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The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca 2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca 2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca 2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca 2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators., (© 2020 International Federation for Cell Biology.)

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility., (© 2019 Samson et al.)

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders. This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP.

Key Points: The extent of myosin regulatory light chain phosphorylation (RLC) necessary for smooth muscle contraction depends on the respective activities of Ca(2+) /calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP), which contains a regulatory subunit MYPT1 bound to the phosphatase catalytic subunit and myosin. MYPT1 showed significant constitutive T696 and T853 phosphorylation, which is predicted to inhibit MLCP activity in isolated ileal smooth muscle tissues, with additional phosphorylation upon pharmacological treatment with the muscarinic agonist carbachol. Electrical field stimulation (EFS), which releases ACh from nerves, increased force and RLC phosphorylation but not MYPT1 T696 or T853 phosphorylation. The conditional knockout of MYPT1 or the knockin mutation T853A in mice had no effect on the frequency-maximal force responses to EFS in isolated ileal tissues. Physiological RLC phosphorylation and force development in ileal smooth muscle depend on myosin light chain kinase and MLCP activities without changes in constitutive MYPT1 phosphorylation., Abstract: Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+) /calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in ileal smooth muscle in adult mice with (1) smooth muscle-specific deletion of MYPT1; (2) non-phosphorylatable MYPT1 containing a T853A knockin mutation; and (3) measurements of force and protein phosphorylation responses to cholinergic neurostimulation initiated by electric field stimulation. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted and relaxed rapidly with moderate differences in sustained responses to KCl and carbachol treatments and washouts, respectively. Similarly, measurements of regulatory proteins responsible for RLC phosphorylation during contractions also revealed moderate changes. There were no differences in contractile or RLC phosphorylation responses to carbachol between tissues from normal mice vs. MYPT1 T853A knockin mice. Quantitatively, there was substantial MYPT1 T696 and T853 phosphorylation in wild-type tissues under resting conditions, predicting a high extent of MLCP phosphatase inhibition. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Electric field stimulation increased RLC phosphorylation and force development in tissues from wild-type mice without an increase in MYPT1 phosphorylation. Thus, physiological RLC phosphorylation and force development in ileal smooth muscle appear to be dependent on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte-vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis., (© 2015 American Heart Association, Inc.)

Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wild-type or MYPT1-deficient tissues. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to inhibit MLCP activity. In summary, MYPT1 deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.

Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca(2+)/calmodulin-dependent myosin light chain kinase. Ca(2+)-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC 20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet shape change through unknown mechanisms. We examined the effects of cAMP signaling on platelet contractile machinery. Prostaglandin E1 (PGE1)-mediated inhibition of thrombin-stimulated shape change was accompanied by diminished phosphorylation of myosin light chain (MLC). Since thrombin stimulates phospho-MLC through RhoA/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent inhibition of MLC phosphatase (MLCP), we examined the effects of cAMP on this pathway. Thrombin stimulated the membrane localization of RhoA and the formation of a signaling complex of RhoA/ROCK2/myosin phosphatase-targeting subunit 1 (MYPT1). This resulted in ROCK-mediated phosphorylation of MYPT1 on threonine 853 (thr(853)), the disassociation of the catalytic subunit protein phosphatase 1δ (PP1δ) from MYPT1 and inhibition of basal MLCP activity. Treatment of platelets with PGE1 prevented thrombin-induced phospho-MYPT1-thr(853) in a protein kinase A (PKA)-dependent manner. Examination of the molecular mechanisms revealed that PGE1 induced the phosphorylation of RhoA on serine(188) through a pathway requiring cAMP and PKA. This event inhibited the membrane relocalization of RhoA, prevented the association of RhoA with ROCK2 and MYPT1, attenuated the dissociation of PP1δ from MYPT1, and thereby restored basal MLCP activity leading to a decrease in phospho-MLC. These data reveal a new mechanism by which the cAMP-PKA signaling pathway regulates platelet function.

