Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

Hoai Bac Nguyen,이신영,박수현,이무열,장인호,명순철 대한비뇨의학회 2013 Investigative and Clinical Urology Vol.54 No.5

Purpose: The location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits. Materials and Methods: Isolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester,7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated. Results: In the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10-9 to 10-4 M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 μM L-NAME or 10 μM 7-nitroindazole) but also by 10 μM atropine and some selective muscarinic receptor antagonists (10-6 M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10-6M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine. Conclusions: Acetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

Activation of the M1 Muscarinic Acetylcholine Receptor Induces GluA2 Internalization in the Hippocampus

Keun Oh Ryu(류근오),Heon Seok(석헌) 한국생명과학회 2015 생명과학회지 Vol.25 No.10

뇌 해마의 콜린성 신경분포는 학습과 기역에 연관성이 있는 것으로 알려져 있으며 이의 작용제인 carbachol 투여 시 장기기억 저하가 유도됨이 알려져 왔다. 그러나 이러한 콜린성 자극에 의한 해마 신경세포의 시냅스 내 변화기작은 완전히 알려지지 않고 있다. 본 연구에서는 아세틸콜린 수용체의 활성에 의하여 유도되는 장기기억저하 현상에 있어 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) 수용체가 후시냅스 표면으로 부터 사라지는 현상과 이의 조절기작에 대하여 알아보고자 한다. 이를 위하여 쥐 해마의 일차세포를 추출하고 체외에서 배양한 성숙 신경세포에 carbachol 을 투여하여 장기기억 저하를 유도 하였으며, 후시냅스의 표면으로부터 AMPA 수용체의 아단위체인 GluA2가 M1 무스카린 수용체의 길항제에 의하여 저해 되었다. 또한 콜린성 자극에 의한 GluA2의 내재화 현상의 작용기작 연구를 위하여 쥐 해마 절편에 carbachol 투여 후 GluA2와 직접적인 상호작용을 하는 Glutam내재화 되었음을 확인하였다. 이러한 현상은 ate receptor-interacting protein 1 (GRIP1)과 clathrine 단백질이 매개하는 세포내이입 작용을 하는 adaptin-α 단백질의 결합 변화를 관찰하였다. GluA2는 carbachol 자극에 의해 세포내이입 과정에서 adaptin-α 와의 결합이 증가하였으며 반대로 GRIP1과는 해리되었다. 이는 아세틸콜린의 수용체의 자극에 의하여 GluA2의 내제화 작용이 수반되며, 이의 작용기작으로 GluA2의 후시냅스 표면 발현시에 결합하고 있는 GRIP1과 해리 되면서 장기기억 저하 현상이 유도됨을 의미한다. Cholinergic innervation of the hippocampus is known to be correlated with learning and memory. The cholinergic agonist carbachol (CCh) modulate synaptic plasticity and produced long-term synaptic depression (LTD) in the hippocampus. However, the exact mechanisms by which the cholinergic system modifies synaptic functions in the hippocampus have yet to be determined. This study introduces an acetylcholine receptor-mediated LTD that requires internalization of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors on the postsynaptic surface and their intracellular mechanism in the hippocampus. In the present study, we showed that the application of the cholinergic agonist CCh reduced the surface expression of GluA2 on synapses and that this reduction was prevented by the M1 muscarinic acetylcholine receptor antagonist pirenzepine in primary hippocampal neurons. The interaction between GluA2 and the glutamate receptor-interacting protein 1 (GRIP1) was disrupted in a hippocampal slice from a rat upon CCh simulation. Under the same conditions, the binding of GluA2 to adaptin-α, a protein involved in clathrin-mediated endocytosis, was enhanced. The current data suggest that the activation of LTD, mediated by the acetylcholine receptor, requires the internalization of the GluA2 subunits of AMPA receptors and that this may be controlled by the disruption of GRIP1 in the PDZ ligand domain of GluA2. Therefore, we can hypothesize that one mechanism underlying the LTD mediated by the M1 mAChR is the internalization of the GluA2 AMPAR subunits from the plasma membrane in the hippocampal cholinergic system.

