• 대한약리학회지
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• 제27권2호
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• pp.89-97
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• 1991

가토 해마(hippocampus)에서 acetylcholine(ACh) 유리에 미치는 cAMP의 역할에 관한 지견을 얻고자하여 $[^3H]-choline$으로 평형시킨 해마 slice 및 시냅토솜(synaptosome)을 사용하여 $[^3H]-ACh$ 유리에 미치는 여러가지 약물들의 영향을 관찰하였다. Adenylate cyclase 활성화제인 forskolin$(0.1{\sim}30{\mu}M)$은 전기 및 고농도 $K^+$ 자극에 의한 $[^3H]-ACh$ 유리를 용량 의존적으로 증가시켰으며, dbcAMP 또한 ACh 유리를 강화시켰다. Muscarine성 흥분제인 oxotremorine$(0.1{\sim}30{\mu}M)$은 전기 및 $K^+$ 자극에 의한 $[^3H]-ACh$ 유리 효과를 용량 의존적으로 감소시켰으며, 이러한 효과는 forskolin 및 atropine에 의하여 차단됨을 관찰할 수 있었다. 한편 $K^+-channel$ 억제제인 glibenclamide$(1,\;10{\mu}M)$는 자체로는 $K^+$ 자극에 의한 $[^3H]-ACh$ 유리를 약간 억제시킴을 관찰할 수 있었으며, 대량의 forskolin$(10\;{\mu}M)$에 의한 $[^3H]-ACh$의 증가효과를 감약시킴을 알 수 있었다. 이상의 실험 결과로 가토 해마의 presynaptic muscarinic autoreceptor를 통한 ACh 유리의 조절에는 세포내 cAMP의 관여가 확실하다 하겠으나, 일부 $K^+-currents$의 관여를 배제할 수는 없을 것으로 사료된다.

• 대한약리학회지
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• 제30권2호
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• pp.145-152
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• 1994

As it has been reported that the depolarization induced acetylcholine(ACh) release is modulated by activation of presynaptic $A_1-adenosine$ heteroreceptor and various lines of evidence indicate the $A_2-receptor$ is present In hippocampus, this study was undertaken to delineate the role of adenosine receptors on hippocampal ACh release. Slices from the rat hippocampus were equilibrated with $[^3H]-choline$ and the release of the labelled product, $[^3H]-ACh$, which evoked by electrical stimulation(3 Hz, $5\;Vcm^{-1}$, 2 ms, rectangular pulses) was measured, and the influence of various agents on the evoked tritium outflow was Investigated. Adenosine$(0.3{\sim}100\;{\mu}M)$ and CPA$(0.1{\sim}30\;{\mu}M)$ decreased the $[^3H]-ACh$ release in a dose-dependent manner without changing the basal rate of release. DPCPX$(1{\sim}10\;{\mu}M)$, a selective $A_1-receptor$, antagonist, increased the $[^3H]-ACh$ release in a dose related fashion with slight increase of basal tritium release. And the effects of adenosine and CPA were significantly inhibited by $DPCPX(2\;{\mu}M)$ treatment. CPCA, a specific $A_2-agonist$, in concentration ranging from 0.3 to 30 ${\mu}M$, decreased the evoked tritium outflow, and these effects were also abolished by $DPCPX(2\;{\mu}M)$ treatment. But the CPCA effects were not affected by $DMPX(2\;{\mu}M)$, a specific Aa-antagonist, treatment. However, CGS 21680c, a recently introduced potent $A_2-agonist$, in concentration ranging from 0.1 to $10{\mu}M$, did not alter the evoked tritium outflow. These results indicate that the decrement of the evoked ACh release by adenosine is mediated by $A_1-heteroreceptor$, but $A_2-adenosine$ receptor is not involved in ACh release in the rat hippocampus.

