• YAKHAK HOEJI
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• v.39 no.2
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• pp.153-160
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• 1995

Receptor binding study was carried out as an in vitro assay to test the anti-ulcer effect for newly synthesized test compounds(IY-80843 and IY-80845) which were reported to have a strong anti-secretory effect in Shay-ligated rats. The histamine H$_{2}$-receptor fraction was prepared from the membranes of the isolated gastric glands in guinea pigs and $^{3}$H-cimetidine was used as a radioligand. The binding of $^{3}$H-cimetidine to the membranes of the isolated gastric glands was found to be time dependent, saturable and confined to a single population of binding sites with $K_{D}$ value of 0.13$\pm$0.03 $\mu{M}$ and B$_{max}$ value of 52.5$\pm$1.5 pmol/mg. From the competition experiments, both IY-80843 and IY-80845 were shown to have a strong blocking effect against binding of $^{3}$H-cimetidine to the histamine H$_{2}$-receptor. The IC$_{50}$, Ki, and Hill coefficient(nH) values for IY-80843 were 0.18$\pm$0.02 $\mu{M}$, 0.16$\pm$0.02 $\mu{M}$, and 0.97$\pm$0.15, respectively and those values for IY-80845 were 0.27$\pm$0.02 $\mu{M}$, 0.24$\pm$0.02 $\mu{M}$, and 0.82$\pm$0.13, respectively. The results demonstrated that the blocking effects of IY-80843 and IY-80845 were 7 and 5 times stronger than that of cimetidine, respectively. Therefore, the newly synthesized compounds, IY-80843 and IY-80845, appeared to be the highly potent competitive inhibitors of histamine on the H$_{2}$-receptor.

• Proceedings of the Ginseng society Conference
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• 2002.10a
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• pp.96-112
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• 2002

In the present study, we have investigated the effects of centrally administered ginsenoside Rc or Rgl on the modulation of NMDA receptor and $GABA_A$ receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using $[^3H]MK-801$ binding, and $GABA_A$ receptor bindings were analyzed by using $[^3H]muscimol\;and\;[^3H]flunitrazepam$ in rat brain slices. Rats were infused with ginsenoside Rc or Rg1 ($10\;{\mu}g/10{\mu}l/hr$, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML), The levels of $[^3H]MK-801$ binding were highly decreased in part of cortex and cingulated by ginsenoside Rc and Rgl. The levels of $[^3H]muscimol$ binding were strongly elevated in almost all regions of frontal cortex by the treatment of ginseoside Rc but decreased by ginsenoside Rg 1. However, the $[^3H]flunitrazepam$ binding was not modulated by ginsenoside Rc or ginsenoside Rgl infusion. These results suggest that prolonged infusion of ginsenoside could differentially modulate $[^3H]MK-801\;and\;[^3H]muscimol$ binding in a region-specific manner. Also, we investigated the influence of centrally administered ginsenoside on the regulation of mRNA levels of the family of NMDA receptor subtypes (NR1, NR2A, NR2B, NR2C) by in situ hybridization histochemistry in the rat brain. The level of NR1 mRNA is significantly increased in temporal cortex, caudate putamen, hippocampus, and granule layer of cerebellum in Rgl-infused rats as compared to control group. The level of NR2A mRNA is elevated in the frontal cortex. In contrast, it was decreased in CAI area of hippocampus in Rgl-infused rats. However, there was no significant change of NR1 and NR2A mRNA levels in Rc-infused rats. The level of NR2B mRNA is elevated in cortex, caudate putamen, and thalamus in both Rc- and Rg-infused rats. In contrast, NR2B level is decreased in CA3 in Rgl-infused rats. The level of NR2C mRNA is increased in the granule layer of cerebellum in only Rg1 but not Rc infused rats. These results show that structure difference of ginsenoside may diversely affect the modulation of expression of NMDA receptor subunit mRNA after infusion into cerebroventricle in rats.

• 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.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.188-195
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• 1993

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.

• Korean Journal of Veterinary Research
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• v.28 no.1
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• pp.17-22
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• 1988

