scholarly journals α1-Adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle

1997 ◽  
Vol 327 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Mitsuo MITA ◽  
P. Michael WALSH
Keyword(s):  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Contractile Response ◽  
Bathing Medium ◽  
Adrenoceptor Agonist ◽  
Phosphoamino Acid Analysis ◽  
Phosphopeptide Mapping ◽  
Rat Tail

The mechanism of α1-adrenoceptor-mediated contraction was investigated in helical strips of the rat-tail artery. Muscle strips with the endothelium removed contracted in response to the α1-adrenoceptor agonist cirazoline, with half-maximal contraction at 0.23 μM. The contractile response to a submaximal concentration of cirazoline (0.3 μM) was biphasic, with a rapid phasic component peaking at approx. 30 s, followed by sustained tonic contraction. Phosphorylation of the 20 kDa light chain of myosin (LC20) in response to 0.3 μM cirazoline was also biphasic and closely matched the time-course of contraction. Resting LC20 phosphorylation levels were 0.22±0.06 mol of Pi/mol of LC20 (n = 3) and reached a maximum of 0.58±0.08 mol of Pi/mol of LC20 (n = 3). Phosphopeptide mapping and phosphoamino acid analysis revealed that LC20 phosphorylation occurred exclusively at serine-19. The sustained phase of contraction was eliminated by removal of extracellular Ca2+ and the phasic response was eliminated by depletion of endogenous Ca2+ stores. Both phases of the contractile response were restored by re-addition of Ca2+ to the bathing medium. LC20 phosphorylation and both phases of the contractile response to 0.3 μM cirazoline were inhibited by the myosin light-chain kinase inhibitor ML-9 (30 μM). Resting LC20 phosphorylation, however, was unaffected by ML-9. Finally, both phasic and tonic responses to 0.3 μM cirazoline were partially inhibited by chloroethylclonidine (50 μM), suggesting the involvement of both α1A and α1B adrenoceptors in these contractile responses.

scholarly journals A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

2008 ◽  
Vol 19 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Yasuhiko Koga ◽  
Mitsuo Ikebe
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Regulatory Mechanism ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Critical Role ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Dependent Cell ◽  
Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

scholarly journals Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

2009 ◽  
Vol 297 (6) ◽  
pp. H2242-H2252 ◽  
Author(s):  
Ravi Goyal ◽  
Ashwani Mittal ◽  
Nina Chu ◽  
Lijun Shi ◽  
Lubo Zhang ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Cerebral Arteries ◽  
Age Groups ◽  
Rho Kinase ◽  
Immunoblot Analysis ◽  
Mek Inhibitor ◽  
Inhibitory Protein ◽  
Regulatory Myosin Light Chain

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

scholarly journals H2O2 mediates Ca2+- and MLC20phosphorylation-independent contraction in intact and permeabilized vascular muscle

2000 ◽  
Vol 279 (3) ◽  
pp. H1185-H1193 ◽  
Author(s):  
Nancy J. Pelaez ◽  
Tracey R. Braun ◽  
Richard J. Paul ◽  
Richard A. Meiss ◽  
C. Subah Packer
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Mesenteric Arteries ◽  
Regulatory Myosin Light Chain ◽  
Pulmonary Arterial ◽  
Dose Dependent ◽  
Arteries And Veins ◽  
Permeabilized Muscle

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H2O2. Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H2O2. A second purpose was to determine whether H2O2-induced contraction is calcium independent. The addition of H2O2 to calcium-depleted (using the Ca2+ ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H2O2 even in zero Ca2+. The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC20) phosphorylation plays a role in H2O2-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H2O2-induced contractions, and the relative amounts of phosphorylated MLC20 were measured. H2O2 caused dose-dependent contractions that were independent of MLC20 phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H2O2 contractile response. In conclusion, H2O2 induces Ca2+- and MLC20 phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.

scholarly journals Zinc exocytosis is sensitive to myosin light chain kinase inhibition in mouse and human eggs

