scholarly journals Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.

1995 ◽  
Vol 130 (3) ◽  
pp. 613-627 ◽  
Author(s):  
Z M Goeckeler ◽  
R B Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Isometric Contraction ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Isometric Tension ◽  
Stress Fibers ◽  
Light Chains ◽  
Myosin Light Chains

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.

Regulation of permeabilized endothelial cell retraction by myosin phosphorylation

1991 ◽  
Vol 261 (1) ◽  
pp. C32-C40 ◽  
Author(s):  
R. B. Wysolmerski ◽  
D. Lagunoff
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Specific Inhibitor ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Inhibitory Peptide ◽  
Endothelial Cell Retraction ◽  
Cell Retraction ◽  
Gamma S

Permeabilized endothelial cell monolayers retracted on exposure to ATP and Ca2+. ADP, inosine triphosphate (ITP), GTP, adenosine 5'-(gamma-thio)triphosphate (ATP-gamma S), and 5'-adenylylimidodiphosphate failed to support retraction. However, ATP gamma S, a substrate for myosin light-chain kinase (MLCK) but not myosin adenosinetriphosphatase (ATPase), combined with ITP, a substrate for myosin ATPase but not MLCK, supported retraction. Two MLCK pseudosubstrate peptides, M5 and SM-1, inhibited endothelial cell retraction equally and more effectively than myosin kinase-inhibitory peptide with a sequence based on the phosphorylated site of myosin light chain. M5 was shown to inhibit thiophosphorylation of endothelial cell myosin light chains. Endothelial cells incubated with exogenous unregulated kinase in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid retracted on addition of ATP. This retraction was accompanied by thiophosphorylation of the 19 kDa myosin light chains in the presence of ATP gamma 35S. The N-ethylmaleimide-modified subfragment 1 of myosin heads, a specific inhibitor of actin-myosin interaction, prevented retraction. These data add support to the proposal of a central role for MLCK activation of myosin in endothelial retraction.

Smooth muscle phosphatases: structure, regulation, and function

1994 ◽  
Vol 72 (11) ◽  
pp. 1427-1433 ◽  
Author(s):  
M. D. Pato ◽  
A. G. Tulloch ◽  
M. P. Walsh ◽  
E. Kerc
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Protein Phosphatases ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Heavy Meromyosin ◽  
Smooth Muscle Contractility

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin by myosin light chain kinase. Secondary mechanisms that might modulate contractility are phosphorylation–dephosphorylation of myosin light chain kinase and thin-filament proteins, caldesmon and calponin. Purification of several protein phosphatases that are active toward myosin light chains and (or) myosin and heavy meromyosin from smooth muscles has been reported. All the cytosolic turkey gizzard smooth muscle phosphatases, termed SMP-I, -II, -III, and -IV, dephosphorylate myosin light chains rapidly, but only SMP-III and -IV are active toward myosin and heavy meromyosin, suggesting that SMP-III and -IV might be directly involved in the relaxation of smooth muscle. SMP-III and -IV exhibit properties typical of type 1 protein phosphatases following tryptic digestion. These enzymes appear to share structural similarity with myofibrillar phosphatase PP1M. Purified calponin phosphatase and caldesmon phosphatase from chicken gizzards are structurally and immunologically identical with SMP-I, a type 2A protein phosphatase. SMP-I dephosphorylates calponin faster than it does caldesmon, and has much higher activity toward these substrates than SMP-II, -III, and -IV. Thus, one role for SMP-I might be to regulate the activities of caldesmon and calponin. Since SMP-I is active toward myosin light chain kinase, it might also modulate this enzyme.Key words: smooth muscle, contractility, protein phosphatases, smooth muscle phosphatases.

