• Molecules and Cells
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• 제27권3호
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• pp.383-390
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• 2009

To investigate the regulatory mechanism underlying the contractile response in the intestinal smooth muscle of the nile tilapia (Orechromis niloticus), we used pharmacologic and molecular approaches to identify the muscarinic subreceptors and the intracellular signaling pathways involved in this motility. Myography assays revealed that an M1- and M3-subtype selective antagonist, but not a M2-subtype selective antagonist, inhibited carbachol HCl (CCH)-induced intestinal smooth muscle contraction. In addition, a phospholipase C inhibitor, but not an adenylate cyclase inhibitor, blocked the contractile response to CCH. We also cloned five muscarinic genes (OnM2A, OnM2B, OnM3, OnM5A, and OnM5B) from the nile tilapia. In the phylogenetic analysis and sequence comparison to compare our putative gene products (OnMs) with the sequences obtained from the near complete teleost genomes, we unexpectedly found that the teleost fish have respectively two paralogous genes corresponding to each muscarinic subreceptor, and other teleost fish, except zebrafish, do not possess muscarinic subreceptor M1. In addition, the expression pattern of the nile tilapia muscarinic subreceptor transcripts during CCH-induced intestinal smooth muscle contraction in the proximal intestinal tissue was analyzed by real-time PCR surveys and it was demonstrated that CCH increased the OnMs mRNA expression rapidly and transiently.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.290-290
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• 1994

The binding characteristic of oxomemazine to muscarinic receptor in the cerebrum, heart, and ileum were compared to those of pirenzepine to investigate whether oxomemazine could classify the muscarinic receptor subtypes. 〔$^3$H〕Quinucl idinyl benzilate(QNB) identified a single class of muscarinic receptors with apparent K$\sub$D/ value of about 60 pM in three tissues. Analysis of the pirenzepine inhibition curve of 〔$^3$H〕QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (Ki=16 nM, M$_1$-receptor) and low (Ki=400 nM, M$_2$-receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with high (Ki=84 nM, On-receptor) and low (Ki=1 4 ${\mu}$M, O$\sub$L/-receptor) affinity in rat cerebral microsome, The percentage population of the M$_1$-and M$_2$-receptors to the total receptors were 61 : 39, and those of the O$\^$H/- and O$\sub$L/-receptors 39 : 61, respectively, However, the Hill coefficients of these two drugs for the inhibition of 〔$^3$H〕QNB binding to the heart and ileum were close to unity which indicated that these drugs bound to a uniform population of receptors in these two tissues. The Ki values for the low affinity sites of pirenzepine and oxomemazine in the cerebrum were similar to those of these drugs in the heart ileum. Both pirenzepine and oxomemazine increased K$\sub$D/ value for 〔$^3$H〕QNB without affecting the binding sites concentration and Hill coefficient for the 〔$^3$H〕QNB binding. Oxomemazine had a 10-fold lower affinity at Ma-receptors than at M$_1$-receptors, and pirenzepine a 8-fold lower affinity at O$\sub$L/-receptors than OH-receptors. Analysis of the shal low competition curves of oxomemazine for the H$_1$ receptors and pirenzepine for the O$\sub$L/-receptors yielded that 69% of the M$_1$-receptors were of the O$\sub$H/-receptors and the remaining 31% of the O$\sub$L/-receptors, and that 29% of the O$\sub$L/-receptors were of the M$_1$-receptors and 71% of the M$_2$-receptors. However, M$_2$ for oxomemazine and O$\sub$H/ for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could discriminatethe muscarnic receptor subtypes and may subclassify the M$_1$-receptors into two subtypes.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.84.1-84.1
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• 2003

Methamphetamine is a powerful stimulant that appears to produce locomotor activity and behavioral sensitization. Previous study has indicated that dopaminergic receptors are implicated in the behavioral responses of methamphetamine. Recently, it has been reported that other receptors, especially, M1 muscarinic acetylcholine receptor (M1R) plays an important role in the regulation of behavioral responses, and this receptor is abundantly expressed in brain regions, including the cerebral cortex, striatum, and the hippocampus of the animal. (omitted)

• 생약학회지
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• 제32권3호통권126호
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• pp.219-225
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• 2001

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.

• International Journal of Oral Biology
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• 제44권2호
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• pp.62-70
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• 2019

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in $Ca^{2+}$ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.147-154
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• 1998