2020 ◽  
Vol 26 (4) ◽  
pp. 228-239 ◽  
Author(s):  
Hoi Chang Lee ◽  
Maxwell E Edmonds ◽  
Francesca E Duncan ◽  
Thomas V O’Halloran ◽  
Teresa K Woodruff
Keyword(s):  
Embryo Development ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Early Embryo ◽  
Egg Activation ◽  
Cortical Granules ◽  
Preimplantation Embryo Development ◽  
Egg Maturation

Abstract Zinc dynamics are essential for oocyte meiotic maturation, egg activation, and preimplantation embryo development. During fertilisation and egg activation, the egg releases billions of zinc atoms (Zn2+) in an exocytotic event termed the ‘zinc spark’. We hypothesised that this zinc transport and exocytosis is dependent upon the intracellular trafficking of cortical granules (CG) which requires myosin-actin-dependent motors. Treatment of mature mouse and human eggs with ML-7, a myosin light chain kinase inhibitor (MLCK), resulted in an 80% reduction in zinc spark intensity compared to untreated controls when activated with ionomycin. Moreover, CG migration towards the plasma membrane was significantly decreased in ML-7-treated eggs compared with controls when activated parthenogenetically with ionomycin. In sperm-induced fertilisation via intracytoplasmic sperm injection (ICSI), ML-7-treated mouse eggs exhibited decreased labile zinc intensity and cortical CG staining. Collectively, these data demonstrate that ML-7 treatment impairs zinc release from both murine and human eggs after activation, demonstrating that zinc exocytosis requires myosin light chain kinase activity. Further, these results provide additional support that zinc is likely stored and released from CGs. These data underscore the importance of intracellular zinc trafficking as a crucial component of egg maturation necessary for egg activation and early embryo development.

Myosin dephosphorylation during rapid relaxation of hog carotid artery smooth muscle

1989 ◽  
Vol 256 (2) ◽  
pp. C315-C321 ◽  
Author(s):  
S. P. Driska ◽  
P. G. Stein ◽  
R. Porter
Keyword(s):  
Smooth Muscle ◽  
Carotid Artery ◽  
Rate Constant ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Force Development ◽  
Relaxation Phase ◽  
Rhythmic Contractions ◽  
Low Levels

Changes in myosin light chain phosphorylation were measured during histamine-induced rhythmic contractions of hog carotid artery smooth muscle strips. Histamine made the muscle strips contract spontaneously every 1-5 min, and this allowed measurement of the time course of phosphorylation in relation to force development under conditions where diffusion of the agonist through tissue would not complicate the interpretation of the data. In the absence of histamine, phosphorylation was low [0.12 +/- 0.04 mol P/mol of the 20,000-Da light chain (LC 20)]. Phosphorylation was slightly (but not significantly) higher in the presence of 10 microM histamine in the relaxed state between contractions (0.20 +/- 0.03 mol P/mol LC 20). In muscle strips frozen during force development, when force had reached half of its peak value, phosphorylation was 0.38 +/- 0.06 mol P/mol LC 20. The highest levels of phosphorylation (0.49 +/- 0.04 mol P/mol LC 20) were found in strips frozen at the peak of the rhythmic contractions. Strips frozen when force had declined to half of the peak force showed low levels of phosphorylation (0.17 +/- 0.07 mol P/mol LC 20), indicating that the myosin light chain phosphatase activity was quite high. Mathematical modeling of the kinase and phosphatase reactions suggested that the apparent first-order phosphatase rate constant was at least 0.08 s-1 under these conditions. To obtain a better estimate of this rate constant, a second series of phosphorylation measurements were made early in the relaxation phase of the rhythmic contractions. The highest phosphatase rate constant obtained from these measurements was 0.23 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of vascular smooth muscle tone

1994 ◽  
Vol 72 (8) ◽  
pp. 919-936 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Muscle Tone ◽  
Myosin Phosphorylation ◽  
Smooth Muscle Tone ◽  
Vascular Smooth Muscle Tone

