• Proceedings of the Korean Society of Applied Pharmacology
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• 1992.05a
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• pp.45-45
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• 1992

Calcium channel에 작용하는 dihydropyridine(DHP) 계열의 calcium channel 효능제와 길항제의 caicium channel에 대한 작용과 muscarinic receptor에 대한 작용과의 관계를 조사하기 위하여 [$^3$H]QNB와 [$^3$H]nitrendipine 결합실험을 시행하고 이를 지표로 하여 칼슘효능제와 길항제의 이들 receptors에 대한 결합성질을 검토하였다. 본 연구결과 칼슘 channel 효능제인 Bay K 8644는 칼슘길항제인 nicardipine 및 nimodipine과 같이 고농도에서 muscarinic receptor에 대한 [$^3$H]QNB결합을 경쟁적으로 억제하였으며 이들 약물의 muscarinic receptor에 대한 Ki치는 각각 16.7 $\mu$M, 3.5 $\mu$M, 및 15.5 $\mu$M이었다. 한편, 이들 약물은 다같이 칼슘 channel의 high affinity DHP결합부위에 대한 [$^3$H]nitrendipine 결합을 억제하였으나 이 부위에 대한 Bay K 8644, nicardipine, 및 nimodipine의 Ki치는 각각 4 nM, 0.1 nM, 및 0.2 nM로서 muscarinic receptor에 대한 Ki치 보다 4,000-75,000배 작았다. 뿐만 아니라 [$^3$H]QNB결합을 완전히 차단하는 고농도의 atropine(1 $\mu$M)에 의해서도 [$^3$H]nitrendipine결합이 전혀 영향을 받지 않았다. 따라서 DHP계 약물의 muscarinic receptor에 대한 작용은 칼슘channel에 대한 이들 약물의 작용을 연구하거나 임상적 치료 목적으로 사용할때는 나타나지 않을 것으로 생각된다.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.55-61
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• 1999

The present experiments were performed to examine the effects of acetylcholine (ACh) and carbachol (CC) on thyroxine ($T_4$) release and any possible relation between inhibition of $T_4$ release and signaling pathway in guinea pig thyroids. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. ACh and CC inhibited the TSH-stimulated $T_4$ release. These inhibition were reversed by atropine, but not by d-tubocurarine. The inhibitory effects of ACh on $T_4$ release were prevented by $M_{1^-}$ and $M_{3^-}$muscarinic antagonists and its inhibition was also slightly reversed by $M_{2^-}$ and $M_{4^-}$muscarinic antagonists. R59022, like ACh and CC, also inhibited the TSH-stimulated $T_4$ release. This inhibition was reversed by protein kinase C inhibitor and $Ca^{2+}$ channel blocker. The present study suggests that cholinergic inhibition of $T_4$ release from thyroids can be induced mainly by activation of the $M_{1^-}$ or $M_{3^-}$ receptors and that it is mediated through the muscarinic receptorstimulated protein kinase C activation.

• Molecules and Cells
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• v.27 no.3
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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.

• YAKHAK HOEJI
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• v.44 no.1
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• pp.52-59
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• 2000

An exceptionally conserved sequence that is shared among most G protein-coupled neurotransmitter receptors is an aspartate-arginine-tyrosine triplet that is located at the second cytoplasmic domain. Using the ml subtype of muscarinic acetylcholine receptors as an example, a point mutation of the arginine residue at position 123 into asparagine was induced. This mutation resulted in a complete blockade of the carbachol-induced increases of PI hydrolysis and intracellular $Ca^2$$^{+}$ level, in spite of the expression of the wild-type and mutant receptors at similar concentrations in Chinese hamster ovary cells. In marked contrast, the muscarinic agonist carbachol induced concentration-dependent enhancement of the activity of NO synthase at mutant ml receptors although the enhancement was significantly smaller than at wild-type ml receptors. These data suggest that this highly conserved arginine residue plays an important role in coupling of muscarinic receptors to the second messenger systems and 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 $Ca^{2+}$.2+/.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.361-368
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• 1994

The nature of the muscarinic receptors in bovine adrenal medulla was investigated in this study. [$^3$H]Quinuclidinyl benzilate(QNB) specifically bound to a single class of muscarinic receptor with a $K_{D}$ value of about 70 pM in bovine adrenal medullary, cardiac ventricular and ileal homogenates. Pirenzepine inhibition curves of [$^3$H]QNB binding to cardiac ventricular and ileal homogenates were steep, indicating the presence of a single class of binding site for pirenzepine with a Ki value of 990 nM and 508 nM, respectively. However, pirenzepine/[$^3$H]QNB competition binding curves in adrenal medulla suggested the presence of two binding sites (Hill coefficient=0.59) with a high( $M_1$) and a low( $M_2$) affinity. Respective Ki values for pirenfepine were 16 nM and 633 nM, with 44% of total sites having a high affinity( $M_1$). Gallamine, which is selective to cardiac $M_2$-receptor, inhibited [$^3$H]QNB binding to adrenal medullary, cardiac ventricular and ileal homogenates with Ki values of 12 $\mu$M, 6 $\mu$M and 13 $\mu$M, respectively. Thus, the binding affinities of pirenzepine and gallamine for $M_2$-receptor in adrenal medulla were similar to those in ileum, which contains the $M_3$-receptor. These results indicate that the $M_1$- and $M_3$- muscarinic receptor subtypes coexist in the bovine adrenal medulla.a.

