Myosin light chain phosphorylation in contraction and relaxation of intact rat thoracic aorta

Myosin light chain phosphorylation in intact rat thoracic aorta was elevated during contraction induced by 0.3 microM norepinephrine, but was not maintained. Addition of 0.5 microM sodium nitroprusside to norepinephrine treated rat aorta strips led to elevation of cyclic GMP levels, relaxation of tension, and dephosphorylation of myosin light chain. Depletion of extracellular calcium or addition of calmodulin antagonists trifluoperazine and W7 diminished the contraction and phosphorylation of myosin light chain by norepinephrine, but did not prevent dephosphorylation by sodium nitroprusside or the elevated levels of cyclic GMP. Isoproterenol, 8-bromo cyclic GMP, and dibutyryl cyclic AMP all caused dephosphorylation of myosin light chain and induced relaxation during the period of development of tone. Eight other proteins had increased phosphorylation following norepinephrine treatment and one protein had less phosphorylation. The different proteins phosphorylated by norepinephrine showed varying degrees of sensitivity to Ca2+-free solution and to the calmodulin antagonists. The pattern of protein phosphorylation caused by sodium nitroprusside was best mimicked by 8-bromo cyclic GMP, rather than isoproterenol and dibutyryl cyclic AMP. These proteins were, generally, unaffected by Ca2+-free solution and the calmodulin antagonists. The present observations support the hypothesis that vasodilators inhibit tone development through myosin light chain dephosphorylation. Furthermore, the nitrovasodilators act through elevation of cyclic GMP and phosphorylation of proteins by cyclic GMP-dependent protein kinase.     

Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca(2+) concentration ([Ca(2+)](i)) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca(2+) mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca(2+)](i) resembled that induced by the Ca(2+) channel blocker nifedipine and were reversed specifically by the Ca(2+) channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca(2+) influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.     

Myosin light chain phosphorylation and evidence for latchbridge formation in norepinephrine stimulated canine veins

The role of 20000 dalton myosin light chain phosphorylation in mediating venous smooth muscle contraction was studied in isolated preparations of canine jugular and femoral vein. One min 10(-5) M norepinephrine-induced contraction was accompanied by significant increases in phosphorylation (jugular - 21 to 46%; femoral - 19 to 54%) which were reversed within 10 min after agonist washout. During 40 min stimulation, phosphorylation and isometric force redevelopment rates declined to near basal levels while force was maintained. These findings implicate light chain phosphorylation as a prerequisite for initial tension development by crossbridge cycling in venous smooth muscle. However, long term tension can be maintained through a process similar to the latchbridge state in tracheal and arterial smooth muscle.     

Myosin light chain phosphorylation in intact human muscle

The phosphate content of the fast (LC2F) and two slow (LC2S and LC2S1) phosphorylatable light chains (P-light chains) in myosin isolated from biopsy samples of rested human vastus lateralis muscle averaged 0.21, 0.28 and 0.25 mol of phosphate per mol of P-light chain, respectively. Following a 10 s maximal contraction, phosphate content was increased by almost 2-fold in the fast and two slow P-light chains. After prolonged, moderate cycling activity phosphate content was only slightly increased in the three P-light chains. These data suggest that, unlike animal skeletal muscle, myosin light chain kinase and phosphatase activities are similar in human fast and slow muscle fibres.     

Myosin light chain phosphorylation and growth cone motility

According to the treadmill hypothesis, the rate of growth cone advance depends upon the difference between the rates of protrusion (powered by actin polymerization at the leading edge) and retrograde F-actin flow, powered by activated myosin. Myosin II, a strong candidate for powering the retrograde flow, is activated by myosin light chain (MLC) phosphorylation. Earlier results showing that pharmacological inhibition of myosin light chain kinase (MLCK) causes growth cone collapse with loss of F-actin-based structures are seemingly inconsistent with the treadmill hypothesis, which predicts faster growth cone advance. These experiments re-examine this issue using an inhibitory pseudosubstrate peptide taken from the MLCK sequence and coupled to the fatty acid stearate to allow it to cross the membrane. At 5-25 microM, the peptide completely collapsed growth cones from goldfish retina with a progressive loss of lamellipodia and then filopodia, as seen with pharmacological inhibitors, but fully reversible. Lower concentrations (2.5 microM) both simplified the growth cone (fewer filopodia) and caused faster advance, doubling growth rates for many axons (51-102 microm/h; p <.025). Rhodamine-phalloidin staining showed reduced F-actin content in the faster growing growth cones, and marked reductions in collapsed ones. At higher concentrations, there was a transient advance of individual filopodia before collapse (also seen with the general myosin inhibitor, butanedione monoxime, which did not accelerate growth). The rho/rho kinase pathway modulates MLC dephosphorylation by myosin-bound protein phosphatase 1 (MPP1), and manipulations of MPP1 also altered motility. Lysophosphatidic acid (10 microM), which causes inhibition of MPP1 to accumulate activated myosin II, caused a contracted collapse (vs. that due to loss of F-actin) but was ineffective after treatment with low doses of peptide, demonstrating that the peptide acts via MLC phosphorylation. Inhibiting rho kinase with Y27632 (100 microM) to disinhibit the phosphatase increased the growth rate like the MLCK peptide, as expected. These results suggest that: varying the level of MLCK activity inversely affects the rate of growth cone advance, consistent with the treadmill hypothesis and myosin II powering of retrograde F-actin flow; MLCK activity in growth cones, as in fibroblasts, contributes strongly to controlling the amount of F-actin; and the phosphatase is already highly active in these cultures, because rho kinase inhibition produces much smaller effects on growth than does MLCK inhibition.     

Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.     

Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments.

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Myosin light chain phosphorylation and pulmonary endothelial cell hyperpermeability in burns

Major cutaneous burns result in not only localized tissue damage but broad systemic inflammation causing organ system damage distal to the burn site. It is well recognized that many problems result from the release of inflammatory mediators that target vascular endothelial cells, causing organ dysfunction. The pulmonary microvessels are particularly susceptible to functional abnormalities as a direct consequence of exposure to burn-induced inflammatory mediators. Traditional therapeutic intervention is quite often ineffective in treating burn patients suffering from systemic problems. A possible explanation for this ineffectiveness may be that because so many mediators are released, supposedly activating numerous signaling cascades that interact with each other, targeting of upstream factors in these cascades on an individual basis becomes futile. Therefore, if an end-point effector responsible for endothelial dysfunction following burn injury could be identified, it may present a target for intervention. In this study, we identified phosphorylation of myosin light chain (MLC) as a required element of burn plasma-induced hyperpermeability across rat lung microvascular endothelial cell monolayers. In addition, pharmacological inhibition of myosin light chain kinase (MLCK) and Rho kinase as well as transfection of MLCK-inhibiting peptide blocked actin stress fiber formation and MLC phosphorylation in response to burn plasma. The results suggest that blocking MLC phosphorylation may provide therapeutic intervention in burn patients with the goal of alleviating systemic inflammation-induced endothelial dysfunction.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

Myosin light chain phosphorylation in fibroblast shape change, detachment and patching

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.