• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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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.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.51-51
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• 2003

The medicinal plant Cimicifuga Racemosa (Black cohosh) has been used to treat many kinds of neuronal and menopausal symptoms, such as arthritis, menopausal depression, nerve pain, etc. Here, we examined the effect of Cimicifugoside (CF), one of triterpene glycosides which have been known as pharmacologically active ingredients of C. Racemosa, on nicotinic acetylcholine receptor (nAChR)-mediated catecholamine (CA) secretion in bovine adrenal chromaffin cell. Cimicifugoside inhibited calcium increase induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nAChR agonist with a half maximal inhibitory concentration (IC50) of 18${\pm}$2${\mu}$M. In contrast, cimicifugoside did not affect the calcium increases evoked by high K$\^$+/, veratridine, and bradykinin. The DMPP-induced sodium increase was also inhibited by cimicifugoside with IC50 of 2${\pm}$0.3${\mu}$M, suggesting that the activity of nAChRs is inhibited by cimicifugoside. Cimicifugoside did not effect on the KCl-induced secretion but markedly inhibited the DMPP-induced catecholamine secretion which was monitored by carbon-fiber amperometry in real time, and by high performance liquid chromatography (HPLC) through electrochemical detection. The results suggest that cimicifugoside selectively inhibits nAChR-mediated response in bovine chromaffin cells.

• 문화기술의 융합
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• 제8권3호
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• pp.291-296
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• 2022

퇴행성 신경질환 치료를 위한 AChE inhibitor 관련 연구로써 생물학적 유용성을 높인 커큐민에 대한 연구를 수행하게 되었다. 본 연구의 목적은 아리큐민(강황추출물)에 대한 in vitro 신경보호 효과를 확인하는데 있다. 신경보호효과를 확인하기 위해 아리큐민(강황추출물)에 대한 AChE inhibition을 평가하였고, HT-22 세포에 대한 세포생존율을 분석하였으며, 산화스트레스(glutamate, H₂O₂) 유발에 따른 HT-22 세포생존을 확인하였다. 아리큐민(강황추출물)의 AChE 저해율 변화결과 아리큐민 39.06㎍/㎖ 이상의 농도에서 약 20% 이상의 AChE 활성을 저해하는 것으로 확인하였다. 그리고 산화스트레스(glutamate 5 mM 및 H₂O₂ 500 µM) 유발 HT-22 cell의 세포 독성을 0.01~0.1 mg/ml 농도에서부터 유의하게 억제하는 것을 확인하였다(p<0.05). 이와 같은 결과로 볼 때 아리큐민(강황추출물)은 신경보호 효과 효능이 우수한 것으로 확인되었다.

• 동의생리병리학회지
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• 제16권2호
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• pp.215-219
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• 2002

Over the past decade there has been a resurgence of interest in botanical products for their medicinal uses. This present study was designed to screen medicinal plants for the treatment of brain diseases such as Alzheimer's disease or aging. We tested the effects of the water extracts from 41 species medicinal plants on acetylcholinesterase (AChE) inhibition and antioxidant activity in vitro. Among them, Euodia rutaecarpa, Rubus coreanus, Achyranthes japonica, Morus alba, Salvia miltiorrhiza, Liriope platyphylla, and Rheum palmatum showed relatively AChE inhibition potency over 80%. Among the 7 medicinal plants investigated, E. rutaecarpa showed an 25%, 65%, and 93% inhibitions on AChE activity at 2.5, 12.5, and 25 ㎍/㎖ of the extract, respectively and was found to be one of most potent AChE inhibitor. The water extracts from 4 species (E. rutaecarpa, R. coreanus, A. iaponica, and M. alba) were tested on their antioxidant capacities using radical scavenging effect against ABTS/sup ㆍ+/. Among the 4 medicinal plants investigated, both R. coreanus and E. rutaecarpa showed significant antioxidant capacity. Therefore, E. rutaecarpa and R. coreanus are expected to ameliorate the clinical symptoms in Alzheimer's disease due to significant AChE inhibition and radical scavenging effect.

