• YAKHAK HOEJI
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• v.36 no.5
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• pp.496-505
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• 1992

The correlation between GABA receptors($GABA_A$ and $GABA_B$ receptor) and benzodiazepine receptor on the saline infusion-induced micturition reflex contraction was studied in the female rat. To investigate the effect of ${\gamma}-aminobutyric$ acid(GABA) on the micturition reflex, exogenous GABA(10 mg/kg) and GABA transaminase inhibitor(aminooxyacetic acid; AOAA $1\;{\mu}g$) were administered intravenously(i.v.) and intracerebroventriculary(i.c.v.), respectively. In result, both GABA and AOAA inhibited the saline induced micturition reflex contraction. This AOAA induced inhibition of micturition reflex was blocked by both bicuculine. $GABA_A$ receptor antagonist, and Ro 15-1788, benzodiazepine receptor antagonist. Muscimol, $GABA_A$ receptor antagonist($0.1\;{\mu}g$ i.c.v., $3\;{\mu}g$ intrathecal; i.t., 1 mg/kg i.v.) and baclofen, $GABA_A$ receptor agonist($1\;{\mu}g$ i.c.v., $3\;{\mu}g$ i.t., 1 mg/kg i.v.) also inhibited the bladder contraction. Pretreatment of bicuculline($1\;{\mu}g$ i.c.v.), but not of 5-aminovaleric acid(AVA, $1\;{\mu}g$ i.c.v.), $GABA_B$ receptor antagonist blocked the central inhibition of muscimol. These inhibitory effects were reversed by Ro15-1788 but were potentiated by flurazepam, benzodiazepine receptor antagonist. On the other hand, the inhibitory effects of baclofen were not affected by Ro 15-1788. Diazepam and flurazepam also inhibited the micturition reflex contraction when they were administered $3\;{\mu}g$ i.c.v., $10\;{\mu}g$ i.t., $10\;{\mu}M$, $30\;{\mu}M$ transurethrally, respectively. In conclusion, these results suggest that the micturition reflex is mediated by $GABA_A$, $GABA_B$ receptor and benzodiazepine receptor. The bezodiazepines increase the receptor binding of GABA to the $GABA_A$ receptor, so that the benzodiiazepines show the synergistic effect on the inhibition of the micturition reflex contraction, but not to the $GABA_B$ receptor.

• The Korean Journal of Pain
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• v.9 no.2
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• pp.318-325
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• 1996

Background: Sympathectomy relieves pain in sympathectically maintained pain, and subcutaneous injection of norepinephrine(NE) can rekindle mechanical allodynia. However, the mechanism of rekindling is not clear. The purpose of this study is to investigate which subtype of $\alpha$-adrenoceptor is involved in NE-induced rekindling of mechanical allodynia in sympathectomized neuropathic rats. Methods: Neuropathic injury was produced by tightly ligating the left L5 and L6 spinal nerves of 36 male Sprague-Dawley rats and bilateral lumbar sympathectomy was done at two weeks postoperatively. Starting at 7 days after sympathectomy, rekindling of mechanical allodynia was induced by NE and clonidine injected into the left paw, which was reversed by pretreatment of phentolamine and idazoxan. Mechanical allocynia was quantified by measuring the frequency of foot lifts to two von Frey filaments applied to the paw. Results: All tested rats displayed well-developed signs of mechanical allodynia at the left paw that were abolished by a bilateral lumbar sympathectomy. Subcutaneous (s.c.) injection of NE (0.05 ${\mu}g$) into the affected paw of sympathectomized neuropathic rats rekindled previous mechanical allodynia. These effects could be mimicked by an ${\alpha}_2$-receptor agonist clonidine, but not by an ${\alpha}_1$-receptor agonist phenylephrine. The NE-induced rekindling of mechanical allodynia was significantly reduced by prior s.c. injection of a mixed $\alpha$-receptor antagonist phentolamine (20${\mu}g$) and ${\alpha}_2$-receptor antagonist idazoxan(20${\mu}g$), but not by a ${\alpha}_1$-receptor antagonist terazosin (20${\mu}g$). The pretreatment of idazoxan produced dose-related inhibition of NE-induced rekindling of mechanical allodynia. The rekindling induced by ${\alpha}_2$-receptor agonist clonidine (5${\mu}g$) was also reversed by prior s.c. injection of ${\alpha}_2$-receptor antagonist idazoxan (20${\mu}g$). Conclusion: Subcutaneous injection of NE into the paw of sympathectomized neuropathic rats rekindles mechanical allodynia, which is reversed by an ${\alpha}_2$-, but not by an ${\alpha}_1$-receptor antagonist. Therefore, rekindling of mechanical allodynia in sympathectomized neuropathic rats is mediated by ${\alpha}_2$-adrenoceptor.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.97-97
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• 2001

