• Journal of Applied Biological Chemistry
• /
• v.62 no.4
• /
• pp.425-431
• /
• 2019

Platelet activation is essential for hemostatic process on blood vessel damage. However, excessive platelet activation can cause some cardiovascular diseases including atherosclerosis, thrombosis, and myocardial infarction. Scoparone is commonly encountered in the roots of genus Artemisia or Scopolia, and has been studied for its potential pharmacological properties including immunosuppression and vasorelaxation, but antiplatelet effects of scoparone have not been reported yet. We investigated the effect of scoparone on human platelet activation prompted by an analogue of thromboxane A₂, U46619. As the results, scoparone dose-dependently increased cyclic adenosine monophosphate (cAMP) levels as well as cyclic guanosine monophosphate (cGMP) levels, both being aggregation-inhibiting molecules. In addition, scoparone strongly phosphorylated inositol 1, 4, 5-triphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP), substrates of cAMP dependent kinase and cGMP dependent kinase. Phosphorylation of IP₃R by scoparone resulted in inhibition of Ca²⁺ mobilization in calcium channels in a dense tubular system, and phosphorylation of VASP by scoparone led to an inability of fibrinogen being able to bind to αIIb/β3. Finally, scoparone inhibited thrombin-induced fibrin clotting, thereby reducing thrombus formation. Therefore, we suggest that scoparone has a strong antiplatelet effect and is highly probable to prevent platelet-derived vascular disease.

• Biomedical Science Letters
• /
• v.16 no.4
• /
• pp.377-380
• /
• 2010

In this study, we investigated the effect of egg yolk lipids (EYL) on collagen ($10\;{\mu}g/ml$)-stimulated platelet aggregation in vivo. Dietary EYL significantly inhibited collagen-induced platelet aggregation, in addition, increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), intracellular $Ca^{2+}$-antagonist as aggregation-inhibiting molecules, in collagen-stimulated platelets. These results suggest that EYL inhibits the collagen-induced platelet aggregation by up-regulating the cAMP and cGMP production. On the other hands, prothrombin time (PT) on extrinsic pathway of blood coagulation was potently prolonged by dietary EYL in vivo. These findings suggest that EYL prolongs the internal time between the conversion of fibrinogen to fibrin. Accordingly, our data demonstrate that EYL may be a crucial tool for a negative regulator during platelet activation and blood coagulation on thrombotic diseases.

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

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

• The Korean Journal of Physiology
• /
• v.26 no.1
• /
• pp.15-25
• /
• 1992

In order to elucidate the properties of the background current whole cell patch clamp studies were performed in rabbit ventricular cells. Ramp pulses of ${\pm}80\;mV$ from holding potential of 40 mV(or 20 mV) at the speed of 0.8 V/sec were given every 30 sec(or 10 sec) and current-voltage diagrams(I-V curve) were obtained. For the activation of the background current isoprenaline, adenosine 3',5'-cyclic monophosphate(dBcAMP), guanosine 3',5'-cyclic monophosphate(cGMP), and $N^6$-2'-o-dibutyryladenosine 3',5'-cyclic monophosphate(dBcAMP) were applied after all known current systems were blocked with 2mM Ba, 1 mM Cd ,5 mM Ni, 10 ${\mu}M$ diltiazem, 10 ${\mu}m$ ouabain, and 20 mM tetraethylammonium(TEA). The conductance of background current in control was $0.65{\pm}0.69$ nS at 0 mV, its I-V curves was almost linear and reversed near 50 mV. When there was no taurine in pipette solution, isoprenaline hardly activated the background current but when taurine existed in pipette solution, isoprenaline activated the larger background current. Cyclic AMP or cyclic GMP alone had little effect on the activation of the background current, while cGMP potentiated cGMP effect. When the background current was activated with cGMP and cAMP, isoprenaline could not further increased the background current. The background current activated by isoprenaline depended on extracellular $Cl^-$ concentration and its reversal potential was shifted according to chloride equilibrium potential. The change of extracellular $Na+$ concentration had little effect on reversal potential of the background current activated by isoprenaline.

• The Korean Journal of Physiology and Pharmacology
• /
• v.16 no.3
• /
• pp.211-217
• /
• 2012

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents ($I_{cap}$). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on $I_{cap}$. Membrane-permeable cGMP analogs, 8-bromoguanosine 3', 5'-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on $I_{cap}$. The PKG inhibitor KT5823 prevented the inhibition of $I_{cap}$ by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.2
• /
• pp.95-100
• /
• 2004

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium $(K_{ATP})$ channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that $K_{ATP}$ channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide $(100\;{\mu}M)$, SNAP $(300\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) $(100\;{\mu}M)$ followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide $(50\;{\mu}M)$, 5-hydroxydecanoate (5-HD) $(100\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) $(100\;{\mu}M)$. These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of $K_{ATP}$ channels in rat ventricular myocytes.

• Biomedical Science Letters
• /
• v.20 no.2
• /
• pp.70-76
• /
• 2014

In this study, we investigated the effect of DMSO, a highly dipolar organic liquid, in collagen ($5{\mu}g/ml$)-stimulated platelet aggregation. DMSO inhibited platelet aggregation at 0.5% by inhibiting production of thromboxane $A_2$ ($TXA_2$) which was associated with blocking cyclooxygenase (COX)-1 activity and $TXA_2$ synthase. In addition, DMSO significantly increased the formation of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). On the other hand, DMSO (0.1~0.5% concentration) did not affect the LDH release which indicates the cytotoxicity. Based on these results, DMSO has anti-platelet effect by regulation of several platelet signaling pathways, therefore we suggest that DMSO could be a novel strategy on many thrombotic disorders.

• Korean Journal of Clinical Laboratory Science
• /
• v.51 no.1
• /
• pp.34-41
• /
• 2019

Platelet aggregation is essential for the formation of a hemostatic plug in the case of blood vessel damage. On the other hand, excessive platelet aggregation may cause cardiovascular disorders, such as thrombosis, atherosclerosis, and myocardial infarction. Scopoletin, which found in the root of plants in the genus Scopolia or Artemisia, has anti-coagulation and anti-malaria effects. This study examined the effects of scopoletin on human platelet aggregation induced by collagen. Scopoletin had anti-platelet effects via the down-regulation of thromboxane $A_2$ ($TXA_2$) production and intracellular $Ca^{2+}$ mobilization ($[Ca^{2+}]_i$), which are aggregation-inducing molecules produced in activated platelets. On the other hand, scopoletin increased both the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels, which are known as intracellular $Ca^{2+}$-antagonists and aggregation-inhibiting molecules. In particular, scopoletin increased the potently cAMP level more than cGMP, which led to suppressed fibrinogen binding to ${\alpha}IIb/{\beta}_3$ in collagen-induced human platelet aggregation. In addition, scopoletin inhibited collagen-elevated adenosine triphosphate (ATP) release in a dose-dependent manner. The results suggest that aggregation amplification through granule secretion is inhibited by scopoletin. Therefore, scopoletin has potent anti-platelet effects and may have potential for the prevention of platelet-derived vascular diseases.

• The Korean Journal of Physiology and Pharmacology
• /
• v.2 no.6
• /
• pp.733-742
• /
• 1998

Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.