• Journal of the Korean Chemical Society
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• v.16 no.2
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• pp.70-73
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• 1972

It has been postulated that acetylcholine exhibits both nicotine and muscarinic activity because of its ability to present two patterns of essential atoms to the receptors. These two patterns arise from the ability of the molecule to exist in more than one preferred conformation. The molecule S(+)-acetyl-${\beta}$-methylacetylcholine exhibits only muscarinic activity. Calculations using molecular orbital theory predict that this molecule prefers only the muscarinic conformation. This is offered as an explanation for the exclusive role of the molecule and as evidence supporting the twoconformation, two-activities hypothesis.

• BMB Reports
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• v.41 no.11
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• pp.803-807
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• 2008

We investigated the expression of muscarinic acetylcholine receptors (mAChRs) and their possible involvement in the regulation of cell proliferation in four colon cancer cell lines (SNU-61, SNU-81, SNU-407, and SNU-1033) derived from Korean colon carcinoma patients. A ligand binding assay showed that all four cell lines expressed mAChRs. Treatment of the four cell lines with the cholinergic agonist carbachol led to the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In SNU-407 cells, carbachol significantly stimulated cell proliferation, which could be abolished by the muscarinic antagonist atropine and the ERK1/2 kinase inhibitor PD98059. These results indicate that mAChRs specifically mediate the proliferation of SNU-407 colon cancer cells via the ERK1/2 pathway.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.206-206
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• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), This triplet is repeated as LYT-LYT in m2 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca$\^$2+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• Archives of Pharmacal Research
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• v.14 no.2
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• pp.181-187
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• 1991

The muscarinic antagonist 1-[benzilic 4, 4'-$[^3H]$ QUINUCLIDINYL BENZILATE $([^3H]$ QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The $K_D\;and\;B_{ max}$ values for $([^3H]$ QNB calculated from analysis of saturation isotherms were 52.5 pM AND 154 fmol/mg, respectively. Chlopheniramine (CHP), histamine $H_1$ blocker, increased $K_D$ vlue for $([^3H]$QNB without affecting the binding site concentrations and Hill coefficient. The $K_i$ value of CHP for inhibition of $([^3H]$QNB binding in ileal membranes was 1.44\mu{M}$ and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with $ED_{50}$ value of $0.11\mu{M}$. CHP caused the rightward shift of the dose-response curve to carbachol. The $pA_2$ value of CHP determined from Schild analysis of carbacholinduced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant $(K_i)$ of CHP obtained in competitive experiments with $([^3H]$ QNB was similar to the $K_A$ value (1.69 \mu{M)}$ of CHP as inhibitor of carbachol induced contraction in rabbit ileum. This result suggest that the binding of $H_i$ blocker. CHP, vs $([^3H]$QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.

• YAKHAK HOEJI
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• v.36 no.3
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• pp.220-229
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• 1992

The muscarinic acetylcholine receptors of the dog unpregant uterus were characterized using $[^3H]quinuclidinyl$ benzilate(QNB) as a radioligand and the binding of muscarinic receptor agonists and antagonists in the uterus was compared to that in the urinary bladder which contains almost exclusively the M2 receptors in order to determine the receptor subtypes in the uterus. $[^3H]QNB$ binding to uterus and bladder was rapid, saturable and reversible. Scatchard analysis of the saturation data gave linear plots and the Hill coefficients were close to unit, which indicated that each preparation contained a single population of specific binding sites for $[^3H]QNB$. The KD values(120 pM) for QNB were almost identical in both organs, whereas the $B_{max}$ value of 256 fmol/mg protein in the uterus was significantly different from that of 563 fmol/mg protein in the bladder. Muscarinic agonists and antagonists inhibited in a competitive manner the $[^3H]QNB$ binding to the same extent in both organs. The competition binding studies using antagonists(atropine and pirenzepine) exhibited a single binding site and this site had a low affinity for pirenzepine with the Ki value of about 330 nM. However, high and low affinity binding sites were observed with carbachol, methacholine and oxotremorine. These binding studies with agonists and antagonists did not show any differences in drug affinities between uterus and bladder. These results indicate that the muscarinic receptors in the uterus are M2 receptors which have a low affinity for pirenzepine.