• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.206-206
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• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), This triplet is repeated as LYT-LYT in m2 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca$\^$2+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• Development and Reproduction
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• v.24 no.4
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• pp.287-296
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• 2020

The absence of functional estrogen receptor α (Esr1) results in an overgrowth of the epididymal fat, as observed in estrogen receptor α knockout (ERαKO) mouse. The present research was aimed to evaluate expression of various molecules associated with adipocyte differentiation and maturation in the epididymal fat of ERαKO mouse at several postnatal ages by using quantitative real-time polymerase chain reaction. The highest transcript levels of all molecules were detected at 12 months of postnatal age, except leptin which the mRNA level was increased at 5 months of age and was unchanged until 12 months of age. The expression levels of CCAAT enhancer binding protein (Cebp) alpha, androgen receptor, and lipoprotein lipase were decreased at 5 months of age but increased at about 8 months of age. The mRNA levels of Cebp gamma and sterol regulatory element binding transcription factor 1 remained steady until 8 months of age. Continuous increases of transcript levels during postnatal period were found in Cebp beta, estrogen receptor (ER) beta, fatty acid binding protein 4, and delta like non-canonical Notch ligand 1. The increases of peroxisome proliferator-activated receptor gamma and adiponectin mRNA levels were detected as early as 8 months of age. The levels of fatty acid synthase and resistin transcript at 5 and 8 months of age were lower than that at 2 months of age. These findings show the aberrant expression patterns of genes related to adipocyte differentiation and maturation in the postnatal epididymal fat pad by the disruption of ER alpha function.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.6 no.2
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• pp.183-188
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• 2005

Two mammalian receptors are reported (MT1A and MT1B). In this experiments, MT1A is expressed at a little enhanced level (about 8 times) in hypothalamus of the 9 hours exposed mice. In other part of the brain, the expression level of the MT1A and MT1B is elevated at nearly same level: 16 times in cerebellum, 128 times in hippocampus and in thalamus, respectively. But MT1B is expressed at very high level (about thousand times) in hypothalamus of the 9 hours exposed mice.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.19-25
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• 1997

The C-terminus ends of the second putative transmembrane domains of both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in $M_1$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposedfashion (LYT-LYT) in the sequence of $M_2$ receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT $M_1$ receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant $M_1$ receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant $M_1$ receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in $M_1$ muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of $M_1$ receptors to the stimulation of phospholipase C.

• Biomolecules & Therapeutics
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• v.4 no.2
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• pp.196-201
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• 1996

The N-methyl-D-aspartate (NMDA) receptor-ion channel complex is activated by the simultaneous presence of L-glutamate and glycine, allowing the binding of MK-801 to the phencyclidine (PCP) site of the receptor. The $[^3H]$MK-801 binding assay system was established for determination of pharmacological functions of test compounds acting at the glycine site of the receptor. The binding in the presence of 0.1 $\mu$M L-glutamate was increased by an agonist (glycine) in a dose-dependent fashion, while decreased by either partial agonist (R-(+)-HA-966) or antagonist (5,7-dichlorokynurenic acid: 5,7-DCKA). To distinguish partial agonism from antagonism, various concentrations of 7-chlorokynurenic acid (7-CKA) were added in the assay to eliminate the interference of the endogenous glycine present in the membrane preparations. The bindings in the presence of L-glutamate (0.1$\muM$) and 7-CKA (1, 5, or 10$\muM$) were increased by R-(+)-HA-966. Being a weak partial agonist, the extent of potentiation was much less than that by the agonist. These binding profiles were clearly distinguishable from those by the antagonist, 5,7-DCKA, which exhibited no intrinsic activity. The binding assays established in the present study are a useful system to classify ligands acting at the glycine site of the NMDA receptor by their pharmacological functions.

• Development and Reproduction
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• v.11 no.1
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• pp.21-29
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• 2007

Previously, we found progesterone receptor membrane component 2 (pgrmc2) was highly expressed in germinal vesicle (GV) stage oocytes. The present study was conducted to characterize the expression of pgrmc2, as well as pgrmc1, in the mouse gonads and embryos according to their developmental stages. We found that these membrane components were expressed in ovaries, testes, and embryos at various developmental stages in addition to oocytes. Progesterone-3-O-carboxymethyl oxime-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was applied to visualize the presence of the progesterone receptor on mouse oocyte membrane, and we confirmed that immobilized progesterone is localized at surface of the oocyte. This is, at our knowledge, the first report regarding the expression of membrane component of progesterone receptor in the mouse oocytes, embryos, and gonads. The function and signal transduction pathway of progesterone receptor membrane components in oocytes requires further studies.