수종 생약추출물의 muscarin성$(M_1\;type)$ acetylcholine 수용체$(mAChR-M_1)$에 대한 친화력 검색

김영섭,김정섭,김성기,허정희,이병의,유시용,Kim, Young-Sup,Kim, Jeoung-Seob,Kim, Seong-Kie,Heor, Jung-Hee,Lee, Byung-Eui,Ryu, Shi-Yong 한국생약학회 2001 생약학회지 Vol.32 No.3

The water extracts of 82 Korean medicinal herbs were examined for the binding affinity on the recombinant human muscarinic acetylcholine receptor subtype 1 $(mAChR-M_1)$ produced from the CHO (Chinese Hamster Ovary) cell line. Of those tested, the extracts of Coptidis Rhizoma, Phellodendri Cortex, Hedyotis Herba and of Terminariae Fructus were found to exhibit a significant competition with $[^3H]$ N-methyl-scopolamine for the specific binding to $mAChR-M_1$ in a dose dependent manner, respectively.

In Vitro Cholinomimetic Effect of Loranthus Ferrugineus in Isolated Guinea Pig Ileum

Omar Ziad Ameer,Ibrahim M. Salman,Mohammad Jamshed Ahmad Siddiqui,Mun Fei Yam,Raghava Naidu Sriramaneni,Amirin Sadikun,Zhari Ismail,Amin Malik Shah,Mohd. Zaini Asmawi 사단법인약침학회 2009 Journal of Acupuncture & Meridian Studies Vol.2 No.4

This study aimed to elucidate the mechanism(s) of the spasmogenic action of Loranthus ferrugineus in isolated guinea pig ileum. Thus the contractile responses of guinea pig ileum to graded additions of either L. ferrugineus methanol extract or its n-butanol fraction were tested in the presence and absence of various pharmacological interventions. The data showed that L. ferrugineus methanol extract and the n-butanol fraction produced a concentration-dependent spasmogenic effect in isolated guinea pig ileum segments. These effects were significantly inhibited in the presence of 1 μM atropine. In contrast, the response to the lowest concentrations of L. ferrugineus methanol extract (0.25, 0.5 and 1 mg/mL) and n-butanol fraction of L. ferrugineus (0.125, 0.25 and 0.5 mg/mL) were considerably enhanced in the presence of 0.05 μM neostigmine. Neither L. ferrugineus methanol extract nor n-butanol fraction contractile responses were affected upon the incubation of the ileal segments with 100 μM hexamethonium. The results of this study show that the spasmogenic effect of L. ferrugineus is possibly mediated through a direct action on intestinal muscarinic receptors. It is suggested that the bioactive constituents of L. ferrugineus serve as a substrate for acetylcholinesterase. This study aimed to elucidate the mechanism(s) of the spasmogenic action of Loranthus ferrugineus in isolated guinea pig ileum. Thus the contractile responses of guinea pig ileum to graded additions of either L. ferrugineus methanol extract or its n-butanol fraction were tested in the presence and absence of various pharmacological interventions. The data showed that L. ferrugineus methanol extract and the n-butanol fraction produced a concentration-dependent spasmogenic effect in isolated guinea pig ileum segments. These effects were significantly inhibited in the presence of 1 μM atropine. In contrast, the response to the lowest concentrations of L. ferrugineus methanol extract (0.25, 0.5 and 1 mg/mL) and n-butanol fraction of L. ferrugineus (0.125, 0.25 and 0.5 mg/mL) were considerably enhanced in the presence of 0.05 μM neostigmine. Neither L. ferrugineus methanol extract nor n-butanol fraction contractile responses were affected upon the incubation of the ileal segments with 100 μM hexamethonium. The results of this study show that the spasmogenic effect of L. ferrugineus is possibly mediated through a direct action on intestinal muscarinic receptors. It is suggested that the bioactive constituents of L. ferrugineus serve as a substrate for acetylcholinesterase.

