• The Korean Journal of Pharmacology
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• v.30 no.2
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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.

• The Korean Journal of Pharmacology
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• v.30 no.2
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• pp.181-190
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• 1994

In rat atrium the characteristics of purinergic receptors were investigated by observing the effects of some purinergic receptor agonists and antagonists on action potential and contractile force. The statistically significant effects of $ATP(10^{-6}{\sim}10^{-3}M)$ and adenosine $(10^{-6}{\sim}10^{-3}M)$ on normal action potential characteristics were a dose-dependent shortening of action potential duration $(APD_{90})$ by both agents and hyperpolarization by $ATP(10^{-4},10^{-3}M)$. $CAP(10^{-8}{\sim}10^{-4}M)$, an $A_1$ adenosine receptor agonist, shortened $(APD_{90})$ markedly in a dose-dependent manner and these effects were almost abolished by $DPCPX\;(10^{-6}\;M), an $A_1$, adenosine receptor antagonist, but not affected by $DMPX(2{\times}10^{-6}\;M)$, an $A_2$ adenosine receptor agonist. On the other hand, CGS $21680(10^{-7}{\sim}10^{-4}M)$, an $A_2$ adenosine receptor agonist, elicited a slight shortening of $(APD_{90})$ and these effects were inhibited by DPCPX but persisted in the presence of DPMX. Adenosine $(10^{-6}{\sim}10{\-4}\;M)$ decreased the basal contraction of atrial muscle in a dose-dependent manner and these effects were not inhibited by DMPX but by DPCPX. These results suggests that purinergic receptor agonists depress the cardiac activity by a short ening of action potential duration and this effect is mostly mediated by $A_1$ adenosine receptors in rat atrium.

• The Korean Journal of Pharmacology
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• v.29 no.2
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• pp.175-182
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• 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 Pharmacology
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• v.32 no.1
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• pp.23-29
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• 1996

According to the traditional receptor model, competitive antagonists share with agonists the ability to bind to a common site on receptors, but they are different from agonist in that they cannot trigger the biological response-i.e., they lack intrinsic efficacy. Recent findings extend the model by indicating that not all antagonists display an intrinsic efficacy of zero but that some display 'inverse agonism'. In the present study we studied the inverse agonism at $A_1$ adenosine receptors in membranes prepared from rat cerebral cortex. Eight commercially available $A_1$ adenosine receptor antagonists (CGS-15943, ADPX, CPT, DPCPX, DPX, N-0840, PACPX and 8-PT) were screened for inverse agonism by measuring the extent of $[^{35}S]guanosine-5'-({\gamma}-thio)$ triphosphate $([^{35}S]GTP_{\gamma}S)$ binding to G proteins. The agonist-induced stimulation of $[^{35}S]GTP_{\gamma}S$ bindings was completely blocked in the presence of $A_1$ adenosine receptor antagonists. Under optimal conditions, two types of antagonists could be distinguished. Seven antagonists including DPCPX decreased the basal $[^{35}S]GTP_{\gamma}S$ binding in the absence of agonist, displaying inverse agonist activity. One (CGS-15943) had no effect on the basal bindings. N-ethylmaleimide treatment reduced the basal bindings as well as agonist-mediated stimulation of $[^{35}S]GTP_{\gamma}S$ bindings, indicating that a substantial amount of this binding reflects an activated state of the C proteins. In good agreement with these findings, 0.1 mM GTP decreased the apparent affinity of the receptors for the agonist PIA, increased that for DPCPX, and had no effect on that for CGS-15943.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.1
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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.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.2
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• pp.135-142
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• 1997

As it has been reported that the depolarization-induced norepinephrine (NE) release is modulated by activation of presynaptic $A_1$-adenosine heteroreceptor and various lines of evidence indicate that $A_2$-adenosine receptor also presents in hippocampus, and that the adenosine effect is magnesium dependent, the present study was undertaken to delineate the role of adenosine receptors in the modulation of hippocampal NE release. Slices from the rat hippocampus were equilibrated with $[^3H]-NE$ and the release of the labelled product, $[^3H]-NE$, was evoked by electrical stimulation (3 Hz, 5 V $cm^{-1}$, 2 ms, rectangular pulses), and the influence of various agents on the evoked tritium outflow was investigated. $N^6-cyclo-pentyladenosine$ (CPA), in concentrations ranging from 0.1 to 10 ${\mu}M$, decreased the $[^3H]-NE$ release in a dose-dependent manner without changing the basal rate of release, and these effects were significantly inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 ${\mu}M$) treatment. When the magnesium concentration was reduced to 0.4 mM or completely removed, the evoked NE release increased along with decreased basal rate of release. In contrast, increasing the magnesium concentrations to 2.4 and 4 mM, decreased the evoked NE release. The CPA effects on evoked NE release were reducedby magnesium removal, but potentiated by 2.4 mM magnesium in the medium. 5-(N-cyclopropyl)-carboxamodiadenosine (CPCA, 1 & 10 ${\mu}M$), an $A_2$-agonist, decreased the evoked tritium outflow, and this effect was also abolished by DPCPX pretreatment. CGS, a powerful $A_2$-agonist, did not affect the evoked NE release. However, the effects of CPCA and CGS on evoked NE release were significantly increased by pretreatment of DPCPX in the magnesium-free medium. These results indicate that inhibitory effect of $A_1$-adenosine receptor on NE release is magnesium-dependent, and $A_2$-receptor may be present in the rat hippocampus.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.657-664
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• 1997