• Archives of Pharmacal Research
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• 제10권4호
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• pp.212-218
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• 1987

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This proparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity ($B_{max}$) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37.deg.C but was not apparent inincubation at $15^{\circ}}C$In addition, it was abolished when the receptors were blocked by atropine. The decline in ($^3H$)N-methylscopolamine (($^3H$)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that 'true' receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist ($^3H$) quinuclidinyl benzilate (($^3H$) QNB) insted of the hydrophilic ligand ($^3H$)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggested that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.

• 동의생리병리학회지
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• 제24권2호
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• pp.228-235
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• 2010

This study was performed to investigate the memory enhancing effect of Poria cocos Wolf (Hoelen cum radix) against scopolamine-induced amnesia in Sprague-Dawley (SD) rats. To induce amnesia, scopolamine (0.75 mg/kg) was intraperitonically injected into SD rats 30 min before starting behavior tests. We have conducted Morris water-maze and Y-maze tests to monitor learning and memory functions. Poria cocos effectively reversed scopolamine-induced memory impairment in SD rats which was represented by an improvement of mean escape latency in water-maze test and spontaneous alterations in Y-maze test. To elucidate possible molecule mechanism, we have measured mRNA as well as protein expression of acetylcholine esterase (AchE), choline acetyltransferase (ChAT), muscarinic acetylcholine receptor (mAchR), and brain-derived neurotrophic factor (BDNF) using RT-PCR and Western blot analysis, respectively. Poria cocos increased mRNA levels of ChAT and mAchR in rat hippocampus compared with those in the scopolamine-injected amnesic group. In addition, protein expression of ChAT and BDNF was also elevated by Poria cocos intake. Furthermore, as an upstrem regulator, the activation of cAMP response element-binding protein (CREB) was assessed by immunohistochemistry. In this immunohistochemical analysis, the phosphorylation of CREB (p-CREB) was reduced by scopolamine injection, which was restored back to control levels by administration of Poria cocos. These results suggest that Poria cocos may improve memory and cognitive deficit in amnesia and have therapeutic potentials through up-regulation of ChAT, mAchR, and BDNF, which seemed to be mediated by activation of CREB.

• Bulletin of the Korean Chemical Society
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• 제32권spc8호
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• pp.2997-3002
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• 2011

A series of hybrid molecules between (R)-lipoic acid (ALA) and the acetylated or methylated polyphenol compounds were synthesized and their in vitro cholinesterase [acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)] inhibition activities were checked. The $IC_{50}$ values of all hybrid molecules for a BuChE inhibition were lower than those of the single parent compounds. Specifically, ALA-acetyl protected caffeic acid (11, ALA-AcCA) was shown as an effective inhibitor of BuChE ($IC_{50}=0.5{\pm}0.2\;{\mu}M$) and also had a great selectivity for BuChE over AChE (more than 800 fold). Inhibition kinetic study indicated that 11 is a mixed inhibition type. Its binding affinity ($K_i$) value to BuChE is $1.52{\pm}0.18\;{\mu}M$.

• The Korean Journal of Physiology and Pharmacology
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• 제9권1호
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• pp.29-35
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• 2005

To prove the buffering contribution of mitochondria to the increase of intracellular $Ca^{2+}$ level ($[Ca^{2+}]_i$) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial $Ca^{2+}$ entry and $Ca^{2+}$ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM ($R_{340/380}$) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh ($10{\mu}M$) increased $R_{340/380}$ by $1.1{\pm}0.15$ ($mean{\pm}S.E.$, n=6). When the external $Na^+$ was totally replaced by $NMDG^+$, $R_{340/380}$ was increased by $1.19{\pm}0.17$ in a reversible manner (n=27). $NMDG^+$-induced $[Ca^{2+}]_i$ increase was followed by oscillatory decay after $[Ca^{2+}]_i$ reached the peak level. The increase of $[Ca^{2+}]_i$ by $NMDG^+$ was completely suppressed by replacement with $Cs^+$. When $1{\mu}M$ CCCP was applied to bath solution, the ratio of $[Ca^{2+}]_i$ was increased by $0.4{\pm}0.06$ (n=31). When $1{\mu}M$ CCCP was used for pretreatment before application of $NMDG^+$, oscillatory decay of $[Ca^{2+}]_i$ by $NMDG^+$ was significantly inhibited compared to the control (p＜0.05). In addition, $NMDG^+-induced$ increase of $[Ca^{2+}]_i$ was highly enhanced by pretreatment with $2{\mu}M$ CCCP by $320{\pm}93.7$%, compared to the control ($mean{\pm}S.E.$, n=12). From these results, it is concluded that mitochondria might have buffering contribution to the $[Ca^{2+}]_i$ increase through regulation of the background NSCC in RAECs.