Rationale: Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level., Objective: We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity., Methods and Results: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1β and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 β/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process., Conclusions: We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I-MYPT1 signaling may be of therapeutic benefit for combating these events.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Background: Uterine contractile activity plays an important role in many and varied reproductive functions including sperm and embryo transport, implantation, menstruation, gestation and parturition. Abnormal contractility might underlie common and important disorders such as infertility, implantation failure, dysmenorrhea, endometriosis, spontaneous miscarriage or preterm birth., Methods: A systematic review of the US National Library of Medicine was performed linking 'uterus' or 'uterine myocyte' with 'calcium ion' (Ca(2+)), 'myosin light chain kinase' and 'myosin light chain phosphatase'. This led to many cross-references involving non-uterine myocytes and, where relevant, these data have been incorporated into the following synthesis., Results: We have grouped the data according to three main components that determine uterine contractility: the contractile apparatus; electrophysiology of the myocyte including excitation-contraction coupling; and regulation of the sensitivity of the contractile apparatus to Ca(2+). We also have included information regarding potential therapeutic methods for regulating uterine contractility., Conclusions: More research is necessary to understand the mechanisms that generate the frequency, amplitude, duration and direction of propagation of uterine contractile activity. On the basis of current knowledge of the molecular control of uterine myocyte function, there are opportunities for systematic testing of the efficacy of a variety of available potential pharmacological agents and for the development of new agents. Taking advantage of these opportunities could result in an overall improvement in reproductive health.

Both rat derived vascular smooth muscle cells (SMC) and human myofibroblasts contain α smooth muscle actin (SMA), but they utilize different mechanisms to contract populated collagen lattices (PCLs). The difference is in how the cells generate the force that contracts the lattices. Human dermal fibroblasts transform into myofibroblasts, expressing α-SMA within stress fibers, when cultured in lattices that remain attached to the surface of a tissue culture dish. When attached lattices are populated with rat derived vascular SMC, the cells retain their vascular SMC phenotype. Comparing the contraction of attached PCLs when they are released from the culture dish on day 4 shows that lattices populated with rat vascular SMC contract less than those populated with human myofibroblast. PCL contraction was evaluated in the presence of vanadate and genistein, which modify protein tyrosine phosphorylation, and ML-7 and Y-27632, which modify myosin ATPase activity. Genistein and ML-7 had no affect upon either myofibroblast or vascular SMC-PCL contraction, demonstrating that neither protein tyrosine kinase nor myosin light chain kinase was involved. Vanadate inhibited myofibroblast-PCL contraction, consistent with a role for protein tyrosine phosphatase activity with myofibroblast-generated forces. Y-27632 inhibited both SMC and myofibroblast PCL contraction, consistent with a central role of myosin light chain phosphatase., (© 2010 Wiley-Liss, Inc.)

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which are an important part of malignant transformation in the most recent model of tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. In this study, we show that cancer cells often failed at cytokinesis because of decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light-chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light-chain phosphorylation was restored to normal levels by phosphatase knockdown, multinucleation and multipolar mitosis were markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light-chain kinase in nonmalignant cells could recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating that these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.

Objective: To investigate the molecular mechanisms leading to edema-induced decreases in intestinal smooth muscle myosin light-chain phosphorylation. Intestinal interstitial edema often develops during abdominal surgery and after fluid resuscitation in trauma patients. Intestinal edema causes decreased intestinal contractile activity via decreased intestinal smooth muscle myosin light-chain phosphorylation, leading to slower intestinal transit. Interstitial edema development is a complex phenomenon, resulting in many changes to the interstitial environment surrounding intestinal smooth muscle cells. Thus, the mechanism(s) by which intestinal edema development causes intestinal dysfunction are likely to be multifactorial., Design: Randomized animal study., Setting: University laboratory., Subjects: Male Sprague-Dawley rats, weighing 250-350 g., Intervention: Studies were performed in a rat model in which a combination of mesenteric venous hypertension and administration of resuscitative fluids induces intestinal edema, mimicking the clinical setting of damage control resuscitation., Measurements and Main Results: Microarray analysis of edematous intestinal smooth muscle combined with an in silico search for overrepresented transcription factor binding sites revealed the involvement of nuclear factor-kappaB in edema-induced intestinal dysfunction. Nuclear factor-kappaB deoxyribonucleic acid binding activity was significantly increased in edematous intestinal smooth muscle compared with controls. Inhibition of nuclear factor-kappaB activation blocked edema-induced decreases in basal intestinal contractile activity. Inhibition of nuclear factor-kappaB activation also attenuated edema-induced decreases in myosin light-chain phosphorylation., Conclusions: We conclude that intestinal edema activates nuclear factor-kappaB, which, in turn, triggers a gene regulation program that eventually leads to decreased myosin light-chain phosphorylation and, thus, decreased intestinal contractile activity.