Calcium-dependent mechanisms of regulation of smooth muscle contraction

1991 ◽  
Vol 69 (12) ◽  
pp. 771-800 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Smooth Muscle Contraction ◽  
Light Chains ◽  
Contractile State ◽  
Calmodulin Binding

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, α-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120–270 to 500–700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca2+-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca2+-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation–dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca2+-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca2+- and phospholipid-dependent protein kinase (protein kinase C).Key words: smooth muscle, Ca2+, myosin phosphorylation, regulation of contraction.

scholarly journals Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

2018 ◽  
Vol 8 (2) ◽  
pp. 204589401876417 ◽  
Author(s):  
Ting Wang ◽  
Mary E. Brown ◽  
Gabriel T. Kelly ◽  
Sara M. Camp ◽  
Joseph B. Mascarenhas ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Human Lung ◽  
Disease Risk ◽  
Spatial Localization ◽  
Cell Periphery ◽  
Nucleotide Polymorphisms

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury

1996 ◽  
Vol 271 (1) ◽  
pp. L121-L125 ◽  
Author(s):  
P. L. Khimenko ◽  
T. M. Moore ◽  
P. S. Wilson ◽  
A. E. Taylor
Keyword(s):  
Endothelial Cell ◽  
Lung Injury ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Ischemia Reperfusion ◽  
Calcium Ionophore ◽  
Microvascular Permeability ◽  
Microvascular Damage ◽  
Pkc Inhibitor

It is generally accepted that microvascular permeability is controlled by intercellular endothelial cell gap size. This process is controlled in endothelial cell monolayers and peripheral blood vessels by calmodulin (CaM)-dependent myosin light-chain kinase (MLCK), which phosphorylates MLC20 with subsequent actin-myosin interaction. In the present study both CaM and MLCK blockers were studied during ischemia-reperfusion (I/R)-induced injury in isolated buffer-perfused rat lungs. The effects of a calcium ionophore (CaI) were tested in isolated intact rat lungs to compare the effects of increasing intracellular Ca2+ to I/R-induced damage. Because protein kinase C (PKC) could also be a mediator of I/R injury, a PKC inhibitor was studied in lungs subjected to either I/R or CaI. In lungs subjected to I/R alone, a fivefold increase in microvascular permeability occurred after 30 min of reperfusion (P < 0.001), and a tenfold increase was present after an additional 60 min of reperfusion (P < 0.01). Pretreatment of the I/R lungs with a CaM inhibitor (trifluoperazine, 100 microM) or with a MLCK inhibitor (ML-7,500 nM) blocked the microvascular damage at both 30 and 90 min of reperfusion. When the CaM inhibitor was introduced into the venous reservoir after 46 min of reperfusion, after the microvascular damage was present, no further increase in microvascular permeability occurred. Pretreatment of the lungs with a PKC inhibitor (staurosporine, 100 nM) did not alter the magnitude of the increased microvascular permeability produced by I/R or the time course of the damage. The calcium ionophore A23187 (7.5 microM) caused increases in Kfc values similar to those produced by I/R. Pretreatment of A23187-treated lungs with a CaM inhibitor produced no protective effect on the microvascular injury at 30 min after administration. Pretreatment of the CaI-challenged lungs with staurosporine significantly increased the microvascular barrier injury at 30 min compared with that occurring with I/R. When a beta-adrenergic receptor agonist (isoproterenol, 10 microM) was introduced to the lung after CaI-induced damage had occurred, no further increase in microvascular permeability was observed, and a trend toward reversal of injury occurred. We conclude from these studies that CaM/MLCK/MLC20 system is involved in our model of I/R-induced rat lung injury but is not involved in lung injury associated with Ca2+ entering the cell.

scholarly journals Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

2008 ◽  
Vol 295 (4) ◽  
pp. C994-C1006 ◽  
Author(s):  
Zoe M. Goeckeler ◽  
Paul C. Bridgman ◽  
Robert B. Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Isometric Tension ◽  
Stress Fiber ◽  
Tension Development ◽  
Basal Tone

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca2+ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

scholarly journals Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

1985 ◽  
Vol 100 (6) ◽  
pp. 2025-2030 ◽  
Author(s):  
H Takano-Ohmuro ◽  
T Obinata ◽  
M Kawashima ◽  
T Masaki ◽  
T Tanaka
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Striated Muscles ◽  
Two Dimensional Gel Electrophoresis ◽  
Embryonic Chicken ◽  
Muscle Myosin

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

scholarly journals Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e59965 ◽  
Author(s):  
Fabeha Fazal ◽  
Kaiser M. Bijli ◽  
Matthew Murrill ◽  
Antony Leonard ◽  
Mohammad Minhajuddin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Critical Role ◽  
Muscle Myosin
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