This Study was attempted to investigate tile effect of dopamine, SKF 81297, a dopamine D$_1$-receptor agonist, and TNPA, a dopamine D$_2$-receptor agonist, on the blood pressure in rat. Dopamine exhibited the hypertensive action in proportion to the doses of 1.0, 3.0 arid 10.0 $\mu\textrm{g}$/kg i.v., these hypertensive action of dopamine was blocked significantly by SCH 23390, a dopamine D$_1$-receptor antagonist, on the other hand, more potentiated by raclopride, a dopamine D$_1$-receptor antagonist. SKF 81297 produced hypertensive action in a dose of 1.0 $\mu\textrm{g}$/kg i.v., wherease hypotensive action in proportion to administered doses 3.0 and 10.0 $\mu\textrm{g}$/kg i.v., these hypertensive action of SKF 81297 in a dose of 1.0 $\mu\textrm{g}$/kg i.v. was not influenced by SCH 23390 or raclopride, but hypotensive action of SKF 81297 in tile doses of 3.0 and 10.0 $\mu\textrm{g}$/kg i.v. was weakened significantly by SCH 23390, but more strenthened by raclopride. TNPA showed the hypotensive action in inverse proportion to administered doses of 1.0, 3.0 and 10.0 $\mu\textrm{g}$/kg i.v., these hypotensive action was reversed to hypertensive action in inverse proportion to the administered doses of TNPA by SCH 23390 and raclopride.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.221-226
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• 2007

Melittin-induced tonic pain model is characterized by local inflammation, edema, spontaneous flinchings, and sustained mechanical hypersensitivity. These nociceptive responses are mediated through selective activation of capsaicin-sensitive primary afferent fibers by melittin. The present study was undertaken to elucidate the role of peripheral 5-hydroxytryptamine(5-HT) receptors in the melittin-induced nociceptive responses. Changes in mechanical threshold, flinching behaviors and paw thickness were measured in rat intraplantarly injected with melittin($40{\mu}g/paw$) alone or treated together with melittin and 5-HT receptor antagonists. WAY-100635($100{\mu}g\;&\;200{\mu}g/paw$), isamoltane hemifumarate($100{\mu}g\;&\;200{\mu}g/paw$), methysergide maleate($60{\mu}g,\;120{\mu}g\;&\;200{\mu}g/paw$) and ICS-205,930($100{\mu}g\;&\;200{\mu}g/paw$) were intraplantarly injected 20 min before melittin injection. All 5-HT receptor antagonists tested in this experiment significantly attenuated the ability of melittin to reduce mechanical threshold and to induce flinching behaviors. 5-HT receptor antagonists, except ICS-205,930, had mild inhibitory effect on melittin-induced edema. These experimental findings suggest that multiple peripheral 5-HT receptors are involved in the melittin-induced nociceptive responses.

• 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.

• YAKHAK HOEJI
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• v.39 no.6
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• pp.645-653
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• 1995

Dibenamine inhibited [$^{3}$H]quinuclidinyl benzilate ([$^{3}$H]QNB) binding in both concentration and incubation time-dependent manners. The $IC_{50}$/ value of dibenamine for the inhibition of the specific binding of 100 pM [$^{3}$'H]QNB following incubation of cerebral microsomes with dibenamine at 37.deg. C for 15 min was 20.mu.M. Dibenamine irreversibly decreased the binding site concentration for [$^{3}$H]QNB binding without affecting the affinity of [$^{3}$H]QNB for the muscarinic receptor. Analysis of the pirenzepine inhibition curve of [$^{3}$H]QNB binding to cerebral microsomes indicated the presence of two receptor subtypes with high(M$_{1}$ receptor, Ki=5nM) and low (M$_{2}$ receptor, Ki=160nM) affinity for pirenzepine. However, dibenamine(20.mu.M) treatment under the condition employed in these experiments caused steepening of the pirenzepine competition curve. The Ki value for pirenzepine in dibenamine treated-microsomes was approximately 120nM. suggesting a selective decrease in the number of M$_{1}$ receptor. Although dibenamine also inhibited [$^{3}$H]QNB binding to ventricular microsomes with $IC_{50}$/ value of 120.mu.M, the sensitivity for dibenamine in the ventricle was much lower than that in the cerebrum. These results indicate that dibenamine at low concentrations welectively inactivates the muscarinic M$_{1}$ receptor.

• 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.

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

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.45-54
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• 1999

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398 of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide synthesizer. The specificity and identification of the antisera were tested by analysis of transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR. MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as endoplasmic reticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity was abolished when anti-MOR sera were preincubated with the peptide against which they were raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which was determined to be within the last amino acid, 391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was observed in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain areas showed the mirrored pattern observed in auto radiographic studies of mu-opioid binding as well as a pattern similar to that seen by is situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future.