• 대한약리학회지
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• 제28권2호
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• pp.129-136
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• 1992

흰쥐 해마(hippocampus)에서 acetylcholine(Ach) 유리에 미치는 $A_1-adenosine$ 수용체의 역할과 post-receptor 기전에 있어서 adenylate cyclase 계의 관여여부에 관한 지견을 얻고자 하여 $[^3H]-choline$으로 평형시킨 해마 slice를 사용하여 $[^3H]-ACh$ 유리에 미치는 여러가지 약물들의 영향을 관찰하였다. $A_1-adenosine$ 수용체 흥분제인 $N^6-cyclopentyladenosine(CPA,\;0.1{\sim}10\;{\mu}M)$은 전기자극$(3Hz,\;5\;Vcm^{-1},\;2\;ms,\;rectangular\;pulses)$에 의한 $[^3H]-ACh$ 유리를 용량 의존적으로 감소시켰다. $A_1-adenosine$ 수용체 차단제인 8-cyclopentyl-1, 3-dipropylxanthine$(DPCPX,\;1{\sim}10\;{\mu}M)$은 용량 의존적으로 $[^3H]-ACh$ 유리를 증가시켰으며, 이때 기저(basal)유리 또한 증가됨을 관찰할 수 있었고, $2\;{\mu}M$ DPCPX 전처리는 CPA의 효과를 길항하여 CPA에 의한 용량반응곡선을 우측으로 이동시킴을 볼 수 있었다. G protein 억제제인 N-ethylmaleimide$(NEM,\;10\;&\;30\;{\mu}M)$는 그 자체에 의하여 자극에 의한 ACh 유리를 증가시켰으며, 기저유리 또한 증가함을 볼 수 있었다. NEM 전처리에 의하여 CPA의 효과는 완전히 소실되었다. 한편 adenylate cyclase 활성화제인 forskolin$(0.3{\sim}10\;{\mu}M)$은 기저유리에 변함없이 용량의존적인 $[^3H]-ACh$ 유리의 증가를 초래하였으며 $3\;{\mu}M$ forskolin 전처리는 대량$(10\;{\mu}M)$의 CPA의 효과를 제외하고는 CPA의 효과를 억제시킴을 관찰할 수 있었다. 이상의 실험 결과로 흰쥐 해마의 choline 작동성신경의 presynaptic $A_1-adenosine$ heteroreceptor는 ACh 유리에 중요한 역할을 하고 있으며, ACh 유리의 조절에 Gi-단백질을 통한 adenylate cyclase 계의 관여가 확실하다 하겠다.

• 대한약리학회지
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• 제29권2호
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• pp.175-182
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• 1993

흰쥐 해마 (hippocampus)에서 acetylcholine(ACh) 유리에 미치는 adenosine 및 이에 미치는 magnesium의 역할에 관한 지견을 얻고자하여 $[^3H]-choline$으로 평형시킨 해마 slice를 사용하여 $[^3H]-ACh$ 유리에 미치는 여러가지 약물들의 영향을 관찰하였다. Adenosine $^*0.3{\sim}100{\mu}M$)은 전기자극 (3 Hz, $5\;Vcm^{-1}$, 2 ms, rectangular pulses)에 의한 $[^3H]-ACh$ 유리를 용량 의존적으로 감소시켰다. $A_1-adenosine$ 수용체 차단제인 8-cyclopentyl-1, 3-dipropylxanthine $(DPCPX,\;1-10\;{\mu}M)$은 용량 의존적으로 $[^3H]-ACh$ 유리를 증가시켰으며, adenosine과 $2{\mu}M$ DPCPX 동시 투여시 adenosine의 효과가 억제됨을 볼 수 있었다. $A_2$-수용체 흥분제인 5-(N-cyclopropyl)-carboxamidoadenosine $(CPCA,\;0.3{\sim}30\;{\mu}M)$은 자극에 의한 $[^3H]-ACh$ 유리를 용량 의존적으로 감소시켰으며, 이 역시 $2\;{\mu}M$ DPCPX 동시 투여시 그 효과가 차단됨을 관찰할 수 있었다. 그러나 또다른 $A_2$-흥분약인 CGS 21680C는 $[^3H]-ACh$ 유리에 별다른 영향을 미치지 못하였다. 한편 관류액내의 magnesium 농도를 변화시켰을때 magnesium 그 자체로는 $[^3H]-ACh$ 유리에 별다른 변화가 없었으며, magnesium을 4 mM로 증가시켰을때 adenosine의 효과가 크게 강화되어 용량 반응 곡선이 좌측으로 이동됨을 볼 수 있었다. 이상의 실험 결과로 adenosine은 흰쥐 해마의 choline 작동성 신경에 presynaptic $A_1-adenosine$ heteroreceptor를 통하여 ACh 유리 감소를 일으키며, 이러한 adenosine 작용은 magnesium이온에 의존적임을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제8권6호
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• pp.301-305
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• 2004

