• 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.

• BMB Reports
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• v.29 no.6
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• pp.525-529
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• 1996

Ligand binding properties of muscarinic acetylcholine receptors (mAChRs) in the nematode Caenorhabditis elegans (C. elegans) were characterized by using filtration binding assays. Scatchard analysis using $[^{3}H]N-methylscopolamine$ ($[^{3}H]NMS$) showed that the dissociation constant ($K_d$) and the maximum binding value ($B_{max}$) were $3.3{\pm}0.8{\times}10^{10}$ M and $9.0{\pm}1.1$ fmol/mg protein, respectively. Binding competition experiments indicated that the affinities of C. elegans mAChRs to atropine, scopolamine, and oxotremorine were similar to those of mammalian mAChRs. Pirenzepine binding experiments revealed that the binding pattern of mAChRs in C. elegans closely resembled that of mAChRs in rat brain, suggesting that the receptors consist primarily of Ml subtype. The affinity of mAChRs for oxotrernorine was significantly affected by guanylylimidodiphosphate (Gpp(NH)p), a non hydrolyzable GTP analog, suggesting that mAChRs in C. elegans might be coupled to G proteins. The data presented here indicate the possibility that C. elegans provides a living animal model to study the action mode of the muscarinic cholinergic system.

• Archives of Pharmacal Research
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• v.10 no.4
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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.

• Molecules and Cells
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• v.25 no.4
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• pp.504-509
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• 2008

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.

• 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+/.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.208-208
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• 1996

In this work we investigated coupling of the m2 and m4 subtypes of muscarinic acetylcholine receptors expressed in chinese hamster ovary (CHO) cells to activation of neuronal nitric oxide synthase (nNOS). Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an index of generation of nitric oxide (NO) in CHO cells. The agonist carbachol induced marked time and concentration-dependent enhancement of the activity of nNOS at m2 receptors. In sharp contrast, the response in CHO cells transfected with the m4 receptor gene was similar in magnitude to that observed in non-transfected cells, suggesting lack of significant coupling of m4 muscarinic receptors to NO signaling. This novel observation of functional divergence of the two muscarinic receptor subtypes at the level of activation of nNOS is quite intriguing, in light of the currently accepted dogma that they belong to the same functional class. This functional selectivity was not due to differential effects on intracellular Ca$\^$2+/ concentration, since activation of both subtypes of muscarinic receptors produced a comparable, albeit quite small, Ca$\^$2+/ signal. Taken together, our present data strongly suggest that the generally assumed functional equivalence of m2 and m4 muscarinic receptors should be carefully reexamined. These data also suggest the presence of alternate mechanisms of activation of nNOS, which might be operative in the absence of large changes in the concentration of cellular Ca$\^$2+/. The latter mechanisms are expected to be activated by m2, but not m4 muscarinic receptors. Both sets of findings are quits important in regards to refining the functional classification of muscarinic receptor subtypes and the cellular mechanisms of activation of NOS.

• 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.

• Biomolecules & Therapeutics
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• v.12 no.2
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• pp.101-107
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• 2004

Repeated administration of psychostimulants such as amphetamines increases locomotor activity in rodents. These drugs, including methamphetamine, enhance dopaminergic neurotransmission and result in hyper-locomotion and behavioral sensitization. It is well known that the existence of a complex balance between the cholinergic and dopaminergic systems in the central nervous system. Thus, behavioral sensitization by methamphetamine may be related to the expression of the M1 muscarinic acetylcholine receptors gene. The present study investigated the changes of M1R mRNA in hyperlocomotor activity and behavioral sensitization by methamphetamine (2 mg/kg) in mice. Our results showed that M1R mRNA expression was increased in the frontal cortex and the hippocampus region (the CA2 region) in the acute methamphetamine administered group compared to the saline administered group. In the chronic group, M1R mRNA expression was increased in the frontal cortex ill1d the hippocampus regions (CA2 and DG regions) in melt1amphetamine administered group compared to saline control group. These results indicate that acute or chronic treatment of mathamphetamine leads to the region-specific changes in mRNA expression levels of M1R. Therefore, Therefore, the present result suggests that M1R may play a role in modulating of methamphetamine-induced behavioral sensitization in mice.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.209-209
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• 1996

Muscarinic acetylcholine receptors contain two highly conserved tyrosine residues which are located within or at the extracellular border of the second transmembrane domain. These tyrosine residues are located at positions 82 and 85 of the sequence of the ml subtype of muscarinic receptors. In this wok, we studied the involvement of these two residues in ligand binding to and agonist-induced activation this receptor subtype. our data suggest an important role for these two tyrosines in these processes, with a more prominent role for the tyrosine residue located at position 82 than that located at position 85. Evidence is also provided that while the aromatic moiety of these tyrosine residues is important for antagonist binding, both this moiety and the tyrosine phenolic hydroxyl group are involved in agonist binding and receptor activation.