It was attempted to clarify the participation of $K^+-channels$ in the post-receptor mechanisms of the muscarinic and $A_1-adenosine$ receptor- mediated control of acetylcholine (ACh) release in the present study. Slices from the rat hippocampus were equilibrated with $[^3H]$choline and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 V/cm, 2 ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Oxotremorine (Oxo, $0.1{\sim}10\;{\mu}M$), a muscarinic agonist, and $N^6-cyclopentyladenosine$ (CPA, $1{\sim}30\;{\mu}M$), a specific $A_1-adenosine$ agonist, decreased the ACh release in a dose-dependent manner, without affecting the basal rate of release. 4-aminopyridine (4AP), a specific A-type $K^+-channel$ blocker ($1{\sim}100\;{\mu}M$), increased the evoked ACh release in a dose-related fashion, and the basal rate of release is increased by 3 and $100\;{\mu}M$. Tetraethylammonium (TEA), a non-specific $K^+-channel$ blocker ($0.1{\sim}10\;{\mu}M$), increased the evoked ACh release in a dose-dependent manner without affecting the basal release. The effects of Oxo and CPA were not affected by $3\;{\mu}M$ 4AP co-treatment, but 10 mM TEA significantly inhibited the effects of Oxo and CPA. 4AP ($10\;{\mu}M$)- and TEA (10 mM)-induced increments of evoked ACh release were completely abolished in Ca^{2+}-free$ medium, but these were recoverd in low Ca^{2+}$ medium. And the effects of $K^+-channel$ blockers in low Ca^{2+}$ medium were inhibited by $Mg^{2+}$ (4 mM) and abolished by $0.3\;{\mu}M$ tetrodotoxin (TTX). These results suggest that the changes in TEA-sensitive potassium channel permeability and the consequent limitation of Ca^{2+}$ influx are partly involved in the presynaptic modulation of the evoked ACh-release by muscarinic and $A_1-adenosine$ receptors of the rat hippocampus.

• 대한약리학회지
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• 제31권3호
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• pp.355-363
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• 1995

Calcium수송에 대한 기전을 추구하기위하여, carbachol을 사용하여 ml muscarinic receptor-transfected RBL-2H3 cell-line에서 다음과 같은 실험결과를 얻었기에 이에 보고한다. 1) Carbachol의 투여로 이들 cell-line에서 $Ca^{2+}$ influx가 농도에 따라 증가하였고, hexosaminidase 분비양도 의의있게 증가하였다. 2) Atropine 투여로 Carbachol의 상승작용이 의의있게 억제되었다. 3) 수종의 금속양이온을 투여하여 carbachol의 $Ca^{2+}$수송에 대한 영향을 관찰한 바, 이들 금속이온들은 $Ca^{2+}$의 influx를 의의있게 억제하였다. 4) PMA(20 nM) 투여로 carbachol의 hexosaminidase의 분비는 억제되지 못했지만 $Ca^{2+}$ influx는 억제되었다. 5) PTx $(0.2\;{\mu}g/ml)$ 투여로 carbachol의 hexosaminidase 분비가 의의있게 억제되었다. 위의 결과로 미루어 보아, 이 세포의 muscarinic receptor가 calcium channel을 통한 calcium수송에 매우 중요한 영향을 나타내는데, 이들 calcium ion channel은 적어도 두 종류가 존재하며, 하나는G-protein-dependent calcium channel에 의하며, 다른 하나는 G-protein-independent calcium channel에 대한 작용에 의한 것으로 생각된다. 또한 이 calcium channel들은 2가 또는 3가의 다른 금속 ion들에 의하여 calcium수송이 억제된다.

• Natural Product Sciences
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• 제19권4호
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• pp.290-296
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• 2013

In preliminary tests, we examined the effect of several fractions isolated from fermented pine needle extract on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from the mouse colon using a whole cell patch clamp technique. Among these fractions, Fraction 3 (F3) elicited the most powerful depolarization of membrane. Therefore, the aim of the present study was to investigate the effect of F3 obtained from fermented extract of Pinus densiflora needle on pacemaker potentials in ICCs and to establish its mechanism of action. Colonic ICCs generated spontaneous periodic pacemaker potentials in the current-clamp mode. F3 depolarized the membrane and decreased the frequency and amplitude of pacemaker potentials in a dose-dependent fashion. The F3-induced effects on pacemaker potentials were blocked by methoctramine, a muscarinic $M_2$ receptor antagonist, and by glycopyrrolate, a muscarinic $M_3$ receptor antagonist. The F3-induced effects on pacemaker potentials were blocked by external $Na^+$-free solution and by flufenamic acid, a non-selective cation channel blocker, as well as by the removal of external $Ca^{2+}$ and in the presence of thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum. Taken together, these results suggest that F3 of pine needle extract modulates the pacemaker activity of colonic ICCs by the activation of non-selective cation channels via muscarinic $M_2$ and $M_3$ receptors. And external $Ca^{2+}$ influx and intracellular $Ca^{2+}$ release are involved in F3 actions on ICCs.

• 대한수의학회지
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• 제39권6호
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• pp.1073-1080
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• 1999

Previous studies suggested that the inhibition of thyroxine ($T_4$) release by ${\alpha}_1$-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of $T_4$ from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of $T_4$. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analog of cAMP, and isobutyl-methylxanthine, a phosphodiesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of $T_4$ from the mouse, rat, and guinea pig thyroids. Methoxamine, an ${\alpha}_1$-adrenoceptor agonist, inhibited the TSH-stimulated release of $T_4$ in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of $T_4$ in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an ${\alpha}_1$-adrenoceptor antagonist or atropine, a muscarinic antagonist or $M_1$- and $M_3$-muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of $T_4$ in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. These findings suggest that the activation of receptor or DAG inhibits TSH-stimulated $T_4$ release through a PKC-dependent mechanism in thyroid gland.

• Molecules and Cells
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• 제39권4호
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• pp.322-329
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• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.