The effects of adenosine, adenosine A1 receptor antagonist (DPCPX), or NMDA receptor antagonist (APV) on the spontaneous release of $[^3H]-5-hydroxytryptamine$ ($[^3H]-5-HT$) during normoxic/normoglycemic or hypoxic/hypoglycemic period were studied in the rat hippocampal slices. The hippocampus was obtained from the rat brain and sliced $400\;{\mu}m$ thickness with the tissue slicer. After 30 min's preincubation in the normal buffer, the slices were incubated for 30 min in a buffer containing $[^3H]-5-HT$ ($0.1\;{\mu}M,\;74{\mu}Ci/8\;ml$) for uptake, and washed. To measure the release of $[^3H]-5-HT$ into the buffer, the incubation medium was drained off and refilled every ten minutes through sequence of 14 tubes. Induction of glucose/oxygen deprivation (GOD; medium depleting glucose and gassed with 95% $N_2/5%\;CO_2$) was done in 6th and 7th tube. The radioactivities in each buffer and the tissue were counted using liquid scintillation counter and the results were expressed as a percentage of the total radioactivities. When slices were exposed to GOD for 20 mins, the spontaneous release of $[^3H]-5-HT$ was markedly increased and this increase of $[^3H]-5-HT$ release was blocked by adenosine ($10\;{\mu}M$) or DL-2-amino-5-phosphonovaleric acid (APV; $30\;{\mu}M$). Adenosine $A_1$ receptor specific antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) exacerbate GOD-induced increase of spontaneous release of $[^3H]-5-HT$. These results suggest that Adenosine may play a role in the GOD-induced spontaneous release of $[^3H]-5-HT$ through adenosine $A_1$ receptor activity.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.13-17
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• 1997

Carbamazepine (CBZ), an anticonvulsant, has beeen reported to displace ligands at adenosine receptors. Several studies have demonstrated that as far as $A_2$adenosine receptors is concerned, CBZ acts as an antagonist. However, the situation with regard to Al receptors is less straightforward. In this study, we describe the effects of one-week CBZ treatment (25 mg/kg/day) on cerebrocortical $A_1$ adenosine receptors. $A_1$ adenosine receptor bindings as determined by using $[^3CH]DPCPX$ was not significantly altered in membranes prepared from CBZ-treated rats. However, there was a significant decrease in the $A_1$ adenosine receptor-mediated stimulation of $[^{35}S]GTP_{\gamma}S$ binding to cerebrocortical membranes prepared from CBZ-treated rats (20.0% decrease in basal activity; 17.8% decrease in maximal activity). The basal and $10^{-4}$ M forskolin-stimulated adenylyl cyclase activities were relatively unaffected by CBZ treatment, but 10 mM NaF-stimulated adenylyl cyclase activity was significantly reduced in CBZ-treated rats. It appears that one-week CBZ treatment caused an uncoupling of adenosine receptors from G proteins without alteration of $A_1$ adenosine receptor molecules, suggesting that CBZ acts as an agonist at $A_1$ adenosine receptors in rat brain.

• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.151-158
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• 1996

Following the subcutaneous administration of $R(-)N^6-(2-phenylisopropyl)adenosine(600\;nmol/kg/hr)$ to rats for 1 week using t$Alzet^{\circledR}$ mini-osmotic pumps, $A_1$ adenosine receptor functions were determined using $[^3H]DPCPX$ binding, $[^{35}S]GTP_{\gamma}S$ binding, and adenylyl cyclase assays. $A_1$ adenosine receptor binding and the inhibition of adenylyl cyclase activity by PIA was not altered in cerebrocortical membranes prepared from PIA-treated rats. However, there was a significant decrease in the $A_1$ adenosine receptor-mediated stimulation of $[^{35}S]GTP_{\gamma}S$ binding to cerebrocortical membranes prepared from PIA-treated rats(22.0% decrease in basal activity; 19.7% decrease in maximal activity). These results suggest that the desensitization of $A_1$ adenosine receptors following chronic administration involves agonist-induced uncoupling of the receptors from G proteins rather than alteration of $A_1$ adenosine receptor molecules. It is also suggested that the determination of stimulation of $[^{35}S]GTP_{\gamma}S$ binding to G proteins is a suitable tool in studying the receptor regulation including desensitization

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