• 대한약리학회:학술대회논문집
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• 2006.10a
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• pp.113.1-113.1
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• 2006

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.5
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• pp.1060-1065
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• 2002

LC20 phosphorylation and PKC α play an important role in modulation of contractile activity of smooth muscle. Besides, myosin phosphatase is also related with smooth muscle contraction in signaling pathways. We previously demonstrated that Crataegi Fructus inhibited phenylephrine-induced contraction and which might be implicated in nitrite formation(Son et al., 2002). In this study, we investigated the effects of butanol fraction of Crataegi Fructus(BFFC) on the localization of α-protein kinease C(PKC α) and myosin phosphatase subnits(MPs) in freshly isolated single ferret potal vein cells, and phosphorylation of LC20 during phenylephrine stimulation. In PKC α and MPs localization, BFFC blocked its translocation from the cytosol to the cell membrane by treatment of phenylephrine. BFFC have also dephosphorylated LC20 phosphorylation by phenylephrine stimulation under basal level, but no significant. These results indicate that the relaxation effect of BFFC is associated with inhibition of PKC α activation and MPs dissociation, and thus myosin phosphatase activity may be increased.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.2
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• pp.461-466
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• 2003

The primary mechanism of smooth muscle contraction is phosphorylation of the 20 kDa myosin light chains(LC20) by a myosin light chain kinase(MLCK). Relaxation, then, is generally the result of dephosphorylation of LC20 by myosin phosphatase(MP). Changes in MP activity is one of the important mechanisms in the regulation of Ca2+-sensitivity. Inhibition of MP activity is linked to an increase in phosphorylated myosin light chain(MLC) without an increase in [Ca/sup 2+/]i-levels. It is now generally accepted that Rho-kinase phosphorylates 130 kDa regulatory and myosin binding subunits(M130, MYPT) of MP, which results in an inhibition of MP activity. In addition Rho-kinase can also directly phosphorylate MLC. In the present study, LC20 phosphorylation and MP subunits translocation to the cell membrane were investigated in freshly isolated ferret portal vein smooth muscle single cells treated with PGF2α. We also examined the effect of Y27632(10-5mol/L), Rho-kinase inhibitor, in the MP subunits localization to compare with butanol fraction of Fructus Crataegi in its effect. Butanol fraction of Fructus Crataegi(BFFC; 1㎎/㎖) was more effective in PGF2α induced contraction than those of phenylephrine in its vasodilation effect. It significantly(P<0.05) dephosphorylated the LC20 at time indicated. In addition, the dissociation of subunits are inhibited by BFCF treatment. The results indicate that, in the smooth muscle cells, the relaxation effect of BFFC is associated with increase of MP activity based on inhibition of dissociation of the catalytic and targeting subunits of the phosphatase, and thus decrease the sensitivity of LC20 phosphorylation for Ca/sup 2+/.

• Biomolecules & Therapeutics
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• v.31 no.2
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• pp.193-199
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• 2023

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.

• Biomolecules & Therapeutics
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• v.30 no.2
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• pp.145-150
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• 2022

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

• Biomolecules & Therapeutics
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• v.32 no.3
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• pp.361-367
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• 2024

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.

• Journal of Life Science
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• v.25 no.5
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• pp.585-593
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• 2015

Stress fiber (SF) alteration is mediated by cellular receptors, which, upon interaction with the extracellular counterpart, signal to the actin cytoskeleton for remodeling. This association is mediated by a variety of scaffold and signaling factors, which control the mechanical and signaling activities of the interaction site. The heterotrimeric transmembrane lymphotoxin α1β2 (LTα1β2), a member of the tumor necrosis factor (TNF) family of cytokines, including soluble homotrimeric lymphotoxin (LT α), plays an important role in lymphoid tissue architecture. Ligation between LTα1β2 and the lymphotoxin β receptor (LTβR) activates signal-cascade in fibroblastic reticular cells (FRCs). We found LTβR stimulation using an agonistic anti-LTβR antibody alone or combined with LTα or TNFα induced changes in the actin and plasticity of cells. To clarify the involvement of myosin underlying the alteration, we analyzed the effect of myosin light chain kinase (MLCK) with an MLCK inhibitor (ML7), the phosphorylation level of myosin light chains (MLC), and the level of phospho-myosin phosphatase target subunit 1 (MYPT1) after treatment with an agonistic anti-LTβR antibody for cytoskeleton reorganization in FRCs. The inhibition of MLCK activity induced changes in the actin cytoskeleton organization and cell morphology in FRC. In addition, we showed the phosphorylation of MLC and MYPT1 was reduced by LTβR stimulation in cells. A DNA chip revealed the LTβR stimulation of FRC down-regulated transcripts of myosin and actin components. Collectively, these results suggest LTβR stimulation is linked to myosin regarding SF alteration in FRC.

• Biomolecules & Therapeutics
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• v.16 no.3
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• pp.249-254
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• 2008

The present study was undertaken to determine whether hypoxia influences on the agonist-induced vascular smooth muscle contraction and, if so, to investigate the related mechanism. The measurement of isometric contractions using a computerized data acquisition system was combined with molecular experiments. Hypoxia significantly inhibited fluoride-induced contraction regardless of endothelial function, but there was no relaxation on thromboxane $A_2$ mimetic U-46619-induced contraction suggesting that other pathway such as $Ca^{2+}$ entry or thin filament regulation was not affected. In addition, hypoxia significantly decreased fluoride-induced increase of phospho-myosin-targeting subunit of myosin light chain phosphatase (pMYPT1). Interestingly, hypoxia didn't inhibit significantly phenylephrine-induced contraction suggesting that myosin light chain kinase (MLCK) activity or thin filament regulation is less important on the hypoxia-induced vasorelaxation in the denuded muscle than Rho-kinase activity. In conclusion, this study provides the evidence and possible related mechanism concerning the vasodilation effect of hypoxia on the agonist-specific contraction in rat aortic rings regardless of endothelial function.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.3
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• pp.201-207
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• 2009

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 $({\mu}M$) or vehicle. We determined the phosphorylation level of the myosin light chain ($MLC_{20}$), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of $MLC_{20}$ phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619 or NaF-induced phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, the downstream effectors of Rho-kinase. In regards to the $Ca^{2+}$-free solution, flavone inhibited the phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, as well as vascular contractions induced by U 46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.1
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• pp.49-57
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• 2024

While arterial tone is generally determined by the phosphorylation of Ser¹⁹ in myosin light chain (p-MLC2), Thr¹⁸/Ser¹⁹ diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-β and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.