• Archives of Pharmacal Research
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• 제24권6호
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• pp.508-513
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• 2001

2-(1-Hexynyl), 2-((E)-1-hexenyl) and $2-(n-hexyl)-N^{6}$-methyladenines were synthesized, starting from 2-amino-6-chloropurine using palladium-catalyzed coupling as a key step as potential adenosineo receptor ligand.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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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
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• 제24권1호
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• pp.145-159
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• 1990

Recently, it has been suggested that the endogenous adenosine may be the mediator for the intercellular communication in the regulation of tubuloglomerular feedback control and renin release. Even though two subclasses of adenosine receptors, A1 and A2, have been described, their functional roles are controversial. The present study was undertaken to clarify the role of adenosine receptors in hypertensive rabbit caused by clamping of renal artery. Experiments were done in two-kidney one clip Goldblatt hypertensive rabbits (2K1GHR) and sham-operated normotensive rabbits. Adenosine, N6-cyclohexyladenosine (CHA) and 5'-N-ethylcarboxamidoadenosine (NECA) were infused into a renal artery. The decreases in urine volume, renal blood flow, glomerular filtration rate and excreted amounts of electrolytes caused by adenosine and CHA were significantly attenuated in 2K1CHR. However, changes in renal function caused by A2 adenosine receptor agonist, NECA, tend to be accentuated in 2K1CHR. These results suggest that the attenuation of renal effect caused by adenosine and A1 adenosine receptor agonist may be due to the modification of adenosine receptor in the kidney in Goldblatt hypertensive rabbits.

• The Korean Journal of Physiology and Pharmacology
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• 제1권1호
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• pp.1-12
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• 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.

• 대한한방소아과학회지
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• 제29권1호
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• pp.1-14
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• 2015

Objectives This experimental study was designed to investigate the effects of Dai-saiko-to (DSH) on differentiation of 3T3-L1 preadipocytes and body weight, serum lipid levels in high-fat diet-induced obese mice. Materials and Methods Cells were incubated with DSH at an indicated concentration (0.01-1 mg/ml) for 24h, then the growth rate was assessed by MTS assay. 3T3-L1 preadipocytes were incubated in DMEM for 2 days with the indicated concentrations of DSH. On Day 6, the cells were fixed and the cellular lipid contents were assessed by Oil-Red-O staining. The expression of peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) and cytidine-cytidine-adenosine-adenosine-thymine (CCAAT)/enhancer-binding proteins ${\alpha}$ ($C/EBP{\alpha}$) as adipocyte-specific proteins were determined by real time RT-PCR and western blotting. Four-weeks old mice (wild-type C57BL/6) were used for all experiments. Body weight gain and serum lipid levels were measured in the obesity-induced mice. Results DSH did not show toxicity even at the concentration of 1 mg/ml and DSH significantly inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. Also, DSH significantly reduced the expressions of $PPAR{\gamma}$ and $C/EBP{\alpha}$ in a dose-dependent manner. Furthermore, DSH significantly reduced body weight gain, serum glucose, total cholesterol and LDL-cholesterol contents in obesity-induced mice. Conclusions These results demonstrated that DSH inhibited 3T3-L1 preadipocyte differentiations and high-fat diet-induced obesity in mice.

• 대한한방소아과학회지
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• 제27권4호
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• pp.17-30
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• 2013

Objectives This experimental study was designed to investigate the effects of Mulberry leaves contained herbal mixture (MLHM) on body weight, serum lipid level and adipocyte differentiation in high fat diet-fed obese mice. Methods Four-week old mice (wild-type C57/BL6) were used for all experiments. Cells were incubated with MLHM at the indicated concentration (0.04-4mg/ml) for 24h, and growth rate was assessed by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3T3-L1 preadipocytes were incubated in DMEM for 2 days with the indicated concentrations of MLHM, and on Day 6, the cells were fixed and the cellular lipid contents were assessed by Oil-Red-O staining. The expression of peroxisome proliferator-activated receptor ${\gamma}$ (PPAR ${\gamma}$) and cytidine-cytidine-adenosine-adenosine-thymine (CCAAT)/enhancer-binding proteins ${\alpha}$ (C/EBP ${\alpha}$) as adipocyte-specific proteins were determined by real time RT-PCR and western blotting. In addition, body weight gain and serum lipid levels were measured in the mice with obesity induced by the high fat-diet for four weeks. Results Though MLHM did not show toxicity even at the concentration of 4mg/ml, MLHM significantly inhibited the differentiation of 3T3-L1 preadipocites in a dose-dependent manner. Also, MLHM significantly reduced the expressions of PPAR ${\gamma}$ and C/EBP ${\alpha}$ in a dose-dependent manner. Furthermore, MLHM significantly reduced body weight gain and LDL-cholesterol contents in high fat diet-fed obese mice. Conclusions These results demonstrate that MLHM exerts anti-obesity effect in 3T3-L1 cells and mice with obesity by high-fat diet.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1229-1238
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• 2016