Vascular smooth muscle tone is regulated primarily by the sarcoplasmic free Ca2+ concentration, which determines the level of myosin phosphorylation. Stimulation of the muscle results in an increase in free [Ca2+], whereupon Ca2+ binds to calmodulin, inducing a conformational change enabling calmodulin to interact with and activate myosin light chain kinase. The active Ca2+∙calmodulin∙myosin light chain kinase complex catalyses the phosphorylation of serine-19 of the two 20-kDa light chains of myosin; this triggers cross-bridge cycling and the development of force. Relaxation follows restoration of free [Ca2+] to the resting level, whereupon calmodulin dissociates from myosin light chain kinase, which is thereby inactivated, and myosin is dephosphorylated by myosin light chain phosphatase and remains detached from actin. Overwhelming evidence now exists in favour of the central role of myosin phosphorylation–dephosphorylation in smooth muscle contraction–relaxation. However, considerable evidence supports the existence of additional, secondary mechanisms that can modulate the contractile state of smooth muscle either by altering the Ca2+ sensitivity of the contractile response or otherwise modulating one of the molecular events occurring downstream of the Ca2+ signal, e.g., the interaction of phosphorylated myosin heads with actin. The interplay of several regulatory elements confers on the contractile response of vascular smooth muscle the high degree of flexibility and adaptability required for the effective regulation of blood pressure.Key words: calcium, myosin, protein kinases, protein phosphatases, signal transduction, regulation of contraction, caldesmon, calponin.

Myosin Light Chain Kinase Inhibitor Inhibits Dextran Sulfate Sodium-Induced Colitis in Mice

2012 ◽  
Vol 58 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Xiaochang Liu ◽  
Jianming Xu ◽  
Qiao Mei ◽  
Liang Han ◽  
Jian Huang
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dextran Sulfate ◽  
Dextran Sulfate Sodium ◽  
Kinase Inhibitor

Contrasting inotropic responses to α1-adrenergic receptor stimulation in left versus right ventricular myocardium

2006 ◽  
Vol 291 (4) ◽  
pp. H2013-H2017 ◽  
Author(s):  
Guan-Ying Wang ◽  
Diana T. McCloskey ◽  
Sally Turcato ◽  
Philip M. Swigart ◽  
Paul C. Simpson ◽  
...  
Keyword(s):  
Light Chain ◽  
Adrenergic Receptor ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Ventricular Myocardium ◽  
Differential Activation ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Myofilament Function ◽  
Inotropic Responses

The left ventricle (LV) and right ventricle (RV) have differing hemodynamics and embryological origins, but it is unclear whether they are regulated differently. In particular, no previous studies have directly compared the LV versus RV myocardial inotropic responses to α1-adrenergic receptor (α1-AR) stimulation. We compared α1-AR inotropy of cardiac trabeculae from the LV versus RV of adult mouse hearts. As previously reported, for mouse RV trabeculae, α1-AR stimulation with phenylephrine (PE) caused a triphasic contractile response with overall negative inotropy. In marked contrast, LV trabeculae had an overall positive inotropic response to PE. Stimulation of a single subtype (α1A-AR) with A-61603 also mediated contrasting LV/RV inotropy, suggesting differential activation of multiple α1-AR-subtypes was not involved. Contrasting LV/RV α1-AR inotropy was not abolished by inhibiting protein kinase C, suggesting differential activation of PKC isoforms was not involved. However, contrasting LV/RV α1-AR inotropic responses did involve different effects on myofilament Ca2+ sensitivity: submaximal force of skinned trabeculae was increased by PE pretreatment for LV but was decreased by PE for RV. For LV myocardium, α1-AR-induced net positive inotropy was abolished by the myosin light chain kinase inhibitor ML-9. This study suggests that LV and RV myocardium have fundamentally different inotropic responses to α1-AR stimulation, involving different effects on myofilament function and myosin light chain phosphorylation.

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