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.     

Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle

Tracheal smooth muscle precontracted with carbachol relaxes upon the addition of 3 μM okadaic add. Although cytosolic Ca²⁺ concentrations decrease, myosin light chain remains highly phosphorylated (50%). In smooth muscle treated with carbachol alone or carbachol plus okadaic acid ³²P is incorporated into a single peptide on myosin light chain which corresponds to the site phosphorylated by myosin light chain kinase. Treatment with okadaic acid alone does not result in myosin light chain phosphorylation or tension development. These results suggest that a cellular mechanism other than myosin light chain phosphorylation can regulate contractile tension.     

Protein phosphorylation during spontaneous contraction of smooth muscle

The relationship between spontaneous contraction and protein phosphorylation of rat uterine smooth muscle was studied. Myometrial strips from estrogen-dominated rats were incubated in [³²P]orthophosphate and then frozen at various levels of isometric tension. Proteins were separated by gel electrophoresis and the incorporation of ³²P was measured. Contraction was associated with the phosphorylation of one major protein (20,000 M_r). This phosphorylation preceded maximal tension development and dephosphorylation preceded complete spontaneous relaxation. Two-dimensional gel electrophoresis indicates that the 20,000-M_r protein is the myosin light chain which has been implicated in the regulation of smooth muscle contraction.     

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

Reversal of endothelin-1-induced contractions by isoproterenol without myosin dephosphorylation in tracheal smooth muscle.

Effects of isoproterenol on isometric force, and 20,000 Da myosin light chain (LC20) phosphorylation were examined in smooth muscle fibre strips from lamb trachea stimulated with endothelin-1 (ET-1). ET-1 induced a rapidly rising isometric tension which was coupled with a multiple site phosphorylation of LC20. Isoproterenol addition at the time of peak isometric force resulted in a brisk relaxation of the fibre strips. Myosin light chain phosphorylation, however, remained unaffected.     

Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber

The Ca²⁺ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca²⁺-activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca²⁺ (10^-5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca²⁺ insensitive tension. In contrast, pretreatment with low Ca²⁺ (10^-8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca²⁺-sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca²⁺-activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP-dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca²⁺-activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.     

Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle

The changes in protein phosphorylation associated with bovine tracheal smooth muscle contraction were studied by labeling intact muscle strips with [32P]PO4(3-) and analyzing the phosphoproteins by two-dimensional gel electrophoresis. Among 20 to 30 phosphoproteins resolvable with the two-dimensional electrophoresis system, the phosphorylation of 12 proteins was reproducibly affected by treatment with carbachol, in a time-dependent manner. Five of these proteins have been identified as 20-kDa myosin light chain, caldesmon, synemin, and two isoelectric variants of desmin. The other 7 are low molecular weight (Mr less than 40,000) cytosolic proteins. One cytosolic protein and myosin light chain are quickly but transiently phosphorylated by carbachol, the peak of myosin light chain phosphorylation being at about 1 min after agonist addition. In contrast, both variants of desmin, synemin, caldesmon, and 5 cytosolic proteins are phosphorylated at varying rates and remain phosphorylated for the duration of carbachol action. These "late" phosphorylation changes occur simultaneously with the dephosphorylation of one cytosolic protein. These carbachol-induced phosphorylation changes, like the contractile response, appear to be calcium-dependent. The addition of 12-deoxyphorbol 13-isobutyrate, a protein kinase C activator, causes a dose-dependent, sustained contraction of tracheal smooth muscle which develops more slowly than that induced by carbachol. This contractile response is associated with the same protein phosphorylation changes as those observed after prolonged carbachol treatment. In contrast, forskolin, an adenylate cyclase activator and a potent smooth muscle relaxant, induces the phosphorylation protein 3 and one variant of desmin. These observations strongly suggest that different phosphoproteins may be mediators of tension development and tension maintenance in agonist-induced contraction of tracheal smooth muscle.     

Substrate based inhibitors of smooth muscle myosin light chain kinase.

