{\mu}g$) or $5-HT_{2A}$ (ketanserin, $30{\mu}g$) receptor antagonist, prevented the anti-allodynic effect of EA. These results suggest that EA may have a significant analgesic action against oxaliplatin-induced neuropathic pain, which is mediated by spinal cholinergic ($M_2$, $M_3$) and serotonergic ($5-HT_3$) receptors.

• Biomolecules & Therapeutics
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• v.8 no.4
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• pp.338-348
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• 2000

The present study was attempted to determine the effect of 5'-(N'-ethylcarboxamido) adenosine (NECA), which is an potent $A_2$-adenosine receptor agonist, on catecholamine (CA) secretion evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat adrenal gland. NECA (20 nM) perfused into the adrenal vein for 60 min produced a time-related inhibition in CA secretion evoked by ACh (5.32x10$^{-3}$ M), high $K^{+}$(5.6x10$^{-2}$ 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 $\beta$,${\gamma}$-methylene adenosine-5'-triphosphate (MATP), which is also known to be a selective $P_{2x}$-purinergic receptor agonist, showed a similar inhibition elf CA release evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid. However, in adrenal glands preloaded with 20$\mu$M NECA for 20 min under the presence of 20$\mu$M 3-isobutyl-1-methyl-xanthine (IBMX), an adenosine receptors antagonist, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were much recovered in comparison to the case of NECA-treatment only. Taken together, these results indicate that NECA causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization. This inhibitory effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells through the adenosine receptor stimulation. Therefore, it is suggested that the inhibitory mechanism of adenosine receptor stimulation may play a modulatory role in regulating CA secretion.n.n.

• Animal Bioscience
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• v.36 no.2
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• pp.200-208
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• 2023

Objective: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes. Methods: We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes. Results: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPAR class="checkNonKBPoint">γ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly. Conclusion: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

• Animal cells and systems
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• v.15 no.2
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• pp.115-121
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• 2011

Previous studies have demonstrated that N-methyl-D-aspartate (NMDA) receptors and acetylcholine receptors are related to learning and memory in rat and mice. In this study, we examined the effects of MK-801, a non-competitive NMDA receptor antagonist, on learning and memory in zebrafish using a passive avoidance test. We further tested whether or not nicotine, a nicotinic acetylcholine receptor agonist, and physostigmine, an acetylcholinesterase inhibitor, reverse the effects of MK-801. Crossing time was increased significantly in the training and test sessions for the controls. When 20 ${\mu}M$ MK-801 was administered prior to the training session, the crossing time did not increase in either session. The MK-801-induced learning deficit was rescued by pretreatment with 20 ${\mu}M$ physostigmine, and crossing time was increased in the training and test sessions compared to the MK-801-treated zebrafish. Further, the MK-801-induced learning deficit was prevented by pretreatment with 20 ${\mu}M$ nicotine, and crossing time was increased in the training session but not in the test session. These results show that MK-801 induced a learning deficit in zebrafish that was prevented by pretreatment with nicotine and physostigmine.

• Bulletin of the Korean Chemical Society
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• v.27 no.11
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• pp.1741-1746
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• 2006

3D-QSARs on the neuroblocking activities by 1-(6-chloro-3-pyridylmethyl)-2-nitroiminoimidazolidine analogues as agonist at the nicotinic acetylcholine receptor (nAChR) were studied quantitatively using CoMFA and CoMSIA methodologies. The statistical results of CoMFA (A5: $r^2\;_{cv.}\;=\;0.707\;&\;r^2\;_{ncv.}$= 0.986) and CoMSIA model (A3: $r^2\;_{cv.}$ = 0.715 & $r^2\;_{ncv.}$ = 0.961) showed the best predictability and fitness for neuroblocking activity based on the cross-validated value and non-cross validated value. The steric and H-bond acceptor nature of a compound were essential for high activity. The study on 3D-QSARs between substrate molecules and their neuroblocking activities appears to be an useful approach for designing better neuroblocking drug development.

• Natural Product Sciences
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• v.13 no.1
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• pp.68-77
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• 2007

The aim of the present study was to examine the effects of green tea extract (CUMS6335) on the release of CA evoked by cholinergic stimulation and direct membrane-depolarization in the perfused model of the adrenal gland isolated from the spontaneously hypertensive rats (SHRs), and to establish the mechanism of action. Furthermore, it was also to test whether there is species difference between animals, and between CUMS6335 and EGCG, one of biologically the most powerful catechin compounds found in green tea. CUMS6335 $(100\;{\mu}g/ml)$, when perfused into an adrenal vein for 60 min, time-dependently inhibited the CA secretory responses evoked by ACh (5.32mM), high $K^+$(56 mM), DMPP $(100\;{\mu}M)$, and McN-A-343 $(100\;{\mu}M)$ from the isolated perfused adrenal glands of SHRs. However, CUMS6335 itself did fail to affect basal catecholamine output. Also, in adrenal glands loaded with CUMS6335 $(100\;{\mu}g/ml)$, the CA secretory responses evoked by Bay-K-8644 $(10\;{\mu}M)$ and cyclopiazonic acid $(10\;{\mu}M)$ were also inhibited in a relatively time-dependent fashion. However, in the Presence of EGCG $(8.0\;{\mu}g/ml)$ for 60 min, the CA secretory response evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were not affected except for last period. Collectively, these results indicate that CUMS6335 inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by direct membrane-depolarization from the perfused adrenal gland of the SHR. It seems that this inhibitory effect of CUMS6335 is exerted by blocking both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of $Ca^{2+}$ into the cytoplasmic calcium store, which are at least partly relevant to the direct interaction with the nicotinic receptor itself. It seems likely that there is much difference in mode of the CA-releasing action between CUMS6335 and EGCG.