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.

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.

Role of myosin light-chain phosphorylation in endothelial cell retraction

1993 ◽  
Vol 265 (6) ◽  
pp. L606-L612 ◽  
Author(s):  
R. Sheldon ◽  
A. Moy ◽  
K. Lindsley ◽  
S. Shasby ◽  
D. M. Shasby
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Extracellular Calcium ◽  
Relative Specificity ◽  
Endothelial Cell Retraction ◽  
Mlc Phosphorylation ◽  
Cell Retraction

Endothelial cells retract centripetally when they are exposed to histamine and when extracellular calcium is chelated. This centripetal retraction implies that a centripetal tension must be expressed in the cells. We asked whether phosphorylation of the light chain of myosin (MLC) was important for the retraction to occur, and, by inference, expression of the tension. In human umbilical vein endothelial (HUVE) cells and in porcine pulmonary artery endothelial (PPAE) cells tryptic peptide maps indicated that MLC was phosphorylated by myosin light-chain kinase (MLCK). Activity of MLCK is inhibited by ML-9, a kinase inhibitor with relative specificity for MLCK, and when MLCK is phosphorylated by the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent kinase. Pretreatment of HUVE cells or PPAE cells with ML-9 or forskolin-aminophylline (to increase cell cAMP) reduced basal MLC phosphorylation and prevented an expected increase in MLC phosphorylation following exposure of HUVE cells to histamine. Pretreatment of HUVE cells with ML-9 or forskolin-aminophylline prevented HUVE cell retraction (measured as an increase in permeability of a monolayer of HUVE cells) in response to histamine. Pretreatment of PPAE cells with ML-9 or forskolin-aminophylline prevented PPAE cell retraction in response to chelation of extracellular calcium. These data support the hypothesis that phosphorylation of MLC is an important component of endothelial cell retraction.

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.

Effect of muscle length on isometric stress and myosin light chain phosphorylation in gallbladder smooth muscle

1991 ◽  
Vol 260 (6) ◽  
pp. G920-G924 ◽  
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
C. L. Dorst ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Length ◽  
Stress Sensitivity ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Myofilament Activation

These experiments were designed to characterize the effect of muscle length on isometric stress, sensitivity to stimulation, and phosphorylation of the 20,000-Da myosin light chains in guinea pig gallbladder smooth muscle. Basal, active, and total isometric stress were determined in acetylcholine- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.6-1.3 times the optimal muscle length (Lo) for isometric stress development. The effect of muscle length on the sensitivity to ACh and K+ was determined in cumulative dose-response experiments (10(-8) to 10(-4) M ACh, 10-80 mM KCl) at 0.7, 1.0, and 1.3 Lo. The effect of muscle length on myosin light chain phosphorylation was determined in ACh- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.7, 1.0, and 1.3 Lo. In gallbladder smooth muscle, 1) active isometric stresses at 0.7 and 1.3 Lo were less than active isometric stress at 1.0 Lo; 2) the sensitivity of developed stress was similar at 1.0 and 1.3 Lo but decreased at 0.7 Lo; 3) the decline in isometric stress and sensitivity at 0.7 Lo was associated with reduced levels of phosphorylated myosin light chain; and 4) the decline in isometric stress at 1.3 Lo was not associated with reduced amounts of phosphorylated myosin light chain. These results suggest that the decline in active stress and sensitivity at short muscle lengths (L less than Lo) in gallbladder smooth muscle is due, at least in part, to decreases in the activation of the myofilaments. The decline in active isometric stress at long muscle lengths (L greater than Lo) is not due to changes in myofilament activation.

Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II

2000 ◽  
Vol 113 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Q. Zeng ◽  
D. Lagunoff ◽  
R. Masaracchia ◽  
Z. Goeckeler ◽  
G. Cote ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Regulatory Light Chain ◽  
Atpase Inhibitor ◽  
Cytoskeletal Rearrangements ◽  
Cell Retraction

The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.

scholarly journals Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

1992 ◽  
Vol 40 (10) ◽  
pp. 1547-1557 ◽  
Author(s):  
R Billeter ◽  
M Messerli ◽  
E Wey ◽  
A Puntschart ◽  
K Jostarndt ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Fiber Type ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Two Dimensional ◽  
Fast Myosin ◽  
Chicken Muscles

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle

1992 ◽  
Vol 263 (1) ◽  
pp. C210-C219 ◽  
Author(s):  
T. B. Vyas ◽  
S. U. Mooers ◽  
S. R. Narayan ◽  
J. C. Witherell ◽  
M. J. Siegman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Quantitative Relationship ◽  
Small Degree ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Maximum Increase ◽  
Rabbit Portal Vein

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

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