• Archives of Pharmacal Research
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• v.26 no.1
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• pp.58-63
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• 2003

Ginseng has been recommended to alleviate the menopausal symptoms, which indicates that components of ginseng very likely contain estrogenic activity. We have examined the possibility that a component of Panax ginseng, $ginsenoside-R_{b1}$ acts by binding to estrogen receptor. We have investigated the estrogenic activity of $ginsenoside-R_{b1}$ in a transient transfection system using estrogen-responsive luciferase plasmids in MCF-7 cells. $ginsenoside-R_{b1}$ activated the transcription of the estrogen-responsive luciferase reporter gene in MCF-7 breast cancer cells at a concentration of 50 $\mu$M. Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of $ginsenoside-R_{b1}$ is estrogen receptor dependent. Next, we evaluated the ability of $ginsenoside-R_{b1}$ to induce the estrogen-responsive gene c-fos by semi-quantitative RT-PCR assays and Western analyses. $ginsenoside-R_{b1}$ increased c-fos both at mRNA and protein levels. However, $ginsenoside-R_{b1}$ failed to activate the glucocorticoid receptor, the retinoic acid receptor, or the androgen receptor in CV-1 cells transiently transfected with the corresponding steroid hormone receptors and hormone responsive reporter plasmids. These data support our hypothesis that $ginsenoside-R_{b1}$ acts a weak phytoestrogen, presumably by binding and activating the estrogen receptor.

• Toxicological Research
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• v.29 no.1
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• pp.21-25
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• 2013

The selective targeting of an integrin ${\alpha}_v{\beta}_3$ receptor using radioligands may enable the assessment of angiogenesis and integrin ${\alpha}_v{\beta}_3$ receptor status in tumors. The aim of this research was to label a peptidomimetic integrin ${\alpha}_v{\beta}_3$ antagonist (PIA) with $^{99m}Tc(CO)_3$ and to test its receptor targeting properties in nude mice bearing receptor-positive tumors. PIA was reacted with tris-succinimidyl aminotriacetate (TSAT) (20 mM) as a PIA per TSAT. The product, PIA-aminodiacetic acid (ADA), was radiolabeled with $[^{99m}Tc(CO)_3(H_2O)_3]^{+1}$, and purified sequentially on a Sep-Pak C-18 cartridge followed by a Sep-Pak QMA anion exchange cartridge. Using gradient C-18 reverse-phase HPLC, the radiochemical purity of $^{99m}Tc(CO)_3$-ADA-PIA (retention time, 10.5 min) was confirmed to be > 95%. Biodistribution analysis was performed in nude mice (n = 5 per time point) bearing receptor-positive M21 human melanoma xenografts. The mice were administered $^{99m}Tc(CO)_3$-ADA-PIA intravenously. The animals were euthanized at 0.33, 1, and 2 hr after injection for the biodistribution study. A separate group of mice were also co-injected with 200 ${\mu}g$ of PIA and euthanized at 1 hr to quantify tumor uptake. $^{99m}Tc(CO)_3$-ADA-PIA was stable in phosphate buffer for 21 hr, but at 3 and 6 hr, 7.9 and 11.5% of the radioactivity was lost as histidine, respectively. In tumor bearing mice, $^{99m}Tc(CO)_3$-ADA-PIA accumulated rapidly in a receptor-positive tumor with a peak uptake at 20 min, and rapid clearance from blood occurring primarily through the hepatobiliary system. At 20 min, the tumor-to-blood ratio was 1.8. At 1 hr, the tumor uptake was 0.47% injected dose (ID)/g, but decreased to 0.12% ID/g when co-injected with an excess amount of PIA, indicating that accumulation was receptor mediated. These results demonstrate successful $^{99m}TC$ labeling of a peptidomimetic integrin antagonist that accumulated in a tumor via receptor-specific binding. However, tumor uptake was very low because of low blood concentrations that likely resulted from rapid uptake of the agent into the hepatobiliary system. This study suggests that for $^{99m}Tc(CO)_3$-ADA-PIA to be useful as a tumor detection agent, it will be necessary to improve receptor binding affinity and increase the hydrophilicity of the product to minimize rapid hepatobiliary uptake.

• Bulletin of the Korean Chemical Society
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• v.32 no.9
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• pp.3215-3218
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• 2011

A new anion receptor utilizing carbamate functionality as hydrogen bonding site has been designed and synthesized. This new receptor 2 has two amide hydrogens and two carbamate hydrogens anchored at 1,8-position of anthracene. These four hydrogens form a concave structure for the anions and the receptor 2 was found to be a selective receptor for acetate among the anions investigated.