• 동의생리병리학회지
• /
• 제17권2호
• /
• pp.461-466
• /
• 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+/.

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

• 대한약리학회지
• /
• 제30권1호
• /
• pp.111-116
• /
• 1994

수용체작동약물과 GTP에 의한 수축단백질의 칼슘이온의 감수성의 증가에 대하여 $Ca^{2+}$/calmodulin-dependent protein kinase II의 역할을 ${\alpha}$-독으로 처리한 토끼장간막동맥에서 조사하였다. $0.3\;{\mu}M$의 칼슘이온은 마이오신의 인산화를 시간의존적으로 증가시켰고 5분 이후부터 평형에 도달하였다. 한편, $10\;{\mu}M$의 norepinephine과 $10\;{\mu}M$의 GTP는 칼슘이온 존재하에 칼슘이온 단독에 의한 것 보다 더 마이오신의 인산화를 증가시켰는데, 5분 후에 최대에 달하였고 그 후는 감소하기 시작하여 20분 후에는 칼슘이온 단독에 의한 마이오신 인산화 증가와 거의 차이가 없었다. $Ca^{2+}$/calmodulin-dependent protein kinase II 억제제인 KN-62를 전처치하여 norepinephrine과 GTP에 의한 마이오신 인산화 증가의 변화를 경시적으로 관찰하였다. $10\;{\mu}M$ KN-62는 1분에서 norepinephrine과 $10\;{\mu}M$ GTP에 의한 마이오신의 인산화의 증가를 억제하였다. 그러나 5분에서 관찰되는 norepinephrine과 GTP에 의한 마이오신 인산화의 증가의 최대치에는 영향이 없었고 그 이후에도 KN-62는 아무 영향을 끼치지 못하였다. 이상과 같은 결과로 볼때 norepinephrine과 GTP에 의하여 일어나는 평활근 수축단백의 칼슘이온의 감수성의 증가는 이미 알려진 바와 같이 마이오신 인산화의 증가에 기인하며 이 증가는 일과성임을 확인하였다. 이때 $Ca^{2+}$/calmodulin-dependent protein kinase II의 역할은 시간의존적으로 norepinephrine과 GTP의 자극 초기에 관여되는 것으로 생각되며 그 이후에는 관여가 없는 것으로 사료된다.

• 생명과학회지
• /
• 제25권5호
• /
• pp.585-593
• /
• 2015

Stress fiber (SF) 변화는 세포외부의 결합인자와 세포 수용체와 결합후 리모델링을 위해 액틴골격에 신호를 전달하며 일어난다. 이 연관은 결합장소에서 기계적 활동과 신호전달활동을 조절하는 다양한 스케폴드들과 신호 전달자에 의해 매게된다. Heterotrimeric transmembrane lymphotoxin α1β2 (LTα1β2)는 용해성 homotrimeric LT α를 포함하는 tumor necrosis factor (TNF) 계로 림프조직을 구성하는데 중요한 역할을 한다. LTα1β2와 LTβR의 결합은 fibroblastic reticular cell (FRC)에서 신호전달을 촉발한다. Agonistic anti-LTβR antibody 단독 혹은 LTα 그리고 TNFα의 조합으로 LTβR 자극은 세포의 액틴과 형태적 변화를 보았다. Agonistic anti-LTβR antibody의 FRC에서 작용을 통한 세포골격 재배열이 myosin과의 관련성을 확인하기위해 myosin light chain kinase (MLCK)의 저해제인 ML-7과 myosin light chains (MLC)와 myosin phosphatase target subunit 1 (MYPT1)의 인산화에 대한 효과를 확인하였다. MLCK 저해는 액틴 세포골격 재배열과 세포형태 변화를 유도하였다. 또한, MLC와 MYPT1인산화가 LTβR 자극에 의해 줄어드는 것을 확인하였다. DNA chip 분석은 myosin and actin 구성선분이 전사체 수준에서도 줄어드는 것을 보였다. 결론적으로 LTβR 자극은 FRC에서 SF변화는 myosin과 관련되어 있다는 것을 제시한다.

• Toxicological Research
• /
• 제25권4호
• /
• pp.181-188
• /
• 2009

Uncontrolled cell growth and increased cell proliferation are major features of cancer that are dependent on the stable structure and dynamics of the cytoskeleton. Since stable cytoskeleton structure and dynamics are partly regulated by myosin light chain kinase (MLCK), many current studies focused on MLCK inhibition as a chemotherapeutic target. As a potent and selective MLCK inhibitor, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazapine hydrochloride] is a promising candidate for an anticancer agent, which would induce apoptosis as well as prevents invasion and metastasis in certain types of cancer cells. This study assessed cytotoxic effects of ML-7 against HL-60 cells and therapeutic efficacy of ML-7 as a potential antileukemia agent. Trypan-blue exclusion assays showed dose- and time- dependent decreases in ML-7 treated HL-60 cells (p<0.05). Comet assays revealed a significant increase in DNA damage in HL-60 cells after treatment with $40{\mu}M$ ML-7 for 2h. Sub-G1 fractions, analyzed by flow cytometry increased in a dose-dependent manner, suggesting that ML-7 can induce apoptotic cell death in HL-60 cells. ML-7 was selectively cytotoxic towards HL-60 cells; not affecting normal human lymphocytes. That selective effect makes it a promising potential anti-leukemia agent. In addition, anticancer efficacy of ML-7 in combination with flavonoids (genistein or quercetin) or anticancer drugs (cisplatin or Ara-C) against HL-60 cells was assessed. Combination of ML-7 with flavonoids increased the anti-cancer effect of ML-7 to a greater extent than combination with the anticancer drugs. This implies that ML-7 in combination with flavonoids could increase the efficacy of anticancer treatment, while avoiding side effects cansed by conventional anticancer drug-containing combination chemotherapy.

