scholarly journals Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

2008 ◽  
Vol 295 (2) ◽  
pp. C358-C364 ◽  
Author(s):  
Yusuke Mizuno ◽  
Eiji Isotani ◽  
Jian Huang ◽  
Hailei Ding ◽  
James T. Stull ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Fluorescent Proteins ◽  
Rho Kinase ◽  
Kinase Activation ◽  
Calphostin C

Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase

2004 ◽  
Vol 287 (1) ◽  
pp. L250-L257 ◽  
Author(s):  
C. Martin ◽  
R. Göggel ◽  
A.-R. Ressmeyer ◽  
S. Uhlig
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Platelet Activating Factor ◽  
Rho Kinase ◽  
Thromboxane Receptor ◽  
Pressor Responses ◽  
Kinase Pathway

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP3)-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP3 receptor (1–10 μM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 μM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 μM ML-7) or of calmodulin kinase kinase (26 μM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 μM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 μM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

2006 ◽  
Vol 290 (3) ◽  
pp. L509-L516 ◽  
Author(s):  
J. Belik ◽  
Ewa Kerc ◽  
Mary D. Pato
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Adult Tissue ◽  
Adult Rat ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Specific Activities

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cδ) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cδ contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

373 Role of myosin light chain kinase and Rho kinase in carbachol-induced contractions of human detrusor smooth muscle

2012 ◽  
Vol 11 (1) ◽  
pp. e373
Author(s):  
C. Protzel ◽  
T. Kirschstein ◽  
K. Porath ◽  
T. Sellmann ◽  
R. Koehling ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Detrusor Smooth Muscle ◽  
Human Detrusor

scholarly journals Inverse relationship of Rho kinase and myosin-light chain kinase expression in the aging human detrusor smooth muscle

BMC Urology ◽  
2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Timo Kirschstein ◽  
Theresa Sahre ◽  
Karoline Kernig ◽  
Chris Protzel ◽  
Katrin Porath ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Inverse Relationship ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Detrusor Smooth Muscle ◽  
Kinase Expression ◽  
Relationship Of ◽  
Human Detrusor

scholarly journals Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition.

1987 ◽  
Vol 105 (1) ◽  
pp. 397-402 ◽  
Author(s):  
B Burnside ◽  
N Ackland
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Phosphorylation ◽  
Calmodulin Binding ◽  
Dependent Protein ◽  
Myosin Interaction ◽  
High Concentrations

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.

Effects of Wortmannin, a Novel Myosin Light-Chain Kinase Inhibitor, on Bile Canalicular Contraction in Vitro and in Vivo

1996 ◽  
Vol 31 (4) ◽  
pp. 391-397 ◽  
Author(s):  
S. Suzuki ◽  
S. Watanabe ◽  
M. Hirose ◽  
T. Kitamura ◽  
H. Oide ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Bile Canalicular Contraction

Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment?

2012 ◽  
Vol 112 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Abdul Raqeeb ◽  
Yuekan Jiao ◽  
Harley T. Syyong ◽  
Peter D. Paré ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Layer ◽  
Radial Force ◽  
Calcium Entry Blocker ◽  
Passive Stiffness

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.

scholarly journals Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in anin VitroModel System

Biochemistry ◽  
2013 ◽  
Vol 52 (47) ◽  
pp. 8489-8500 ◽  
Author(s):  
Feng Hong ◽  
Kevin C. Facemyer ◽  
Michael S. Carter ◽  
Del R. Jackson ◽  
Brian D. Haldeman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Kinase Activation ◽  
Muscle Myosin ◽  
Kinetics Of
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