• Journal of Physiology & Pathology in Korean Medicine
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• 제17권2호
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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+/.

• The Korean Journal of Pharmacology
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• 제30권1호
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• pp.111-116
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• 1994

The role of $Ca^{2+}$/calmodulin-dependent protein kinase II in the increase of myofilament $Ca^{2+}$ sensitivity by agonist and GTP was investigated in rabbit mesenteric ${\alpha}-toxin$ permeabilized artery. $0.3{\mu}M\;Ca^{2+}$ increased myosin light chain phosphorylations monotonically. $10\;{\mu}M$ norepinephrine and $10\;{\mu}M$ GTP potentiated increase of myosin light chain phosphorylations by $0.3{\mu}M\;Ca^{2+}$, which reaches a peak at 5 min and gradually declines to the $Ca^{2+}$ alone level at 20 min. At the early phase (1 min), $10\;{\mu}M$ KN 62, the inhibitor of $Ca^{2+}$/calmodulin-dependent protein kinase II , decreased myosin light chain phosphorylation levels by $10\;{\mu}M$ norepinephrine and $10\;{\mu}M$ GTP in the presence of $0.3{\mu}M\;Ca^{2+}.\;However\;10\;{\mu}M$ KN-62 did not affect the myosin light chain phosphorylations by $10\;{\mu}M$ norepinephrine and $10\;{\mu}M$ GTP in the presence of $0.3{\mu}M\;Ca^{2+}$ at the peak (5 min) and plateau phases (20 min). From these results, the role of $Ca^{2+}$/calmodulin-dependent protein kinase II may be different depending on time, which may play a role in increase of myofilamint $Ca^{2+}$ sensitivity by norepinephrine and GTP resulting from increase of myosin light chain phosphorylations at the early phase. However, at plateau phase, $Ca^{2+}$/calmodulin-dependent protein kinase II may not be involved in the increase of myofilament $Ca^{2+}$ sensitivity by norepinephrine and GTP in rabbit mesenteric ${\alpha}-toxin$ permeabilized artery.

• The Korean Journal of Physiology and Pharmacology
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• 제21권6호
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• pp.609-616
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• 2017

Ardipusilloside-I is a natural triterpenoid saponin, which was isolated from Ardisia pusilla A. DC. The aim of the study was to evaluate the stimulation of ardipusilloside-I on gastrointestinal motility in vitro and in vivo. The experiment of smooth muscle contraction directly monitored the contractions of the isolated jejunal segment (IJS) in different contractile states, and the effects of ardipusilloside-I on myosin were measured in the presence of $Ca^{2+}$-calmodulin using the activities of 20 kDa myosin light chain ($MLC_{20}$) phosphorylation and myosin $Mg^{2+}$-ATPase. The effects of ardipusilloside-I on gastro emptying and intestinal transit in constipation-predominant rats were observed, and the MLCK expression in jejuna of constipated rats was determined by western blot. The results showed that, ardipusilloside-I increased the contractility of IJS in a dose-dependent manner and reversed the low contractile state (LCS) of IJS induced by low $Ca^{2+}$, adrenaline, and atropine respectively. There were synergistic effects on contractivity of IJS between ardipusilloside-I and ACh, high $Ca^{2+}$, and histamine, respectively. Ardipusilloside-I could stimulate the phosphorylation of $MLC_{20}$ and $Mg^{2+}$-ATPase activities of $Ca^{2+}$- dependent phosphorylated myosin. Ardipusilloside-I also stimulated the gastric emptying and intestinal transit in normal and constipated rats in vivo, respectively, and increased the MLCK expression in the jejuna of constipation-predominant rats. Briefly, the findings demonstrated that ardipusilloside-I could effectively excite gastrointestinal motility in vitro and in vivo.

• Biomolecules & Therapeutics
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• 제16권3호
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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.

• 대한약리학회:학술대회논문집
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• 대한약리학회 2006년도 The 6th Congress of the Federation of Asian and Oceanian Physiological Societies
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• pp.96.2-96.2
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• 2006

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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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.

• Proceedings of the Korean Biophysical Society Conference
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.24-24
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• 2001

The present study was undertaken to determine whether p38 mitogen-activated protein kinase participates in the regulation of vascular smooth muscle contraction by endothelin-I (ET-1) in rat thoracic aorta. ET-1 induced a sustained contraction. In contrast, both the intracellular Ca$\^$2＋/ and myosin light chain (MLC) phosphorylations were not sustained.(omitted)

• Journal of Life Science
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• 제25권5호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제26권6호
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• pp.479-499
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• 2022

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7687-7692
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• 2014

Aim: To observe the effects of a novel all-trans retinoid acid (ATRA) derivative, N-(3-trifluoromethyl-phenyl)-retinamide (ATPR), on lung adenocarcinoma A549 cells and to explore the potential mechanism of ATPR inhibiting of A549 cell migration. Materials and Methods: The cytotoxicity of ATRA and ATPR on A549 cells was assessed using MTT assay. Wound healing assays were used to analyze the influences of ATRA, ATPR, ML-7 (a highly selective inhibitor of myosin light chain kinase (MLCK)), PMA (an activator of MAPKs) and PD98059 (a selective inhibitor of ERK1/2) on the migration of A549 cells. Expression of MLCK and phosphorylation of myosin light chain (MLC) were assessed by Western blotting. Results: ATRA and ATPR inhibited the proliferation of A549 cells in a dose- and time-dependent manner, and the effect of ATPR was much more remarkable compared with ATRA. Relative migration rate and migration distance of A549 cells both decreased significantly after treatment with ATPR or ML-7. The effect on cell migration of PD98059 combining ATPR treatment was more notable than that of ATPR alone. Moreover, compared with control groups, the expression levels of MLCK and phosphorylated MLC in A549 cells were both clearly reduced in ATRA and ATPR groups. Conclusions: ATPR could suppress the migration and invasion of A549 cells, and the mechanism might be concerned with down-regulating the expression of MLCK in the ERK-MAPK signaling pathway, pointing to therapeutic prospects in lung cancer.