• The Korean Journal of Pharmacology
• /
• v.27 no.2
• /
• pp.89-97
• /
• 1991

As it has been reported that the depolarization-induced release of acetylcholine(ACh) is diminished by activation of presynaptic muscarinic autoreceptor in rabbit hippocampus and various lines of evidence indicate the involvement of adenylate cyclase system in ACh release, it was attempted to delineate the role of cAMP in the muscarinic autoreceptor-mediated control of ACh release. Slices and synaptosomal preparations from rabbit hippocampus were incubated with $[^3H]-choline$ and the release of the labelled products was evoked either by electrical stimulation or by $high-K^+$, and the influence of various agents on the evoked tritium release was investigated. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from $0.1\;to\;30\;{\mu}M$, increased the $[^3H]-ACh$ release in a dose-dependent manner and also dbcAMP increased the tritium outflow. The responses to oxotremorine, a specific muscarinic agonist, were characterized by decrement of ACh release in dose range of $0.1-30\;{\mu}M$, and the oxotremorine effects were inhibited either by forskolin or by atropine. Glibenclamide, a specific $K^+-channel$ inhibitor, in concentration of $1{\sim}10\;{\mu}M$, decreased the evoked ACh release slightly and inhibited the enhancing effect of evoked ACh-release of a large dose$(10\;{\mu}M)$ of forskolin. These results indicate that the cAMP might play a role in the muscarinic ACh receptor-mediated control of ACh rlease in the rabbit hippocampus and suggest that certain potassium currents may also be participated in the post-receptor mechanism of ACh release.

• The Korean Journal of Pharmacology
• /
• v.30 no.2
• /
• pp.145-152
• /
• 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.

• The Korean Journal of Pharmacology
• /
• v.28 no.2
• /
• pp.129-136
• /
• 1992

As it has been reported that the depolarization-induced acetylcholine (ACh) release is modulated by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus and various lines of evidence indicate the involvement of adenylate cyclase system in $A_1-adenosine$ post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the $A_1-receptor-mediated$ control of ACh release in this study. Slices from rat hippocampus were incubated with $[^3H]-choline$ and the release of the labelled products was evoked by electrical stimulation $(3\;Hz,\;5\;Vcm^{-1},\;2\;ms,\;rectangular\;pulses)$, and the influence of various agents on the evoked tritium-outflow was investigated. $N^6-cyclopentyladenosine$ (CPA), a specific $A_1-adenosine$ receptor agonist, in concentrations ranging from 0.1 to $10\;{\mu}M$, decreased the $[^3H]-ACh$ release in a dose-dependent manner without the changes of basal rate of release. 8-cyclopentyl-1,3-dipropylxanthine $(DPCPX,\;1{\sim}10\;{\mu}M)$, 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 CPA effects were significantly inhibited by DPCPX $(2\;{\mu}M)$ pretreatment and the dose-response curve produced by CPA was shifted to the right. The responses to N-ethylmaleimide $(NEM,\;10\;&\;30\;{\mu}M)$, a SH-alkylating agent of G-protein, were characterized by increments of the evoked ACh-release and the basal release, and the CPA effect were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.3 to $10\;{\mu}M$, increased the evoked ACh-release in a dose-dependent manner and the CPA effects were inhibited by forskolin. These results indicate that the $A_1-adenosine$ heteroreceptor plays an important role in ACh-release via nucleotide-binding protein Gi in the rat hippocampus and that the adenylate cyclase system might be participated in this process.

• The Korean Journal of Pharmacology
• /
• v.29 no.2
• /
• pp.175-182
• /
• 1993

As it has been reported that the depolarization-induced ACh release is modulated by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus and various lines of evidence indicate the adenosine effect is magnesium dependent, the present study was undertaken to delineate the role of endogenus adenosine as a modulator of hippocampal acetylcholine release in this study. Slices from the rat hippocampus were equilibrated with $[^3H]-choline$ and the release of the labelled product, $[^3H]-ACh$, was evoked by electrical stimulation(3Hz, $5\;V\;cm^{-1},$ 2ms, rectangular pulses), and the influence of various agents on the evoked tritium outflow was investigated. Adenosine, in concentrations ranging from $0.3\;to\;100\;{\mu}M$, 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 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 evoked tritium outflow with increase of basal rate of tritium release, and these effects were also abolished by $DPCPX(2{\mu}M)$ pretreatment. But, $CGS(0.1{\sim}10{\mu}M)$, a recently introduced potent $A_2-agonist$, did not alter the evoked tritium outflow. When the magnesium concentration of the medium was reduced to 0 mM, there was no change in evoked ACh release by adenosine. In contrast, increasing the magnesium concentration to 4 mM, the inhibitory effects of adenosine were significantly potentiated. These results indicate that $A_1-adenosine$ heteroreceptor is involved in ACh-release in the rat hippocampus and the inhibitory effects of adenosine mediated by $A_1-receptor$ is magnesium-dependent.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.6
• /
• pp.301-305
• /
• 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
• /
• v.2 no.1
• /
• pp.31-39
• /
• 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.

• The Korean Journal of Pharmacology
• /
• v.30 no.3
• /
• pp.263-272
• /
• 1994

Since it was been reported that the depolarization-induced ACh release is inhibited by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus, a large body of experimental data on the post-receptor mechanism of this process has been accumulated. But, the post-receptor mechanism of presynaptic $A_1-adenosine$ receptor on the ACh release has not been clearly elucidated yet. Therefore, it was attempted to clarify the post-receptor mechanisms of the $A_1-adenosine$ receptor-mediated control of ACh release in this study. Slices from rat hippocampus were equilibrated with $^3H-choline$ and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 $VCm^{-1}$, 2ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Adenosine, in concentrations ranging from $0.3{\sim}300\;{\mu}M$, decreased the ACh release in a dose-dependent manner, without affecting the basal rate of release. The adenosine effects were significantly inhibited by $DPCPX\;(2\;{\mu}M)$, a selective $A_1-receptor$ antagonist. The responses to N-ethylmaleimide $(10&30{\mu}M)$, a SH-alkylating agent of G-protein, were characterized by increments of the evoked ACh-release and the basal release, and the adenosine effects were completely abolished by NEM pretreatment. PDB $(1{\sim}10\;{\mu}M)$, a specific protein kinase C (PKC) activator, increased, whereas PMB $(0.03{\sim}1\;mg)$, a PKC inhibitor, decreased the evoked ACh-release, and the adenosine effects were not affected by these agents. Nifedipine $(1\;{\mu}M)$, a $Ca^{2+}\;-channel$ blocker of dihydropyridine analogue, significantly inhibited the adenosine effect, but glibenclamide, a $K^+-channel$ blocker, did not. Finally, 8-bromo cyclic AMP $(100\;&\;300{\mu}M)$, a membrane-permeable analogue of cAMP, did not alter the ACh release, but adenosine effects were inhibited by pretreatment with large dose of 8-br-cAMP $(300\;{\mu}M)$. These results indicate that the decrement of the evoked ACh-release by $A_1-adenosine$ receptor is mediated by the G-protein, and nifedipine-sensitive $Ca^{2+}-channel$ and adenylate cyclase system are coupled partly to this effect, and that protein kinase C and glibenclamide-sensitive $K{^+}-channel$ are not involved in this process.

• The Korean Journal of Pharmacology
• /
• v.32 no.2
• /
• pp.127-138
• /
• 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
• /
• v.1 no.1
• /
• pp.1-12
• /
• 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
• /
• v.41 no.3
• /
• pp.119-123
• /
• 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.