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

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) but Ca2+-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCKSMKO) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCKSMKO mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCKSMKO mice may be a useful model of vascular failure and hypotension.

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Role of non-kinase activity of myosin light-chain kinase in regulating smooth muscle contraction, a review dedicated to Dr. Setsuro Ebashi

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.11.096 ◽

2008 ◽

Vol 369 (1) ◽

pp. 135-143 ◽

Cited By ~ 16

Author(s):

Akio Nakamura ◽

Ce Xie ◽

Yue Zhang ◽

Ying Gao ◽

Hong-Hui Wang ◽

...

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Kinase Activity ◽

Smooth Muscle Contraction

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00145.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. L509-L516 ◽

Cited By ~ 13

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.

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Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.142298599 ◽

2002 ◽

Vol 99 (14) ◽

pp. 9556-9561 ◽

Cited By ~ 24

Author(s):

J. Bao ◽

K. Oishi ◽

T. Yamada ◽

L. Liu ◽

A. Nakamura ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Muscle Cells ◽

Down Regulation ◽

Cultured Smooth Muscle Cells

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

AJP Cell Physiology ◽

10.1152/ajpcell.90645.2007 ◽

2008 ◽

Vol 295 (2) ◽

pp. C358-C364 ◽

Cited By ~ 69

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.

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