• Biomedical Science Letters
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• v.27 no.4
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• pp.270-276
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• 2021

Physiological agents trigger a signaling process called "inside-out signaling" and activated platelets promote adhesion, granule release, and conformational changes of glycoprotein IIb/IIIa (αIIb/β₃). Activated αIIb/β₃ interacts with fibrinogen and initiates a second signaling step called "external signaling". These two signaling pathways can cause hemostasis or thrombosis, and thrombosis is a possible medical problem in arterial and venous vessels, and platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, modulating platelet activity is important for platelet-mediated thrombosis and cardiovascular disease. Esculetin is a coumarin-based physiologically active 6,7-dihydroxy derivative known to have pharmacological activity against obesity, diabetes, renal failure and CVD. Although some studies have confirmed the effects of esculetin in human platelet activation and experimental mouse models, it is not clear how esculetin has antiplatelet and antithrombotic effects. We confirmed the effect and mechanism of action of escultein on human platelets induced by collagen. As a result, esculetin decreased Ca²⁺ recruitment through upregulation of inositol 1, 4, 5-triphosphate receptor. In addition, esculetin upregulates cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-dependent pathways and inhibits fibrinogen binding and thrombus contraction. Our results demonstrate the antiplatelet effect and antithrombotic effect of esculetin in human platelets. Therefore, we suggest that esculetin could be a potential phytochemical for the prevention of thrombus-mediated CVD.

• Korean Journal of Veterinary Research
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• v.22 no.1
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• pp.1-8
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• 1982

The effect of acetylcholine, oxytocin and prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$) on cyclic nucleotide levels in estrogen-primed rabbit whole uterus were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiestrase inhibitor, and indomethacin, a prostagandin inhibitor. In the absence of MIX, acetylcholine increased guanosine 3', 5'-cyclic monophosphate (cGMP), but had no effect on adenosine 3', 5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin had no influence on cGMP, but decreased cAMP levels. $PGF_{2{\alpha}}$ increased cGMP and decreased cAMP levels. MIX increased both cAMP and cGMP levels. Oxytocin and $PGF_{2{\alpha}}$ further increased cGMP levels, indicating activation of guanylate cyclase activity. The ratio of cAMP/cGMP was decreased by uterine stinulants both in presence and absence of MIX. Indomethacin elevated cAMP and cGMP revels. The effects of uterine stimulants in the presence of indomethacin on cyclic nucleotide levels were varied from tissue to tisse. In general, oxytocin decreased cGMP and $PGF_{2{\alpha}}$ increased cAMP/cGMP levels, but the effects were statisically nonsignicficant. The cAMP/cGMP ratio was increased by uterine stimulant in the presence of indomethacin. In conclusion, uterine stimulants eased cAMP/cGMP ratio which indicates that the uterine stimulants have opposing effects on adenylate cyclase and guanylate cyclase activities. The endometrium plays a role in the regulation of cyclic nucleotide levels and uterine contraction by means of PG synthesis. Indomethacin has an unknown activities besides both of PG synthetase and phosphodiesterase inhibitions.

• BMB Reports
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• v.36 no.3
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• pp.299-304
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• 2003

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the co-precipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.257-262
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• 1992

Virginiae butanolide (VB) is an autoregulator which triggers virginiamycin production in Strefltomyces virginiae. VB-C binding protein activity was investigated under various additives. The VB-C binding protein was almost fully observed in sotubte fraction (>90%) and the binding activity was optimum at pH 7.0. The VB-C binding activity was increased about 15% in 0.5 M KCI, whereas decreased about 60% in 20 mM $Mo^{6+}$. Chelating reagents (ethylenediarnine tetraacetic acid, ethyleneglycol bis(2-aminoethylether) tetraacetic acid, 8-hydroxyquinoline) and SH protecting reagents (rnercaptoethanol, dithiothreitol, thioglycerol) inhibited the VB-C binding activity about 30~55% and 3~20%, respectively. Serine protease inhibitor (phenyl methane sulfonyl fluoride), nucleotides (guanosine 5'-monophosphate, adenosine 3',5'-cyclic monophosphate), and phosphatases (alkaline, acid phosphatase) increased the VB-C binding activity about 17%, 6~20%, and 4- 13%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1570-1574
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• 2004