평골근(平滑筋)으로 된 자궁근(子宮筋)은 자동성(自動性)을 가지고 있어서 신경지배(神徑支配)와 관계(關係)없이 근자체(筋自體)로 운동(運動)을 하게 된다. 그러나 이러한 자궁근(子宮筋)의 형태적(形態的) 및 기능적(機能的) 정상상태유지(正常狀態維持)에는 estrogen의 작용(作用)이 불가결(不可缺)한 요소(要素)로 되어 있으며 이 estrogen의 작용(作用)에 의하여 histamine의 자궁근(子宮筋)에 대한 (작용)作用이 수용체(受容體)의 어떤 기전에 의한 것인지를 알기 위하여 본(本) 연구(硏究)는 histamine과 5-hydroxytryptamine 및 이들 길항물질(拮抗物質)들의 자궁근(子宮筋) 운동성(運動性)에 대한 수축(收縮) 및 이완작용(弛緩作用)을 조사(調査)하였다. 자궁근(子宮筋)의 운동성(運動性)은 physiograph를 통(通)해 자궁수축(子宮收縮)의 빈도(頻度)와 크기를 기록하여 아래와 같은 결론(結論)을 얻었다. 1. 5-hydroxytryptamine에 대한 phenoxybenzamine의 억제작용(抑制作用)은 phenoxybenzamine의 길항성(拮抗性)의 결과(結果)이다. 2. histamine은 $H_1$-receptor를 통해서 흰쥐의 자궁평활근(子宮平滑筋)의 운동성(運動性)은 증가(增加)한다. 3. 반면 histamine은 $H_2$-receptor를 통해서는 자궁평활근(子宮平滑筋)의 운동성(運動性)을 이완(弛緩)시켰다. 4. 흰쥐의 자궁근(子官筋)에서 $H_2$-receptor 차단제(遮斷劑)가 $H_1$-receptor 차단제(遮斷劑)의 작용(作用)보다 더욱 강하였다.

• The Korean Journal of Pharmacology
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• v.20 no.2
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• pp.23-34
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• 1984

To investigate diurnal variations of opiate receptor binding and its modification by experimental condition or treatment of various centrally-acting drugs, the amount of maximum $^3H-morphine$ binding in rat midbrain homogenates was measured at 4 hour intervals for 24 hours. Animals were conditioned under the controlled L : D, 12 : 12 cycle or D: D, 12 : 12 cycle, for 3 weeks and treated with 0.5 ml of physiological saline or drugs for 2 weeks. A highly significant diurnal rhythm with peak at 22 hour of early dark phase with an amplitude$(0.68{\pm}0.06\;pmole/mg\;protein)$ of +51.1% and nadir $(0.33{\pm}0.03\;mole/mg\;prtein)$ at 18 hour of late light phase with an amplitude of -26.6% was found in control group. 24 tour mean of $^3H-morphine$ binding was $0.45{\pm}0.03\;pmole/mg$ protein respectively. Constant dark adaptation or treatment of reserpine, pargyline, imipramine, amphetamine and chlorpromazine modified the diurnal rhythm in the time of peak and nadir binding shape, phase, amplitude of the diurnal curve and 24 hour mean of $^3H-morphine$ binding. However, Kd values were not changed in all experimental groups : Statistical analysis at times of least and great binding indicates that the differences in $^3H-morphine$ binding were due to changes not in the affinity, but in the number of binding sites. The results are interpreted with regard to the diurnal rhythm of opiate receptor finding. The modes of action of psychoactive drugs are closely related to postulated changes of receptor sensitivity in neuropharmacological aspects.

• Bulletin of the Korean Chemical Society
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• v.27 no.11
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• pp.1791-1794
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• 2006

We have introduced a new enantioselective receptor 1 by incorporation of chiral building blocks to the previously reported carboxylate selective receptor 2. Binding studies carried out using $^1H$ NMR revealed that the receptor 1 showed moderate enantioselectivity with a general preference for D-amino acids and a highest enantioselectivity for leucine among the amino acids we investigated.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.11
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• pp.7575-7581
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• 2015

Corticotropin-releasing factor(CRF), one of the stress driven neuropeptides, was widely proposed to influence hair loss and re-growth. For the development of receptor antagonists, the screening system based on intracellular calcium signal process was developed and optimized. The aequorin parental cells were transfected with CRF1 receptor and alpha 16 promiscuous G protein cDNA to establish HEK293a16/hCRF1, a stable cell line for the human CRF1 receptor. In HEK293a16/hCRF1 cells, the range of sauvagine dose response was 12-fold higher($EC_{50}:15.21{\pm}1.83nM$) than in the transiently expressed cells, hence essential conditions for the antagonist screening experiments such as the robust signals and high solvent tolerance were secured. The standard antagonists for the CRF1 receptor, antalarmin and CP154526, resulted $IC_{50}$ values of $414.1{\pm}5.5$ and $290.7{\pm}1.9nM$, respectively. Similar results were presented with frozen HEK293a16/hCRF1 cells. Finally, our HEK293a16/hCRF1 cells with the aequorin based cellular functional assay can be a model system for the development of functional cosmetics and modulators that can have a clinical efficacy on hair re-growth.

• Molecules and Cells
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• v.43 no.5
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• pp.459-468
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• 2020

Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.