This study examined the effects of N-methyl-D-aspartate (NMDA), ${\alpha}-amino$-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate on basal and electrically-evoked release of acetylcholine (ACh) from the rat hippocampal and striatal slices which were preincubated with $[^3H]choline$. Unexpectedly, the basal and evoked ACh release were not affected at all by the treatment with NMDA $(3{\sim}100{\mu}M)$, AMPA $(1{\sim}100{\mu}M)$ or kainate $(1{\sim}100{\mu}M)$ in hippocampal slices. However, in striatal slices, under the $Mg^{2+}-free$ medium, $30{\mu}M$ NMDA increased the basal ACh release with significant decrease of the electrically-evoked releases. The treatment with $1{\mu}M MK-801 not only reversed the $30{\mu}M$ NMDA-induced decrease of the evoked ACh release, but also attenuated the facilitatory effect of $30\;{\mu}M$ NMDA on the basal ACh release. The treatment with either $30\;{\mu}M$ AMPA or $100\;{\mu}M$ kainate increased the basal ACh release without any effects on the evoked release. The treatment with $10{\mu}M$ NBQX abolished the AMPA- or kainate-induced increase of the basal ACh release. Interestingly, NBQX significantly attenuated the evoked release when it was treated with AMPA, although it did not affect the evoked release alone without AMPA. These observations demonstrate that in hippocampal slices, ionotropic glutamate receptors do not modulate the ACh release in cholinergic terminals, whereas in striatal slices, activations of ionotropic glutamate receptors increase the basal ACh release though NMDA may decrease the electrically-evoked ACh release.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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• pp.31-39
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• 1998

As it has been reported that the depolarization induced acetylcholine (ACh) release is modulated by activation of presynaptic $A_1$ adenosine heteroreceptor and various lines of evidence suggest the $A_2$ adenosine receptor is present in the hippocampus. The present study was undertaken to delineate the role of adenosine receptors on the hippocampal ACh release. Slices from the rat hippocampus were equilibrated with $[^3H]choline$ and then the release amount of the labelled product, $[^3H]ACh$, which was evoked by electrical stimulation (rectangular pulses, 3 Hz, 2 ms, 24 mA, $5\;V/cm^{-1}$, 2 min), was measured, and the influence of various adenosine receptor-related agents on the evoked tritium outflow was investigated. And also, the drug-receptor binding assay was performed in order to confirm the presence of $A_1$ and $A_2$ adenosine receptors in the rat hippocampus. N-ethylcarboxamidoadenosine (NECA), a potent adenosine receptor agonist with nearly equal affinity at $A_1$ and $A_2$ adenosine receptors, in concentrations ranging from $1{\sim}30\;{\mu}M$, decreased the electrically-evoked $[^3H]ACh$ release in a concentration-dependent manner without affecting the basal rate of release. And the effect of NECA was significantly inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 ${\mu}M$), a selective $A_1$ adenosine receptor antagonist, but was not influenced by 3,7-dimethyl-1-propargylxanthine (DMPX, 5 ${\mu}M$), a specific $A_2$ adenosine receptor antagonist. $N^6-cyclopentyladenosine$ (CPA), a selective $A_1$ adenosine receptor agonist, in doses ranging from 0.1 to 10 ${\mu}M$, reduced evoked $[^3H]ACh$ release in a dose-dependent manner without the change of the basal release. And the effect of CPA was significantly inhibited by 2 ${\mu}M$ DPCPX treatment. 2-P-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680C), a potent $A_2$ adenosine receptor agonist, in concentrations ranging from 0.1 to 10 ${\mu}M$, did not alter the evoked ACh release. In the drug-receptor binding assay, the binding of $[^3H]2-chloro-N^6-cyclopentyladenosine$ ($[^3H]$CCPA) to the $A_1$ adenosine receptor of rat hippocampal membranes was inhibited by CPA ($K_i$ = 1.22 nM), NECA ($K_i=10.17 nM$) and DPCPX ($K_i=161.86 nM$), but not by CGS-21680C ($K_i=2,380 nM$) and DMPX ($K_i=22,367 nM$). However, the specific binding of $[^3H]CGS-21680C$ to the $A_2$ adenosine receptor was not observed. These results suggest that the $A_1$ adenosine heteroreceptor play an important role in evoked ACh release, but the presence of $A_2$ adenosine receptor is not confirmed in this study.

