• 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.

• 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+/.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.1
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• pp.47-53
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• 2001

Drinking excessive alcohol has been recognized as a risk factor for hypertension. However, the mechanism by which alcohol intake causes hypertension still remains elusive. We tested the hypothesis that ethanol itself acts as a stress factor on vasculature and indirectly modulates vascular contractility. After end of exposure to 1, 2.5 and 5% ethanol for 45 min, rat aortic strips were subjected to contractile responses, immunoblot for Hsp70 and the measurement of levels of myosin light chain phosphorylation. Exposure to 5% ethanol not only augmented contractions to KCl or phenylephrine, but also increased expression of Hsp70 and the levels of myosin light chain phosphorylation. There were no significant differences in contractions produced by $1\;{\mu}mol/L$ phorbol 12,13-dibutyrate, a protein kinase C activator, whether the tissues were exposed to 5% ethanol or not. This is the first report to show that even short exposure to ethanol has a delayed effect to increase vascular smooth muscle contractility through a modulation of thick filament regulation. It may be a mechanism by which ingestion of alcohol induces hypertension.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.1
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• pp.33-39
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• 2002

It has been suggested that $Ca^{2+}$ sensitization mechanisms might contribute to myogenic tone, however, specific mechanisms have not yet been fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced $Ca^{2+}$ sensitization in myogenic tone of the rabbit basilar vessel. Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 $Ca^{2+}$ signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ only in the presence of extracellular $Ca^{2+}$. Stretch evoked greater contraction than high $K^+$ at a given $[Ca^{2+}]_i.$ The stretch-induced increase in $[Ca^{2+}]_i$ and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent $Ca^{2+}$ channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing $[Ca^{2+}]_i.$ Immunoblotting using isoform-specific antibodies showed the presence of $PKC_{\alpha}$ and $PKC_{\varepsilon}$ in the rabbit basilar artery. $PKC_{\alpha},$ but not $PKC_{\varepsilon},$ and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of $PKC_{\alpha}$ and/or RhoA may be key mechanisms for the $Ca^{2+}$ sensitization associated with myogenic tone in basilar vessels.

• Food Science of Animal Resources
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• v.30 no.3
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• pp.385-391
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• 2010

This study was conducted to compare proteins expressed in M. longissimus from Hanwoo and Holstein steers immediately after slaughter. Two-dimensional electrophoresis (2DE)/LC-MS/MS analysis revealed that the total number of detectable protein spots from longissimus muscle tissues was slightly higher in Hanwoo ($575{\pm}65$) than Holstein ($534{\pm}13$) steers, but that these numbers were not statistically significant due to large variation between replicates. A total of twelve protein spots did not match between sample groups, eight of which were expressed in the Hanwoo sample and four that were expressed in the Holstein sample. The protein spots detected in the Hanwoo sample included smooth muscle and non-muscle myosin alkali light chain 6B isomers, ${\alpha}B$ crystallin isomers, hemoglobin ${\beta}$-A chains, slow myosin heavy chains, and slow skeletal muscle troponin T chains. Collectively, these proteins are a class of slow-twitch muscle fiber and mirror that Hanwoo muscle tissue sampled for the current study contained more slow-twitch muscle fibers than Holstein one. Conversely, proteins detected from the Holstein sample included ankyrin repeat domain 2 and creatin kinase isomers. Given that creatin kinase isomers are related to the fast-twitch muscle, these results likely indicate that Holstein muscle tissue sampled for the current study contained more fast-twitch muscle fibers than Hanwoo beef.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.2
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• pp.315-320
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• 2008

This study investigated the signaling mechanisms contributed to the vasodilatory effects of HMC05, a herbal prescription. HMC05 acted in an endothelium-independent manner. To elucidate the fundamental mechanisms of its vascular actions, we focused on the signaling molecules involved in actin-myosin filament regulation including 20 kDa myosin light chains (LC20), Rho-associated kinase (ROCK), PKC, JNK and extracellular signal-regulated protein kinase (ERK) in the endothelium-denuded thoracic aorta or isolated smooth muscle cells (SMCs). It lowered the phosphorylation level of LC20 and showed that ROCK, ERK, JNK and $PKC{\alpha}$ pathways played important roles in the effects, as confirmed by the observations with a specific inhibition or activation, and with the activity and the subcellular localization of these molecules. In particular, HMC05 dramatically inhibited the activity of ERK and the downstream signaling of ROCK. It also changed the subcellular localization of the phophorylated $PKC{\alpha}$ as well as the amount of phosphorylation. Taken together, these data indicate that the vascular relaxation effects of HMC05 are attributed to the regulation of these signaling mechanisms.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.171-177
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• 2011

Tonic smooth muscle exhibit the latch phenomenon: high force at low myosin regulatory light chains (MRLC) phosphorylation, shortening velocity (Vo), and energy consumption. However, the kinetics of MRLC phosphorylation and cellular activation in phasic smooth muscle are unknown. The present study was to determine whether $Ca^{2+}$-stimulated MRLC phosphorylation could suffice to explain the agonist- or high $K^+$-induced contraction in a fast, phasic smooth muscle. We measured myoplasmic [$Ca^{2+}$], MRLC phosphorylation, half-time after step-shortening (a measure of Vo) and contractile stress in rabbit urinary bladder strips. High $K^+$-induced contractions were phasic at both $22^{\circ}C$ and $37^{\circ}C$: myoplasmic [$Ca^{2+}$], MRLC phosphorylation, 1/half-time, and contractile stress increased transiently and then all decreased to intermediate values. Carbachol (CCh)-induced contractions exhibited latch at $37^{\circ}C$: stress was maintained at high levels despite decreasing myoplasmic [$Ca^{2+}$], MRLC phosphorylation, and 1/half-time. At $22^{\circ}C$ CCh induced sustained elevations in all parameters. 1/half-time depended on both myoplasmic [$Ca^{2+}$] and MRLC phosphorylation. The steady-state dependence of stress on MRLC phosphorylation was very steep at $37^{\circ}C$ in the CCh- or $K^+$-depolarized tissue and reduced temperature flattend the dependence of stress on MRLC phosphorylation compared to $37^{\circ}C$. These data suggest that phasic smooth muscle also exhibits latch behavior and latch is less prominent at lower temperature.