We demonstrate that in the zebrafish hindbrain, cell shape, rhombomere morphogenesis and, unexpectedly, brain ventricle lumen expansion depend on the contractile state of the neuroepithelium. The hindbrain neural tube opens in a specific sequence, with initial separation along the midline at rhombomere boundaries, subsequent openings within rhombomeres and eventual coalescence of openings into the hindbrain ventricle lumen. A mutation in the myosin phosphatase regulator mypt1 results in a small ventricle due to impaired stretching of the surrounding neuroepithelium. Although initial hindbrain opening remains normal, mypt1 mutant rhombomeres do not undergo normal morphological progression. Three-dimensional reconstruction demonstrates cell shapes within rhombomeres and at rhombomere boundaries are abnormal in mypt1 mutants. Wild-type cell shape requires that surrounding cells are also wild type, whereas mutant cell shape is autonomously regulated. Supporting the requirement for regulation of myosin function during hindbrain morphogenesis, wild-type embryos show dynamic levels of phosphorylated myosin regulatory light chain (pMRLC). By contrast, mutants show continuously high pMRLC levels, with concentration of pMRLC and myosin II at the apical side of the epithelium, and myosin II and actin concentration at rhombomere boundaries. Brain ventricle lumen expansion, rhombomere morphology and cell shape are rescued by inhibition of myosin II function, indicating that each defect is a consequence of overactive myosin. We suggest that the epithelium must ;relax', via activity of myosin phosphatase, to allow for normal hindbrain morphogenesis and expansion of the brain ventricular lumen. Epithelial relaxation might be a widespread strategy to facilitate tube inflation in many organs.

Purpose: The purpose of the present study was to clarify the role of Rho-kinase and/or protein kinase C in the resting tension of the isolated pig iris sphincter muscle., Materials and Methods: The motor activity of the isolated pig iris sphincter muscle was measured isometrically., Results: EGTA, a chelator of extracellular Ca(2+), significantly reduced the resting tension. Y27632, a Rho-kinase inhibitor, significantly reduced the resting tension in a concentration-dependent manner. The resting tension diminished by Y27632 was significantly recovered by the addition of calyculin A, a myosin light chain phosphatase (MLCP) inhibitor, in a concentration-dependent manner. GF109203X, a protein kinase C inhibitor, had no effect on the resting tension., Conclusion: These results suggest that, in the isolated pig iris sphincter muscle, Rho-kinase plays an important role in the generation of spontaneous tone in the resting phase via the inhibition of MLCP activity.

H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A (PKA). In this study, we found that H89 reduced the phosphorylation of the myosin regulatory light chain (MRLC) at Thr-18/Ser-19 and induced disassembly of stress fibers in HeLa cells. In addition, we found that H89 induced not only reduction of the MRLC phosphorylation but also cell growth inhibition in several human cancer cell lines. Recently H89 has been found to inhibit Rho-kinase with potency similar to or greater than that for inhibition of PKA. Indeed, the effects of H89 on both the MRLC phosphorylation and actin cytoskeleton organization were nearly identical to those of Rho-kinase inhibitor Y-27632. However, unlike H89, Y-27632 did not affect cell growth of HeLa cells. Further, when the myosin phosphatase targeting subunit 1 (MYPT1) expression was silenced by RNA interference in HeLa cells, the suppressive effect of H89 on the MRLC phosphorylation was not affected, while H89-induced cell growth inhibition was blocked. These results suggest that H89-induced reduction of the MRLC phosphorylation results from inhibition of Rho-kinase and that H89-induced cell growth inhibition is independent of reduction of the MRLC phosphorylation.