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.     

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.     

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.     

Angiotensin II stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells

The vasoactive peptide angiotensin II stimulates phosphorylation of myosin light chain in 32P-labeled confluent cultures of vascular smooth muscle cells derived from rat mesenteric arteries. Myosin light chain was identified and its 32P-phosphorylation level quantitated following selective immunoprecipitation with an antiserum raised against purified human uterine smooth muscle myosin. Following exposure to 0.1 nM angiotensin II, phosphorylation of the light chain peaked at 4 min and then slowly decreased. The stimulation of light chain phosphorylation at 4 min is half-maximal at approximately 0.2 mM angiotensin II; the maximal response is approximately 210% of the unstimulated level. Basal myosin light chain phosphorylation was markedly reduced by incubation of cells with dibutyryl cyclic AMP or the calmodulin-inhibitor chlorpromazine. These data suggest that angiotensin II-mediated contraction in intact blood vessels involves phosphorylation of the myosin light chain, and that phosphorylation is inhibited by a cAMP-mediated process and may be calmodulin-dependent.     

Intracellular calcium and smooth muscle contraction

Excitation-contraction coupling in smooth muscle involves many processes, some of which are outlined in this article. The total amount of Ca2+ released on excitation is considerably in excess of the free Ca2+ concentration and this implies a high capacity, high affinity Ca2+ buffer system. The two major Ca2+-binding proteins are calmodulin and myosin. Only calmodulin has the appropriate binding affinity to act as a component of the Ca2+-buffer system. The Ca2+-calmodulin complex activates myosin light chain kinase and thus is involved in the regulation of contractile activity. Phosphorylation of myosin stabilizes an active conformation and promotes cross bridge cycling and is essential for the initiation of contraction. During the initial contractile response phosphorylation correlates to tension development and velocity of shortening. However, as contraction continues the extent of myosin phosphorylation and velocity often decreases but tension is maintained. In general, the Ca2+ transient is reflected by the extent of phosphorylation that in turn correlates with shortening velocity. Maintenance of tension at low phosphorylation levels is not accounted for within our understanding of the phosphorylation theory and thus alternative regulatory mechanisms have been implicated. Some of the possibilities are discussed.     

Selected contribution: roles of focal adhesion kinase and paxillin in the mechanosensitive regulation of myosin phosphorylation in smooth muscle.

The increase in intracellular Ca(2+) and myosin light chain (MLC) phosphorylation in response to the contractile activation of tracheal smooth muscle is greater at longer muscle lengths (21). However, MLC phosphorylation can also be stimulated by Ca(2+)-insensitive signaling pathways (19). The cytoskeletal proteins paxillin and focal adhesion kinase (FAK) mediate a Ca(2+)-independent length-sensitive signaling pathway in tracheal smooth muscle (30). We used alpha-toxin-permeabilized tracheal smooth muscle strips to determine whether the length sensitivity of MLC phosphorylation can be regulated by a Ca(2+)-insensitive signaling pathway and whether the length sensitivity of active tension depends on the length sensitivity of myosin activation. Although active tension remained length sensitive, ACh-induced MLC phosphorylation was the same at optimal muscle length (L(o)) and 0.5 L(o) when intracellular Ca(2+) was maintained at pCa 7. MLC phosphorylation was also the same at L(o) and 0.5 L(o) in strips stimulated with 10 microM Ca(2+). In contrast, the Ca(2+)-insensitive tyrosine phosphorylation of FAK and paxillin stimulated by ACh was higher at L(o) than at 0.5 L(o). We conclude that the length-sensitivity of MLC phosphorylation depends on length-dependent changes in intracellular Ca(2+) but that length-dependent changes in MLC phosphorylation are not the primary mechanism for the length sensitivity of active tension.     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.     

Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells

Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.     

Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation

The inhibition of myosin phosphatase evokes smooth muscle contraction in the absence of Ca(2+), yet the underlying mechanisms are not understood. To this end, we have cloned smooth muscle zipper-interacting protein (ZIP) kinase cDNA. ZIP kinase is present in various smooth muscle tissues including arteries. Triton X-100 skinning did not diminish ZIP kinase content, suggesting that ZIP kinase associates with the filamentous component in smooth muscle. Smooth muscle ZIP kinase phosphorylated smooth muscle myosin as well as the isolated 20-kDa myosin light chain in a Ca(2+)/calmodulin-independent manner. ZIP kinase phosphorylated myosin light chain at both Ser(19) and Thr(18) residues with the same rate constant. The actin-activated ATPase activity of myosin increased significantly following ZIP kinase-induced phosphorylation. Introduction of ZIP kinase into Triton X-100-permeabilized rabbit mesenteric artery provoked a Ca(2+)-free contraction. A protein phosphatase inhibitor, microcystin LR, also induced contraction in the absence of Ca(2+), which was accompanied by an increase in both mono- and diphosphorylation of myosin light chain. The observed sensitivity of the microcystin-induced contraction to various protein kinase inhibitors was identical to the sensitivity of isolated ZIP kinase to these inhibitors. These results suggest that ZIP kinase is responsible for Ca(2+) independent myosin phosphorylation and contraction in smooth muscle.     

Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress

The mechanism by which vascular smooth muscle (VSM) cells modulate their contractility in response to structural cues from extracellular matrix remains poorly understood. When pulmonary VSM cells were cultured on increasing densities of immobilized fibronectin (FN), cell spreading, myosin light chain (MLC) phosphorylation, cytoskeletal prestress (isometric tension in the cell before vasoagonist stimulation), and the active contractile response to the vasoconstrictor endothelin-1 all increased in parallel. In contrast, MLC phosphorylation did not increase when suspended cells were allowed to bind FN-coated microbeads (4.5-microm diameter) or cultured on micrometer-sized (30 x 30 microm) FN islands surrounded by nonadhesive regions that support integrin binding but prevent cell spreading. Cell spreading and MLC phosphorylation also both decreased in parallel when the mechanical compliance of flexible FN substrates was raised. MLC phosphorylation was inhibited independently of cell shape when cytoskeletal prestress was dissipated using a myosin ATPase inhibitor in fully spread cells, whereas it increased to maximal levels when microtubules were disrupted using nocodazole in cells adherent to FN but not in suspended cells. These data demonstrate that changes in cell-extracellular matrix (ECM) interactions modulate smooth muscle cell contractility at the level of biochemical signal transduction and suggest that the mechanism underlying this regulation may involve physical interplay between ECM and the cytoskeleton, such that cell spreading and generation of cytoskeletal tension feed back to promote MLC phosphorylation and further increase tension generation.     

Phosphorylation of the 20,000-dalton light chain of myosin of intact arterial smooth muscle in rest and in contraction.

The 20,000-dalton myosin light chain of intact pig carotid arteries was found to be rapidly labeled when the arterial muscle was incubated in physiological salt solution at 37 degrees C containing [32P]orthophosphate. Light chain phosphorylation in the intact muscle had a marked requirement for Ca2+ and was dependent upon the passive tension applied to the muscle. Norepinephrine- or KCl-induced contractures were associated with concomitant increases in light chain phosphorylation.     

平滑肌肌球蛋白B的超沉淀与肌球蛋白轻链磷酸化

本文报导了牛胃肌球蛋白B(天然肌动球蛋白)的超沉淀性质。当钙离子、钙调蛋白和ATP存在时,肌球蛋白B出现超沉淀,在pH6.8和7.5处,有两个峰值。Ca~(2+)(PCa值8-4)对超沉淀影响的浓度-反应曲线呈典型的S形,表明当Ca~(2+)浓度处于微摩尔水平时产生超沉淀。伴随超沉淀发生了肌球蛋白调节轻链磷酸化。这说明肌球蛋白轻链的Ca~(2+)-CaM依赖性磷酸化可能包含在脊椎动物平滑肌收缩活动的调节机制中。