• Nutrition Research and Practice
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• 제17권2호
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• pp.192-205
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• 2023

BACKGROUND/OBJECTIVES: It is known that the renin-angiotensin system (RAS) in the brain could regulate cognitive functions as well as blood pressure. Inhibition of RAS for the improvement of cognitive function may be a new strategy, but studies so far have mostly reported on the effects of RAS inhibition by drugs, and there is no research on cognitive improvement through RAS inhibition of food ingredients. Therefore, this study investigated the effect of curcumin on blood pressure and cognitive function and its related mechanism in spontaneously hypertensive rat/Izm (SHR/Izm). MATERIALS/METHODS: Six-week-old SHR/Izm rats were divided into 5 groups: control group (CON), scopolamine group (SCO, drug for inducing cognitive deficits), positive control (SCO and tacrine [TAC]), curcumin 100 group (CUR100, SCO + Cur 100 mg/kg), and curcumin 200 group (CUR200, SCO + Cur 200 mg/kg). Changes in blood pressure, RAS, cholinergic system, and cognitive function were compared before and after cognitive impairment. RESULTS: The SCO group showed increased blood pressure and significantly reduced cognitive function based on the y-maze and passive avoidance test. Curcumin treatments significantly improved blood pressure and cognitive function compared with the SCO group. In both the CUR100 and CUR200 groups, the mRNA expressions of angiotensin-converting enzyme (ACE) and angiotensin II receptor type1 (AT1), as well as the concentrations of angiotensin II (Ang II) in brain tissue were significantly decreased. The mRNA expression of the muscarinic acetylcholine receptors (mAChRs) and acetylcholine (ACh) content was significantly increased, compared with the SCO group. CONCLUSIONS: The administration of curcumin improved blood pressure and cognitive function in SCO-induced hypertensive mice, indicating that the cholinergic system was improved by suppressing RAS and AT1 receptor expression and increasing the mAChR expression.

• Biomolecules & Therapeutics
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• 제7권4호
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• pp.307-314
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• 1999

The human muscarinic acetylcholine receptor (mAChR) subtypes Hml, Hm2 and Hm3 have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus expression system. Expression of relevant DNA, transcript and receptor proteins was identified by PCR, Northern blotting and [$^{3}H$]QNB binding, respectively. As assessed by [$^{3}H$]QNB binding sites, yields of muscarinic receptors in membrane preparations in this study were as about 5-20 times high as those in mammalian cells reported in previous studies. The [$^{3}H$]QNB competition binding studies with well-known subtype-selective mAChR antagonists showed that the receptors expressed in Sf9 cells retain the pharmacological characteristics expected for the ml , m2 and m3 muscarinic receptors. The ml-selective antagonist, pirenzepine, displayed a considerably higher affinity for Hml by 110-fold and 35-fold than for Hm2 and Hm3, respectively, The m2-selective methoctramine displayed a significantly higher affinity for Hm2 than for Hml and Hm3 (10- and 26-fold, respectively). p-F-HHSiD exhibited high affinity for Hm3 that is not significantly different from those for Hml, but 66-fold higher than its affinity for Hm2. The functional coupling of the recombinant receptors to second messenger systems was also examined. While both Hml and Hm3 stimulated phosphoinositide hydrolysis upon activation by carba-chol, Hm2 produced no response. On the other hand, activation of mAChRs induced the inhibition of forsko-lin-stimulated cyclic AMP formation in Hm2-expressing cells, whereas the significant dose-dependent increase in or poor response on cyclic AMP formation were produced in Hml or Hm3-expressing cells, respectively. These results indicate the differential coupling of recombinant Hml, Hm2 and Hm3 receptors expressed in SF9 cells to intracellular signalling system.