In airway smooth muscles, kinase/phosphatase-dependent phosphorylation and dephosphorylation of the myosin light chain (MLC) have been revealed by many authors as important steps in calcium (Ca(2+)) signalling pathway from the variation of Ca(2+) concentration in cytosol to the force development. Here, a theoretical analysis of the control action of MLC-kinase (MLCK) and MLC-phosphatase (MLCP) in Ca(2+) signalling is presented and related to the general control principles of these enzymes, which were previously studied by Reinhart Heinrich and his co-workers. The kinetic scheme of the mathematical model considers interactions among Ca(2+), calmodulin (CaM) and MLCK and the well-known 4-state actomyosin latch bridge model, whereby a link between them is accomplished by the conservation relation of all species of MLCK. The mathematical model predicts the magnitude and velocity of isometric force in smooth muscles upon transient biphasic Ca(2+) signal. The properties of signal transduction in the system such as the signalling time, signal duration and signal amplitude, which are reflected in the properties of force developed, are studied by the principles of the metabolic control theory. The analysis of our model predictions confirms as shown by Reinhart Heinrich and his co-workers that MLCK controls the amplitude of signal more than its duration, whereas MLCP controls both. Finally, the simulations of elevated total content of MLCK, a typical feature of bronchial muscles of asthmatic subjects and spontaneously hypertensive rats as well as potentiation of MLCP catalytic activity, are carried out and are discussed in view of an increase in the force magnitude.

Endothelial hyperpermeability is regulated by a myosin light chain-2 (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Cytoskeletal dynamics, regions of paracellular flux, and MLC2-phosphorylation of ECs were visualized by digital three-dimensional imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2 phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase dependent. These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction.

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.

(-)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to be a potent chemopreventive agent. Recently, 67-kDa laminin receptor (67LR) has been identified as a cell surface receptor for EGCG that mediates the anticancer activity of EGCG. Indeed, expression of 67LR confers EGCG responsiveness to tumor cells; however, the molecular basis for the anticancer activity of EGCG in vivo is not entirely understood. Here we show that (i) using a direct genetic screen, eukaryotic translation elongation factor 1A (eEF1A) is identified as a component responsible for the anticancer activity of EGCG; (ii) through both eEF1A and 67LR, EGCG induces the dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) at Thr-696 and activates myosin phosphatase; and (iii) silencing of 67LR, eEF1A, or MYPT1 in tumor cells results in abrogation of EGCG-induced tumor growth inhibition in vivo. Additionally, we found that eEF1A is up-regulated by EGCG through 67LR. Overall, these findings implicate both eEF1A and MYPT1 in EGCG signaling for cancer prevention through 67LR.

Aims: High-risk atheromatous plaques contain significant extra- and intracellular lipid deposits and very low smooth muscle cell numbers in the intima. However, the mechanisms inducing vessel wall remodelling and high-risk plaque composition are unknown. Low-density lipoproteins (LDLs) infiltrate the vessel wall and become retained and aggregated (agLDL) in the intima by binding to extracellular matrix proteoglycans. The cellular responses triggered by agLDL are not fully understood. This study was designed to investigate the effects of agLDL on vascular remodelling and repair, specifically studying human coronary vascular smooth muscle cell (VSMC) functions., Methods and Results: Using a wound repair VSMC model system, we have shown that agLDL significantly impair cell migration. Proteomic analysis revealed a differential phenotypic pattern of myosin light chain and lower levels of phosphorylated myosin regulatory light chain (P-MRLC) in agLDL-exposed VSMC. LDL also induced changes in the subcellular localization of P-MRLC, with dephosphorylation strongly evident on the front edge of agLDL-treated migrating cells. PMA, a strong inducer of myosin light chain (MLC) phosphorylation, significantly reduced the effects of agLDL in VSMC migration. Inhibition of MLC kinase with ML9 did not affect MRLC dephosphorylation already induced by agLDL., Conclusion: Our results indicate that LDLs impair human VSMC migration and wound repair after injury. agLDL, and to a lesser extent nLDL, induce dephosphorylation of MRLC and striking changes in the subcellular localization of P-MRLC, a cytoskeleton protein involved in VSMC migration kinetics.

