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

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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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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Myosin light chain kinase expression during smooth muscle development

Bioscience Reports ◽

10.1007/bf01137965 ◽

1993 ◽

Vol 13 (5) ◽

pp. 289-295 ◽

Cited By ~ 2

Author(s):

Luciano Dalla Libera ◽

John H. Collins

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

High Performance ◽

Myosin Light Chain ◽

Muscle Development ◽

Primary Sequence ◽

Post Translational Modifications ◽

Antigenic Properties ◽

Kinase Expression

The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00420.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. L250-L257 ◽

Cited By ~ 18

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.

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