This study was conducted to determine the relationships of five intragenic single nucleotide polymorphism (SNP) markers (protein kinase adenosine monophosphate-activated ${\gamma}3$ subunit [PRKAG3], fatty acid synthase [FASN], calpastatin [CAST], high mobility group AT-hook 1 [HMGA1], and melanocortin-4 receptor [MC4R]) and meat quality traits of Duroc breeding stocks in Korea. A total of 200 purebred Duroc gilts from 8 sires and 40 dams at 4 pig breeding farms from 2010 to 2011 reaching market weight (110 kg) were slaughtered and their carcasses were chilled overnight. Longissimus dorsi muscles were removed from the carcass after 24 h of slaughter and used to determine pork properties including carcass weight, backfat thickness, moisture, intramuscular fat, $pH_{24h}$, shear force, redness, texture, and fatty acid composition. The PRKAG3, FASN, CAST, and MC4R gene SNPs were significantly associated with the meat quality traits (p<0.003). The meats of PRKAG3 (A 0.024/G 0.976) AA genotype had higher pH, redness and texture than those from PRKAG3 GG genotype. Meats of FASN (C 0.301/A 0.699) AA genotype had higher backfat thickness, texture, stearic acid, oleic acid and polyunsaturated fatty acid than FASN CC genotype. While the carcasses of CAST (A 0.373/G 0.627) AA genotype had thicker backfat, and lower shear force, palmitoleic acid and oleic acid content, they had higher stearic acid content than those from the CAST GG genotype. The MC4R (G 0.208/A 0.792) AA genotype were involved in increasing backfat thickness, carcass weight, moisture and saturated fatty acid content, and decreasing unsaturated fatty acid content in Duroc meat. These results indicated that the five SNP markers tested can be a help to select Duroc breed to improve carcass and meat quality properties in crossbred pigs.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.211-219
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• 2004

The effects of adenosine 5'-triphosphate (ATP) and ATP analogs, P/sub 2y/ purinoceptor agonists, on growth of normal mouse mammary epithelial cells (NMuMG) were examined. Cells were plated onto 24 well plates in DMEM supplemented with 10 % fetal calf serum. After serum starvation for 24 hours, ATP, P/sub 2y/ purinoceptor agonists (AdoPP[NH]P, ATP-α-S, ATP-γ-S, β, γ-me-ATP and 2me-S-ATP), P/sub 2u/ purinoceptor agonist (UTP) and P/sub 2y/ purinoceptor antagonists (Reactive Blue 2, more selective to P/sub 2y/ receptor than PPADS; PPADS) were added. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA (1 hour pulse with 1 μ Ci/ml, 18～19 hours after treatment). ATP, Adopp[NH]P, ATP-α-S or ATP-γ-S, significantly increased DNA synthesis at 1, 10 and 100 μM concentrations with dose-dependency (P<0.05), and the maximum responses of ATP and ATP analogs were shown at 100 μM concentration (P<0.05). The potency order of DNA synthesis was ATP≥ATP- γ -S>Adopp [NH]P>ATP-α-S. β, γ -me-ATP, 2me-S-ATP and UTP did not increase DNA synthesis. In autoradiographic analysis of percentage of S-phase cells, similar results were observed to those of DNA synthesis. Addition of 1, 10 or 100 μM Reactive Blue 2 or PPADS significantly decreased ATP (100 μM)-induced DNA synthesis, however, PPADS was less effective than Reactive Blue 2. In Elvax 40P implant experiment, ATP directly stimulated mammary endbud growth in situ suggesting the physiological regulator of ATP in mammary growth. ATP 100 μM rapidly increased MAPK activity, reaching a maximum at 5 min and then gradually decreasing to the base level in 30 min. ATP analogs, Adopp[NH]P and ATP-γ-S also increased MAPK activity, however, β, γ-me-ATP and 2me-S-ATP did not. The inhibitor of the upstream MAPK kinase (MEK), PD 98059 (25 μM), effectively reduced ATP (100 μM) or EGF(10 ng/ml, as positive control)-induced MAPK activity and DNA synthesis (P<0.05). These results indicate that ATP-induced DNA synthesis was prevented from the direct inhibition of MAPK kinase pathway. Overall results support the hypothesis that the stimulatory effects of normal mouse mammary epithelial growth by addition of ATP or ATP analogs are mediated through mammary tissue specific P/sub 2y/ purinoceptor subtype, and MAPK activation is necessary for the ATP-induced cell growth.