Our previous research showed that Rho kinase took part in the regulation of vascular hyporeactivity after shock. The objective of the present study was to investigate its mechanism. With isolated superior mesenteric artery (SMA) from hemorrhagic shock rats, we studied the relationship of Rho kinase regulating vascular reactivity to calcium sensitivity and myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK). The vascular reactivity and calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative norepinephrine (NE) and calcium under depolarizing condition (120 mM K(+)) with an isolated organ perfusion system. Hypoxia-treated vascular smooth muscle cells (VSMCs) were used to study the effects of Rho kinase on the activity of MLCP and MLCK and the phosphorylation of 20-kDa myosin light chain (MLC(20)). Myosin light chain (20 kDa) phosphorylation of VSMC in mesenteric artery was detected by immunoprecipitation and Western blotting. The activity of MLCP and MLCK was assayed by enzymatic catalysis. The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin through transwell. The results indicated that the vascular reactivity and calcium sensitivity of SMA to NE and calcium following hemorrhagic shock and the contractile response of VSMC to NE following hypoxia were significantly decreased. Angiotensin II (Ang-II), the Rho kinase stimulator, significantly improved hypoxia or hemorrhagic shock-induced decrease of vascular reactivity and calcium sensitivity. These effects of Ang-II on vascular reactivity were abolished by Y-27632, the specific Rho kinase inhibitor. Calyculin A, the MLCP inhibitor, further enhanced Ang-II-induced increase of calcium sensitivity, but ML-9, the MLCK inhibitor, had no effect. Further studies showed Ang-II reversed the hypoxia-induced increase of MLCP activity and increased the hypoxia-induced decrease of MLC(20) phosphorylation in VSMC. It was suggested that Rho kinase played an important role in the regulation of vascular reactivity after hemorrhagic shock. The mechanisms may be related to its calcium sensitivity regulation. Rho kinase up-regulates calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC(20).

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca(2+) sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.

In isolated guinea pig vas deferens, prior addition of norepinephrine (NE) significantly potentiated the contractile responses to adenosine-5'-triphosphate (ATP) and diadenosine tetraphosphate (AP4A) in a dose-dependent manner up to 240% of the control purine dose. The myosin light chain phosphatase (MLCP) inhibitor cantharidin at a dose of 10 micromol/l caused significant enhancement of ATP at concentrations of 1 and 3 mmol/l by 91 and 95% respectively. Similarly, cantharidin enhanced the contraction to AP4A, 30 and 100 micromol/l by 92 and 100% respectively. Inhibition of protein kinase C (PKC) by the use of chelerythrine (10 micromol/l), incubated at the vas deferens for 60 min, inhibited the NE-induced enhancement of purine-induced contraction. Chelerythrine reversed the NE-ATP and NE-AP4A synergism back close to control ATP and AP4A contraction values respectively. It can be concluded that postjunctional synergism becomes evident not only for adenine mononucleotides and NE but also for diadenosine polyphosphates presented here by AP4A in the guinea pig vas deferens. This synergism involves receptor-mediated activation of PKC and possibly PKC-induced inhibition of MLCP., (Copyright (c) 2007 S. Karger AG, Basel.)