A flock of AA breed chickens were reared in peterstme brood-vait chamber and were provided with high energy pelleted feed. At 14 d of age, a total of 350 birds were randomly divided into 3 groups as follows: 100 birds were exposed to normal ambient temperature of 20$^{\circ}C$ for control group; 150 birds were exposed to lower ambient temperature of 11$^{\circ}C$ to induce ascites (treatment I); and another group of 100 birds were exposed to lower ambient temperature of 11$^{\circ}C$ and fed diet containing 1% L-arginine for ascitic prophylactic treatment (treatment II). Samples were collected from blood and abdominal fluid of chicken at 3, 4, 5, 6 and 7 wk of age subsequently, to analysis the contents of plasma endothelin (ET-1), angiotensin II (Ang II), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP). The results indicated that the contents of cAMP, cGMP, and Ang II in reatment I and ascitic broilers were higher than the corresponding control group (p<0.01, p<0.05), ET-1 of preascitic broilers were control group (p<0.05), while there was an insignificant difference with later ascitic broilers. The contents of cAMP and cGMP in treatment II were higher than the treatment I and control groups (p<0.01, p<0.05), whereas, the contents of Ang II were gradually decreased compared to the control group (p<0.05), the contents of ET-1 were insignificantly different. On further analysis, the increased plasma Ang II at low ambient temperature condition in broilers made endothelium cell secretion of increased ET-1, cAMP, cGMP and decreased NO. Therefore, low temperature accelerated ascites syndrome in broilers. Supplemently L-arginine can decrease ET-1, and increase cAMP and cGMP. It is concluded that cAMP mediated in broilers pulmonary hypertension syndrome.

• Biomolecules & Therapeutics
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• v.7 no.2
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• pp.105-111
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• 1999

Nitric oxide (NO), a cellular messenger synthesized from L-arginine by NO synthase (NOS, EC.1.14.13.39), is considered to be a regulator of gastric secretion. In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat gastric chief cells. Treatment of chief cells with carba-chol resulted in an increase in the arginine conversion to citrulline, the amount of $NO_{x}$, the release of pepsine-gen, and the level of cGMP Especially, carbachol-stimulated increase of arginine to citrulline transformation, the amount of $NO_{x}$, cGMP level and the release of pepsinogen were partially reduced by the natural NOS inhibitor, $N^{G}$-monomethyl-L-arginine (MMA) and $N^{G}$, $N^{G}$-dimethyl-L-arginine (DMA). Furthermore, MMA- and DMA-induced decrease of pepsinogen secretion showed dose-dependent patters. Activation of NOS is one of the early events in receptor-mediated cascade of reactions in gastric chief cells and NO, not completely, but partially mediates gastric secretion. Agonist-stimulated pepsinogen secretion in chief cells has been considered to be mediated in adenosine 3',5'-cyclic monophosphate pathway and/or guanosine 3', 5'-cyclic monophosphate (cGMP) pathway. Taken together, the above results suggest that partial decrease of exocrine secretion following treatment of NOS inhibitor may result from the inactivation of NOS and subsequent guano- late cyclase, and NO/cGMP pathway may play a pivotal role in exocrine secretion.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.9-14
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• 2016

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the $Na^+-K^+$-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain ($3.0{\mu}mol/L$) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 ($3.0{\mu}mol/L$), an inhibitor for reverse mode of $Na^+-Ca^{2+}$ exchangers (NCX), but did not by L-type $Ca^{2+}$ channel blocker nifedipine ($1.0{\mu}mol/L$) or protein kinase A (PKA) selective inhibitor H-89 ($3.0{\mu}mol/L$). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline ($100.0{\mu}mol/L$), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP ($0.5{\mu}mol/L$) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 ($30{\mu}mol/L$), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