• Nutrition Research and Practice
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• 제9권6호
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• pp.606-612
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• 2015

BACKGROUND/OBJECTIVES: Several medicinal properties of Smilax china L. have been studied including antioxidant, anti-inflammatory, and anti-cancer effects. However, the antiobesity activity and mechanism by which the water-soluble fraction of this plant mediates its effects are not clear. In the present study, we investigated the lipolytic actions of the water-soluble fraction of Smilax china L. leaf ethanol extract (wsSCLE) in 3T3-L1 adipocytes. MATERIALS/METHODS: The wsSCLE was identified by measuring the total polyphenol and flavonoid content. The wsSCLE was evaluated for its effects on cell viability, lipid accumulation, glycerol, and cyclic adenosine monophosphate (cAMP) contents. In addition, western blot analysis was used to evaluate the effects on protein kinase A (PKA), PKA substrates (PKAs), and hormone-sensitive lipase (HSL). For the lipid accumulation assay, 3T3-L1 adipocytes were treated with different doses of wsSCLE for 9 days starting 2 days post-confluence. In other cell experiments, mature 3T3-L1 adipocytes were treated for 24 h with wsSCLE. RESULTS: Results showed that treatment with wsSCLE at 0.05, 0.1, and 0.25 mg/mL had no effect on cell morphology and viability. Without evidence of toxicity, wsSCLE treatment decreased lipid accumulation compared with the untreated adipocyte controls as shown by the lower absorbance of Oil Red O stain. The wsSCLE significantly induced glycerol release and cAMP production in mature 3T3-L1 cells. Furthermore, protein levels of phosphorylated PKA, PKAs, and HSL significantly increased following wsSCLE treatment. CONCLUSION: These results demonstrate that the potential antiobesity activity of wsSCLE is at least in part due to the stimulation of cAMP-PKA-HSL signaling. In addition, the wsSCLE-stimulated lipolysis induced by the signaling is mediated via activation of the ${\beta}$-adrenergic receptor.

• 대한약리학회지
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• 제24권1호
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• pp.31-42
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• 1988

본 연구에서 한국산 인삼의 활성성분의 하나인 Panaxadiol(PD)에 대한 가토적출부신에서 카테콜아민(CA)의 분비작용과 작용기전을 파악하고자 실험을 시행하여 다음과 같은 결과를 얻었다. $PD(400{\mu}g)$을 가토적출부신에 투여하였을 때 카테콜아민의 분비를 의의있게 증가시켰다. PD의 이러한 CA 분비작용은 atropine 처치로 현저히 억제되었다. Physostigmine 전처치시 PD 뿐만 아니라 Ach의 CA 분비작용은 뚜렷이 증가되었다. 그러나 chlorisondamine 전처치로 PD나 Ach의 분비 효과는 억제되었다. 또한 $PD(400{\mu}g/30\;min)$을 주입한 후에 Ach의 CA 분비 효과는 오히려 강화되었다. PD나 Ach의 작용은 adenosine 전처러시 현저히 증강되었다. EGTA(5mM)와 함께 Ca-free Krebs액으로 30분 주입한 경우에 Ach의 분비작용은 거의 전적으로 차단되었으며, PD의 작용도 약화되었다. 이상의 실험결과로 보아, PD는 가토적출부신에서 $Ca^{++}$ 의존적으로 CA분비를 증가시키며, 이러한 작용은 cholinergi muscarinic 및 nicotinic receptor의 흥분작용에 기인하며, chromaffincell 대한 일부 직접작용도 개재되어 나타나는 것으로 사료된다.