Epidemiologic studies have shown that aging accounts significantly for the prevalence of erectile dysfunction (ED). The pathophysiology of ED during aging and its underlying molecular mechanisms are largely unknown. We hypothesized that increased RhoA/Rho-kinase signaling is a major factor in the pathogenesis of age-associated ED and the mechanism involves increased penile smooth muscle contractility through inhibition of myosin light chain phosphatase. Male Fischer 344 young (4 month old) and aged (20-22 month old) rats underwent erectile function testing in vivo by measuring intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) upon electrical stimulation of the cavernous nerve. The data demonstrated that erectile function was significantly lower in aged rats than that in young rats at all voltages tested (P<0.05). Western blot analysis results showed that there were no significant changes in protein expressions of RhoA, Rho-kinase-alpha and -beta isoforms, and myosin light chain phosphatase target subunit (MYPT1); however, membrane-bound RhoA and phosphorylated MYPT1 were increased in aged rat penes by 95 +/- 15 and 56 +/- 8% (P<0.05), respectively, indicating enhanced RhoA and Rho-kinase activity. Inhibition of Rho-kinase with Y27632 maximally increased ICP/MAP to 0.72 +/- 0.05 in aged rats vs. 0.47 +/- 0.06 in young rats (P<0.05). Gene transfer of adeno-associated virus (AAV) encoding dominant negative RhoA (T19NRhoA) to penes of aged and young rats for 7 days markedly improved erectile function in aged rats when compared with that in young rats (P<0.05). These observations were also supported by Rho-kinase activity assay results showing that basal Rho-kinase activity in aged rat penes receiving AAV vehicle treatment was twofold greater than that in young rat penes receiving AAV vehicle treatment, while it was reduced to a level similar to that in young rat penes after gene therapy of T19NRhoA (P<0.05). Taken together, these data suggest that impaired erectile function during the aging process involves increased RhoA/Rho-kinase signaling, and this pathway may be exploited for the treatment of age-associated ED.

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.

Transient transfection of NIH3T3 cells with various constructs of myosin phosphatase target subunit (MYPT1) and GFP showed distinct cellular localizations. Constructs containing the N-terminal nuclear localization signals (NLS), i.e. full-length MYPT1 and N-terminal MYPT1 fragments, were concentrated in the nucleus. Full-length chicken and human MYPT1-GFP showed discrete nuclear foci. Deletion of the N-terminal NLS or use of central or C-terminal MYPT1 fragments did not show unique nuclear distributions (C-terminal NLS are present). Transient transfection of NIH3T3 cells (in the presence of serum) with full-length MYPT1-GFP caused a marked decrease in number of attached cells, an apparent block in the cell cycle prior to M phase and signs of increased apoptosis. Under conditions of serum starvation the unique nuclear localization of MYPT1-GFP was not found and there was no marked decrease in the number of attached cells (after 48 h). Stable transfection of HEK 293 cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1.

Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. We show that this phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and that it requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. We suggest that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration.

Objective: The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions., Methods and Results: Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 micromol/L TAT-MYPT1(1-374), a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+]i elevation. The deletion of the Tat peptide, MYPT1(1-374), abolished the augmenting effect. TAT-MYPT1(1-296) demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1(1-171), TAT-MYPT1(39-374), TAT-MYPT1(39-296), and TAT-MYPT1(297-374) had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1(1-374) compared with the control., Conclusions: Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Vascular smooth muscle contraction plays a defining role in the regulation and maintenance of blood pressure, and its deregulation is associated with many clinical syndromes including hypertension, coronary vasospasm and congestive heart failure. Over the past 20 years, there has been a growing understanding of the regulation of 20 kDa myosin light chain phosphorylation by myosin light chain kinase and myosin light chain phosphatase, the role of splice-variant isoforms of both the myosin heavy chain and the essential myosin light chain, as well as the signaling pathways involved in smooth muscle contraction under normal and pathophysiological conditions. This review will attempt to recapitulate the data in the field, primarily focusing on the contractile response of smooth muscle, and the molecular determinants responsible for the regulation of vascular tone.