Proteomic analysis of muscarinic acetylcholine receptor-mediated proliferation in HT-29 human colon cancer cells

이수연,이아름,안지영,Jung Ho Ko,Lyon Lee,Janet Han,Young-Chang Kim,Yang-Hoon Kim 대한독성 유전단백체 학회 2018 Molecular & cellular toxicology Vol.14 No.2

Backgrounds: Muscarinic acetylcholine receptors (mAChRs) are members of G-protein-coupled receptors. They can induce agonist-dependent neoplastic transformation and facilitate colon cancer proliferation via promoting rapid expression of a variety of early responsive genes. Methods: In this study, we used 2-dimensional gel electrophoresis(2-DE) approach with subsequent mass spectrometry (MS) to identify up- and down-regulated proteins (a total of 23 protein spots) involved in mAChRs-related signaling pathway, energy metabolism, transcription/translation, oxidative stress metabolism and cytoskeleton organization in agonist carbachol stimulated HT-29 human colon cells. Results: We found that the increased expression of adenocarcinoma biomarker, annexin A5 (ANXA5) induced by carbachol treatment, which was confirmed by immunoblot. This study contributes to the understanding of mechanisms underlying mAChRs agonist-induced expression of whole proteins in HT-29 colon cancer cells. Conclusion: Our results indicated that ANXA5 might serve as a potential biomarker for the diagnosis of colon cancer.

Proteomic analysis of muscarinic acetylcholine receptor-mediated proliferation in HT-29 human colon cancer cells

Lee, Soo Youn,Lee, Ar-Reum,Ahn, Ji-Young,Ko, Jung Ho,Lee, Lyon,Han, Janet,Kim, Young-Chang,Kim, Yang-Hoon THE KOREAN SOCIETY OF TOXICOGENOMICS AND TOXICOPRP 2018 MOLECULAR AND CELLULAR TOXICOLOGY Vol. No.

Backgrounds: Muscarinic acetylcholine receptors (mAChRs) are members of G-protein-coupled receptors. They can induce agonist-dependent neoplastic transformation and facilitate colon cancer proliferation via promoting rapid expression of a variety of early responsive genes. Methods: In this study, we used 2-dimensional gel electrophoresis(2-DE) approach with subsequent mass spectrometry (MS) to identify up- and down-regulated proteins (a total of 23 protein spots) involved in mAChRs-related signaling pathway, energy metabolism, transcription/translation, oxidative stress metabolism and cytoskeleton organization in agonist carbachol stimulated HT-29 human colon cells. Results: We found that the increased expression of adenocarcinoma biomarker, annexin A5 (ANXA5) induced by carbachol treatment, which was confirmed by immunoblot. This study contributes to the understanding of mechanisms underlying mAChRs agonist-induced expression of whole proteins in HT-29 colon cancer cells. Conclusion: Our results indicated that ANXA5 might serve as a potential biomarker for the diagnosis of colon cancer.

Infliximab ameliorates AD-associated object recognition memory impairment

Kim, D.H.,Choi, S.M.,Jho, J.,Park, M.S.,Kang, J.,Park, S.J.,Ryu, J.H.,Jo, J.,Kim, H.H.,Kim, B.C. Elsevier/North-Holland Biomedical Press 2016 Behavioural brain research Vol.311 No.-

Dysfunctions in the perirhinal cortex (PRh) are associated with visual recognition memory deficit, which is frequently detected in the early stage of Alzheimer's disease. Muscarinic acetylcholine receptor-dependent long-term depression (mAChR-LTD) of synaptic transmission is known as a key pathway in eliciting this type of memory, and Tg2576 mice expressing enhanced levels of Aβ oligomers are found to have impaired mAChR-LTD in this brain area at as early as 3 months of age. We found that the administration of Aβ oligomers in young normal mice also induced visual recognition memory impairment and perturbed mAChR-LTD in mouse PRh slices. In addition, when mice were treated with infliximab, a monoclonal antibody against TNF-α, visual recognition memory impaired by pre-administered Aβ oligomers dramatically improved and the detrimental Aβ effect on mAChR-LTD was annulled. Taken together, these findings suggest that Aβ-induced inflammation is mediated through TNF-α signaling cascades, disturbing synaptic transmission in the PRh, and leading to visual recognition memory deficits.

