• The Korean Journal of Physiology and Pharmacology
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• v.26 no.3
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• pp.175-182
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• 2022

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

• 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.

• Biomolecules & Therapeutics
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• v.26 no.4
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• pp.374-379
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• 2018

In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to determine the underlying mechanism of relaxation. Isometric contractions of denuded aortic muscles from male rats were recorded, and the data were combined with those obtained in western blot analysis. Pelargonidin significantly inhibited fluoride-, thromboxane A2-, and phorbol ester-induced vascular contractions, regardless of the presence or absence of endothelium, suggesting a direct effect of the compound on vascular smooth muscles via a different pathway. Pelargonidin significantly inhibited the fluoride-dependent increase in the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation at Thr-855 and the phorbol 12,13-dibutyrate-dependent increase in the level of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at Thr202/Tyr204, suggesting the inhibition of Rho-kinase and mitogen-activated protein kinase kinase (MEK) activities and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxation effect of pelargonidin on agonist-dependent vascular contractions includes inhibition of Rho-kinase and MEK activities, independent of the endothelial function.

• Biomolecules & Therapeutics
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• v.26 no.2
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• pp.139-145
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• 2018

The present study was undertaken to investigate the influence of hypothermia on endothelium-independent vascular smooth muscle contractility and to determine the mechanism underlying the relaxation. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Hypothermia significantly inhibited fluoride-, thromboxane $A_{2-}$, phenylephrine-, and phorbol ester-induced vascular contractions regardless of endothelial nitric oxide synthesis, suggesting that another pathway had a direct effect on vascular smooth muscle. Hypothermia significantly inhibited the fluoride-induced increase in pMYPT1 level and phorbol ester-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxing effect of moderate hypothermia on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activities.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.51-57
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• 2010

It was hypothesized that NaF induces calcium sensitization in $Ca^{2+}$-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with $\beta$-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to $Ca^{2+}$ (decreased $EC_{50}$ and increased $E_{max}$). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in $Ca^{2+}$-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and $GTP{\gamma}S$-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a $Ca^{2+}$ dependent manner in $\beta$-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

• 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.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+/.

• Molecules and Cells
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• v.42 no.6
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• pp.470-479
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• 2019

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5'-diphosphate-${\beta}$-S and pre-treatment with $Ca^{2+}$-free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate ($IP_3$), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the $IP_3$ receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, $IP_3$-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular $Ca^{2+}$ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.

• BMB Reports
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• v.44 no.6
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• pp.415-420
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• 2011

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane $A_2$ agonist, via augmentation of the phosphorylation of MLC20$MLC_{20}$, MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.31-36
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• 2013

The present study was undertaken to investigate the influence of eupatilin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Eupatilin more significantly relaxed fluoride-induced vascular contraction than thromboxane $A_2$ or phorbol ester-induced contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, eupatilin significantly inhibited fluoride-induced increases in pMYPT1 levels. On the other hand, it didn't significantly inhibit phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving the primarily inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1. This study provides evidence regarding the mechanism underlying the relaxation effect of eupatilin on agonist-induced vascular contraction regardless of endothelial function.