• The Korean Journal of Physiology and Pharmacology
• /
• 제28권1호
• /
• pp.49-57
• /
• 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.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권14호
• /
• pp.5835-5842
• /
• 2015

Background: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. Materials and Methods: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). Results: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Conclusions: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

• Journal of Photoscience
• /
• 제4권2호
• /
• pp.35-40
• /
• 1997

The sensory transduction processes of blue light in guard cells have been suggested the involvement of Ca$^{2+}$/calmodulin-dependent myosin light chain kinase (MLCK) or MLCK-like proteins. The source of Ca$^{2+}$ required for the signal transduction process was investigated in guard cell protoplasts (GCPs). The GCPs showed the typical H$^+$ pumping activity by blue light (200 $\mu$mol m$^{-2}$ s$^{-1}$) and fusicoccin (10 $\mu$M) under background red light (600 $\mu$mol m$^{-2}$ s$^{-1}$). The blue light-dependent H$^+$ pumping was not significantly affected by the externally changed Ca$^{2+}$ concentrations. The addition of 1 mM Ca$^{2+}$ in the bathing medium ratherly inhibited the H$^+$ pumping. In contrast, the blue light-dependent H$^+$ pumping was inhibited by caffeine and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitor of C$^{2+}$-ATPase in endoplasmic reticulum (ER) without inhibiting the H $^+$ pump. The inhibition by caffeine and BHQ was fully reversible. The extent of inhibition by caffeine and BHQ was larger when they were added together than when added separately. The results suggest that Ca$^{2+}$ required for the blue light-dependent H$^+$ pumping may be released from the intracellular Ca$^{2+}$ stores, probably ER in guard cells.

• The Journal of Korean Physical Therapy
• /
• 제19권5호
• /
• pp.65-76
• /
• 2007

Purpose: It is generally accepted that smooth muscle contraction is triggered by intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) released from intracellular $Ca^{2+}$ stores such as sarcoplasmic teticulum (SR) and from the extracellular space. The increased $[Ca^{2+}]^i$ can phosphorylate the 20,000 dalton myosin light chain $(MLC_{20})$ by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the $[Ca^{2+}]_i$MACK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and others, play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of depletion of SR $Ca^{2+}$ in mouse gastric smooth muscle strips is not still clear. Methods: To investigate the rotes of $Ca^{2+}$ influx and SR $Ca^{2+}$ release channel on gastric motility, isometric contraction and $[Ca^{2+}]_i$ were examined in mouse gastric smooth muscle strips. Results: High KCl, ryanodine, an activator of $Ca^{2+-}$induced $Ca^{2+}$ release channel, and cyclopiazonic acid (CPA), an inhibitor of SR $Ca^{2+-}$ATPase evoked a sustained increase in muscle contraction and $[Ca^{2+}]_i$. These increases induced by high KCl, ryanodine, and CPA were partially blocked by application of verapamil ($10{\mu}M$), a L-type $Ca^{2+}$ channel inhibitor. Additionally, in $Ca^{2+-}$free solution (1 mM EGTA), ryanodine and CPA had no effect contraction and $[Ca^{2+}]_i$ in fundic muscle strips. Conclusion: These results that extracellular $Ca^{2+}$ influx and depletion of SR trigger $Ca^{2+}$ influx through verapamil-sensitive $Ca^{2+}$ channel, and extracellular and SR $Ca^{2+}$ store may functionally involve in the subcellular $Ca^{2+}$ mobilization in mouse gastric muscle.

• Journal of Yeungnam Medical Science
• /
• 제38권1호
• /
• pp.27-33
• /
• 2021

The gut is a complex organ that has played an important role in digestion, absorption, endocrine functions, and immunity. The gut mucosal barriers consist of the immunologic barrier and nonimmunologic barrier. During critical illnesses, the gut is susceptible to injury due to the induction of intestinal hyperpermeability. Gut hyperpermeability and barrier dysfunction may lead to systemic inflammatory response syndrome. Additionally, gut microbiota are altered during critical illnesses. The etiology of such microbiome alterations in critical illnesses is multifactorial. The interaction or systemic host defense modulation between distant organs and the gut microbiome is increasingly studied in disease research. No treatment modality exists to significantly enhance the gut epithelial integrity, permeability, or mucus layer in critically ill patients. However, multiple helpful approaches including clinical and preclinical strategies exist. Enteral nutrition is associated with an increased mucosal barrier in animal and human studies. The trophic effects of enteral nutrition might help to maintain the intestinal physiology, prevent atrophy of gut villi, reduce intestinal permeability, and protect against ischemia-reperfusion injury. The microbiome approach such as the use of probiotics, fecal microbial transplantation, and selective decontamination of the digestive tract has been suggested. However, its evidence does not have a high quality. To promote rapid hypertrophy of the small bowel, various factors have been reported, including the epidermal growth factor, membrane permeant inhibitor of myosin light chain kinase, mucus surrogate, pharmacologic vagus nerve agonist, immune-enhancing diet, and glucagon-like peptide-2 as preclinical strategies. However, the evidence remains unclear.