Muscarinic acetylcholine receptors mediate eIF4B phosphorylation in SNU-407 colon cancer cells

Liu, Ziyu,Cho, Nam Jeong Academic Press 2016 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>We have previously shown that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. To gain insight into the molecular mechanisms underlying this event, we examined whether mAChRs regulate the phosphorylation of eIF4B (eukaryotic initiation factor 4B), an essential component of the translation machinery. When SNU-407 cells were treated with the cholinergic agonist carbachol, eIF4B was phosphorylated in a dose- and time-dependent manner. This carbachol effect was almost completely blocked by the muscarinic antagonist atropine, demonstrating that mAChRs specifically mediate the phosphorylation of eIF4B. Carbachol-stimulated eIF4B phosphorylation was significantly reduced by the MEK1/2 inhibitor U0126, indicating that the MEK1/2-ERK1/2 pathway plays an important role in mAChR-mediated eIF4B phosphorylation. However, treating the cells with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the mTORC1 (mammalian target of rapamycin complex 1) inhibitor rapamycin had little effect on carbachol-stimulated eIF4B phosphorylation, suggesting that PI3K and mTORC1 are not the key participants in this process. We also observed that the inhibition of protein kinase C (PKC) by GF109203X greatly diminished carbachol-stimulated eIF4B phosphorylation. Together, our data show that mAChRs modulate eIF4B phosphorylation via the ERK1/2 and PKC signaling pathways in SNU-407 colon cancer cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Muscarinic acetylcholine receptors (mAChRs) mediate eIF4B phosphorylation. </LI> <LI> The ERK1/2 signaling pathway is required for mAChR-mediated eIF4B phosphorylation. </LI> <LI> The PI3K-mTORC1 pathway is not important for mAChR-mediated eIF4B phosphorylation. </LI> <LI> Protein kinase C is involved in mAChR-mediated eIF4B phosphorylation. </LI> </UL> </P>

Activation of Neuronal Nitric Oxide Synthase by M2 Muscarinic Receptors Associated with a Small Increase in Intracellular Calcium

Wang, Shou-Zhen,Lee, Seok-Yong,Zhu, Sheng-Zu,Wotta, Diane R.,Parsons, Ann M.,El-Fakahany, Esam E. 성균관대학교 약학연구소 1998 成均藥硏論文集 Vol.10 No.1

We investigated the coupling of the M2 muscarinic acetylcholine receptors expressed in Chinese hamster ovary cells to activation of neuronal nitric oxide(NO) synthase. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an indirect measure of the generation of NO in Chinese hamster ovary cells. The muscarinic agonist carbachol induced marked time-and concentration-dependent enhancement of the activity of NO synthase. Activation of neuronal NO synthase by M2 muscarinic receptors was associated with a small increase in the concentration of intracellular Ca^2+. These data suggest the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular Ca^2+. These findings help to understand the mechanisms of activation of NO synthase.

기니피그 담낭 평활근에서 무스카린 수용체 아형의 특성

이종균(Jong Kyun Lee),이풍렬(Poong Lyul Rhee),강동묵(Tong Mook Kang),김재준(Jae Jun Kim),고광철(Kwang Cheol Koh),백승운(Seung Woon Paik),이종철(Jong Chul Rhee) 대한소화기학회 1998 대한소화기학회지 Vol.30 No.1

N/A Background/Aims: Acetylcholine(Ach) is the most irnportant rnediator in vagal-induced and cholecystokinin-induced gallbladder contraction. There are several rnuscarinic receptor subtypes characterized by pharmacologic study. The majority of smooth muscles from many species exhibit heterogenous populations of muscarinic receptors and different subtypes activate different signal transduction pathways. Therefore, it is important and essential to characterize the muscarinic receptor subtypes for understanding the mechanism of muscle contraction in a certain tissue. The aim of the present study was to characterize the muscarinic receptor subtypes in guinea pig gallbladder smooth muscle using relatively selective receptor subtype antagonists. Methods: Gallbladder muscle strips were prepared and mounted in an organ bath. Isometric contractions were recorded on a polygraph. 50% inhibitory concentrations(Icqp) and pA (-log KB) values of pirenze- pine, methoctramine, and 4-DAMP(M1, M2, and M3 antagonist, respectively) were compared on Ach-induced gallbladder muscle contraction. Results: Icsp of pirenzepine, methoctramine, and 4-DAMP on 1pM Ach-induced contraction were 7.8 > 10 'M, 2.'7 x 10 M, and 3,7 x 10 M, respectively. pA, values of pirenzepine, methoctramine, and 4-DAMP were 6.8, 5.0 and 11.4, respectively. Conclusions: M3 subtype may play the most important role in Ach-induced contraction of guinea pig gallbladder muscle. (Korean J Gastroenterol 1997; 30:90 - 97)