• 대한약리학회지
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• 제30권3호
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• pp.263-272
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• 1994

흰쥐 해마(hippocampus)에서 acetylcholine (ACh) 유리에 미치는 $A_{1}-adenosine$ 수용체의 post-receptor 기전에 관한 지견을 얻고자 하여 $^3H-choline$으로 평형시킨 해마 slice를 사용하여 $^3H-ACh$ 유리에 미치는 여러가지 약물들의 영향을 관찰하였다. Adenosine $(0.3{\sim}300\;{\mu}M)$은 전기자극(3Hz, 2 ms, 5 $VCm^{-1}$, 구형파)에 의한 ACh 유리를 용량 의존적으로 감소 시켰으며, 이러한 효과는 $A_1-adenosine$ 수용체의 선택적 차단제인 8-cyclopentyl-1, 3-dipropylxanthine $(2\;{\mu}M)$에 의해 차단됨을 볼 수 있었다. G-단백 억제제인 N-ethylmaleimide (NEM, 10과 $30\;{\mu}M$)는 그 자체에 의하여 자극유발성 ACh 유리를 증가시켰으며, adenosine의 효과는 NEM 전처리에 의하여 완전히 소실되었다. Protein kinase C 활성화제인 $4{\beta}-phorbol$ 12, 13-dibutyrate (PDB, $1{\sim}10\;{\mu}M$)는 ACh 유리 증가를 일으켰으며 억제제인 polymyxin B (PMB, $0.03{\sim}1\;mg$)는 감소를 일으켰으나, adenosine에 의한 ACh 유리 감소효과는 PDB 및 PMB에 의해 영향을 받지 않았다. $Ca^{++}$-통로 차단제인 nifedipine $(1\;{\mu}M)$은 adenosine의 효과를 길항함을 볼 수 있었으나 $K{^+}$-통로 차단제인 glibenclamide는 adenosine의 효과에 영향을 미치지 못하였다. 8-Bromo-cAMP (100과 $300{\mu}M$) 그 자체로는 ACh 유리에 별다른 영향을 미치치 못하였으나 $300\;{\mu}M$ 8-bromo-cAMP 전처리에 의하여 $30\;{\mu}M\;adenosine$의 효과가 억제됨을 볼 수 있었다. 이상의 실험결과로 흰쥐 해마에서 $A_1-adenosine$ 수용체를 통한 adenosine의 ACh유리 감소는 G-단백에 의존적이며, 이러한 효과에 nifedipine에 예민한 $Ca^{++}$-통로와 adenylate cyclase계가 일부 관여함은 확실하나 proteinkinase C 및 glibenclamide에 예민한 $K{^+}$통로는 관여하지 않는 것으로 사료된다.

• 대한약리학회지
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• 제32권2호
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• pp.127-138
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• 1996

The effects of adenosine analogues on the electrically-evoked acetylcholine(ACh) release and the influence of ischemia on the effects were studied in the rat hippocampus. Slices from the rat hippocampus were equilibrated with $0.1{\mu}M$ $[^3H]-choline$ and the release of the labelled product, $[^3H]-ACh$, was evoked by electrical stimulation(3 Hz, 2 ms, 5 $VCm^{-1}$ and rectangular pulses for 2 min), and the influence of various agents on the evoked tritiumoutflow was investigated. Ischemia(10 min with 95% $N_2$ + 5% $CO_2$) increased both the basal and evoked ACh release. These increases were abolished by glucose addition into the superfused medium, and they significantly inhibited either by 0.1 & $0.3{\mu}M$ TTX pretreatment or by removing $Ca^{++}$ in the medium. MK-801($1{\sim}10{\mu}M$), a specific NMDA receptor antagonist, and glibenclamide $(1{\mu}M)$, a $K^+-channel$ inhibitor, did not alter the evoked ACh release and nor did they affect the ischemia-induced increases In ACh release. However, polymyxin B(0.03 mg), a specific protein kinase C inhibitor, significantly inhibited the effects of ischemia on the evoked ACh release. Adenosine and $N^6-cyclopentyladenosine$ decreased the ACh release in a dose dependent manner in ischemic condition, though the magnitude of inhibition was far less than those in normal(normoxic) condition. However, the treatment with $5{\mu}M$ DPCPX, a potent $A_1-adenosine$ receptor antagonist, potentiated the ischemia-effect. These results indicate that the evoked-ACh release is potentiated by ischemia, and this process being most probably mediated by protein kinase C, and that the decreased effect of ACh release mediated by $A_1-adenosine$ receptor is significantly inhibited in ischemic state.