• Journal of Ginseng Research
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• v.44 no.2
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• pp.341-349
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• 2020

Background: The replicative senescence of human dermal fibroblasts (HDFs) is accompanied by growth arrest. In our previous study, the treatment of senescent HDFs with Rg3(S) lowered the intrinsic reactive oxygen species (ROS) levels and reversed cellular senescence by inducing peroxiredoxin-3, an antioxidant enzyme. However, the signaling pathways involved in Rg3(S)-induced senescence reversal in HDFs and the relatedness of the stereoisomer Rg3(R) in corresponding signaling pathways are not known yet. Methods: We performed senescence-associated β-galactosidase and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP) as well as the mitochondrial DNA copy number, nicotinamide adenine dinucleotide (NAD)⁺/1,4-dihydronicotinamide adenine dinucleotide (NADH) ratio, and NAD-dependent sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of phosphatidylinositol 3-kinase/Akt, 5' adenosine monophosphate-activated protein kinase (AMPK), and sirtuin 1/3, including cell cycle regulatory proteins, were examined by immunoblot analysis. Results: Ginsenoside Rg3(S) reversed the replicative senescence of HDFs by restoring the ATP level and NAD⁺/NADH ratio in downregulated senescent HDFs. Rg3(S) recovered directly the cellular levels of ROS and the NAD⁺/NADH ratio in young HDFs inactivated by rotenone. Rg3(S) mainly downregulated phosphatidylinositol 3-kinase/Akt through the inhibition of mTOR by cell cycle regulators like p53/p21 in senescent HDFs, whereas Rg3(R) did not alter the corresponding signaling pathways. Rg3(S)-activated sirtuin 3/PGC1α to stimulate mitochondrial biogenesis. Conclusion: Cellular molecular analysis suggests that Rg3(S) specifically reverses the replicative senescence of HDFs by modulating Akt-mTOR-sirtuin signaling to promote the biogenesis of mitochondria.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.6
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• pp.503-510
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• 2009

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high $K^+$ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High $K^+$ (50 mM) produced sustained tonic contraction, and ACh $(10\;{\mu}M)$ produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high $K^+$-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine $(1\;{\mu}M)$, inhibitor of voltage-dependent L-type calcium current $(VDCC_L)$, almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of $VDCC_L$.

• Journal of Korean Medicine for Obesity Research
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• v.20 no.2
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• pp.109-121
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• 2020

Objectives: Tanshinone I is a bioactive constituent in Salvia miltiorrhiza. At present, the anti-obesity effect and mechanism of tanshinone I are not fully understood. Here we investigated the effect of tanshinone I on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Methods: Lipid accumulation and triglyceride (TG) content in 3T3-L1 cells were determined by Oil Red O staining and AdipoRed assay, respectively. The expression and phosphorylation levels of adipogenic/lipogenic proteins in 3T3-L1 cells were evaluated by Western blotting. The messenger RNA (mRNA) expression levels of adipogenic/lipogenic markers and leptin in 3T3-L1 cells were measured by reverse transcription polymerase chain reaction (RT-PCR). Lipid accumulation in zebrafish was assessed by LipidGreen2 staining. Results: Tanshinone I at 5 μM largely blocked lipid accumulation and reduced TG content in differentiating 3T3-L1 cells. Furthermore, tanshinone I decreased the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells. In addition, tanshinone I increased the phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP)-activated protein kinase (AMPK) while decreased the intracellular adenosine triphosphate (ATP) content with no change in the phosphorylation and expression of liver kinase-B1 in differentiating 3T3-L1 cells. Importantly, tanshinone I also reduced the extent of lipid deposit formation in developing zebrafish. Conclusions: These findings demonstrate that tanshinone I has strong anti-adipogenic effects on 3T3-L1 cells and reduces adiposity in zebrafish, and these anti-adipogenic effect in 3T3-L1 cells are mediated through control of C/EBP-α, PPAR-γ, STAT-3, FAS, ACC, perilipin A, and AMPK.