• The Korean Journal of Physiology and Pharmacology
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• 제1권1호
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• pp.1-12
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• 1997

As it has been reported that the depolarization induced acetylcholine (ACh) release is modulated by activation of presynaptic $A_1$ adenosine heteroreceptor and various evidence suggest that indicate the $A_2$ adenosine receptor is present in the striatum, this study was undertaken to delineate the role of adenosine receptors on the striatal ACh release. Slices from the rat striatum were equilibrated with $[^3H]$choline and then the release amount of the labelled product, $[^3H]$ACh, which was evoked by electrical stimulation (rectangular pulses, 3 Hz, 2 ms, 24 mA, $5\;Vcm^{-1}$, 2 min), was measured, and the influence of various agents on the evoked tritium outflow was investigated. And also, quantitative receptor autoradiography and drug-receptor binding assay were performed in order to confirm the presence and characteristics of $A_1$ and $A_2$ adenosine receptors in the rat striatum. Adenosine $(10{sim}100\;{mu}M)$ and $N^6$-cyclopentyladenosine (CPA, $1{sim}100\;{mu}M)$ decreased the $[^3H]$ACh release in a dose-dependent manner without changing the basal rate of release in the rat striatum. The reducing effects of ACh release by adenosine and CPA were abolished by 8-cyclopentyl-1,3-dipropy-Ixanthine (DPCPX, 2 ${mu}M$), a selective $A_1$, adenosine receptor antagonist, treatment. The effect of adenosine was potentiated markedly by 3,7-dimethyl-1-propargylxanthine (DMPX, 10 ${mu}M$), a specific $A_2$ adenosine receptor antagonist. 2-P-(2-carboxyethyl)phenethylamimo-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680C), in concentrations ranging from 0.01 to 10 ${mu}M$, a recently introduced potent $A_2$ adenosine receptor agonist, increased the $[^3H]$ACh release in a dose related fashion without changing the basal rate of release. These effects were completely abolished by DMPX $(10\;{mu}M)$. In autoradiograrhy experiments, $[^3H]$2-chloro-$N^6$-cyclopentyladenosine ($[^3H]$ CCPA) bindings were highly localized in the hippocampus and the cerebral cortex. Additionally, lower levels of binding were found in the striatum. However, $[^3H]$CGS-21680C bindings were highly localized in the striatal region with the greatest density of binding found in the caudate nucleus and putamen. Lower levels of binding were also found in the nucleus accumbens and olfactory tubercle. In drug-receptor binding assay, binding of $[^3H]$ CCPA to $A_1$ adenosine receptors of rat striatal membranes was inhibited by CPA ($K_i$ = 1.6 nM) and N-ethylcarboxamidoadenosine (NECA, $K_i$ = 12.9 nM), but not by CGS-21680C ($K_i$ = 2609.2 nM) and DMPX ($K_i$ = 19,386 nM). In contrast, $[^3H]$CGS-21680C binding to $A_2$ denosine receptors was inhibited by CGS-21680C ($K_i$ = 47.6 nM) and NECA ($K_i$ = 44.9 nM), but not by CPA ($K_i$ = 2099.2 nM) and DPCPX ($K_i$ = 19,207 nM). The results presented here suggest that both types of $A_1$ and $A_2$ adenosine heteroreceptors exist and play an important role in ACh release in the rat striatal cholinergic neurons.

• International Journal of Oral Biology
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• 제41권3호
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• pp.119-123
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• 2016

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic $Ca^{2+}([Ca^{2+}]_i)$, transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular $Ca^{2+}$ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent $Ca^{2+}$ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced $[Ca^{2+}]_i$ increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.