Thin-filament linked regulation of smooth muscle myosin

Phosphorylation of the regulatory light chain subunit of smooth muscle myosin is sufficient, but not necessary for muscle contraction. It has been suggested that thin-filament regulation may also contribute to the regulation of contraction. A hallmark feature of regulated thin filaments, previously described for vertebrate skeletal muscle, is the capacity of strong-binding or rigor-like cross bridges to turn-on the actin filament. Turned-on thin filaments stimulate cross-bridge attachment even in the absence of calcium. The present study utilized an in vitro sliding-filament motility assay to test for thin-filament regulation of both unphosphorylated and phosphorylated smooth muscle myosins. Regulated thin-filaments were reconstituted from skeletal muscle actin and chicken gizzard smooth muscle tropomyosin (Tm_CG), and then turned-on either (1) by rigor cross bridges at low concentrations of MgATP, or (2) by adding N-ethyl-maleimide-modified skeletal subfragment S1(NEM-S1), which forms rigor-like bonds in the presence of MgATP. For control actin·Tm_CG filaments, force production by unphosphorylated myosin was 0.5% of that produced by thiophosphorylated myosin. The force exerted on actin·Tm filaments by both unphosphorylated and phosphorylated myosins was increased by reducing the [MgATP] to 10–100 M MgATP (rigor-dependent activation). Force was also increased by actin·Tm_CG filaments that had been turned-on by NEM-S1 binding, with force production by unphosphorylated myosin increased 80-fold vs. 2.3-fold for thiophosphorylated myosin. Tm_CG was required for increased force production with both low MgATP and NEM-S1. Unloaded filament velocity for NEM-S1-activated thin filaments was 0.72 m/sec with unphosphorylated myosin compared to 1.24 m/sec with thiophosphorylated myosin. Taken together, these results suggest that thin-filament regulation may play a role in the activation of both unphosphorylated and phosphorylated smooth muscle myosins and suggest a possible mechanism for activation of slowly cycling unphosphorylated cross bridges (i.e. latch-state) during tonic contractions of smooth muscle.     

Skinned smooth muscle: calcium-calmodulin activation independent of myosin phosphorylation

In chemically skinned chicken gizzard smooth muscle fibers investigated shortly after preparation, a contraction may be induced by calcium and calmodulin which is independent of myosin phosphorylation at intermediate Ca²⁺-concentrations. However, fibers stored for a prolonged period also contract in the absence of exogenous calmodulin and exhibit a close relationship between force development and myosin phosphorylation.     

Ca binding of intestinal smooth muscle myosin B.

By means of both centrifugation and filtration techniques, the Ca binding activity of intestinal myosin B was studied. The binding capacity of myosin B was Ca dependent and was approximately linear when the concentration of Ca in the medium ranged from 10(-4) to 10(-7) M. The Ca sensitivity of ATPase activity in the same range of Ca concentration exhibited a sigmoid curve. The Scatchard plot of Ca binding showed that intestinal myosin B had at least two types of binding sites. One of these was defined as a high affinity site with an apparent affinity constant of 2.5 x 10(6) M-1. The other was supposed to be a low affinity site of Ca binding. Mild trypsin treatment reduced the Ca binding capacity of intestinal myosin B by 1.45-2.44 nmol/mg protein. These values are approximately the concentration of the high affinity Ca binding sites in the intestinal myosin B. A major concern regarding the effect of trypsin is that the reduction of Ca binding surely accompanied the elimination of Ca sensitivity of myosin B ATPase activity. From these results, it seems likely that the high affinity sites of Ca binding identified in this study are based on the troponin-like component included in intestinal myosin B.     

Length-dependent myosin phosphorylation and contraction of arterial smooth muscle

Ca²⁺, calmodulin-dependent myosin light chain phosphorylation is generally considered to be an important regulatory mechanism of smooth muscle contraction. We investigated the length dependence of myosin phosphorylation and active stress induced by K⁺ depolarization in arterial smooth muscle by measuring the two variables in the swine carotid media held at three steady-state tissue lengths — optimal length for contraction (L ₀), 1.5 L ₀, and slack length. We found that the length dependence of peak and steady-state myosin phosphorylation with respect to tissue length was different. Peak myosin phosphorylation was highest at L ₀ but lower at both slack length and 1.5 L ₀, whereas steady-state myosin phosphorylation was similar at both L ₀ and 1.5 L ₀, but lower at slack length. Stretching tissues to 1.5 L ₀ did not significantly change the steady-state myosin phosphorylation induced by K⁺ depolarization, but releasing tissues to slack length was associated with a 42% decrease in the steady-state myosin phosphorylation induced by K⁺ depolarization. These data indicated that one or more steps coupling membrane depolarization and Ca²⁺-dependent myosin phosphorylation were length sensitive. Additional data from skinned tissue experiments indicated that the length-sensitive step was not the coupling between Ca²⁺ and myosin phosphorylation. Therefore, these data together suggest that one or more steps coupling membrane depolarization and the increase in cytosolic Ca²⁺ concentration are length sensitive.     

Cable properties of smooth muscle

1. The cable properties of smooth muscle of guinea-pig taenia coli were studied by intracellular recording of electrotonic potentials produced by square current pulses and alternating current applied with external electrodes.2. An electrical model of the smooth muscle was constructed to test how the junctional resistance between cells affected the cable properties. The model consisted of a series of short cables (representing cells) which were connected by junctional resistances.3. It was concluded, from the experiments on the living tissue and on the model, that the electrotonic potential in smooth muscle can be expressed by the ordinary cable equation used for nerve and skeletal muscle fibres, even though the junctional resistance is of the same order of magnitude as that of the myoplasmic resistance.4. The cable equation was used to analyse the membrane parameters from the electrotonic potential, from the time course of the foot of the spike and from the conduction velocity. The analysis indicates that the smooth muscle has a membrane capacity of 2-3 muF/cm(2) and a membrane resistance of 30-50 kOmega cm(2).     

An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms

Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing onlyin a seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility than heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymerization of myosin on activation of rat anococcygeus smooth muscle

The in vivo state of assembly ofmyosin in vertebrate smooth muscle is controversial. In vitrostudies on purified smooth muscle myosin show that it ismonomeric (10S) under relaxing conditions and filamentous undercontraction conditions. Electron microscopic and antibodylabelling studies of intact smooth muscles, on the other hand,suggest that myosin is filamentous in the relaxed as well as thecontracting state and that 10S myosin occurs only in traceamounts. However, birefringence, conventional EM and X-raydiffraction evidence suggests that in certain smooth muscles invivo (e.g. rat anococcygeus), while myosin filaments exist in therelaxed state, their number increases on contraction. Here, wehave used low temperature electron microscopic techniques (rapidfreezing followed by freeze-substitution), which preserve labilecomponents in close to their in vivo state, to detect any changein filament number on contraction. The results from ratanococcygeus have been compared with those from guinea pig taeniacoli, in which other techniques have revealed no change infilament number. In the anococcygeus, we find evidence for a 23%increase in filament density in transverse sections ofcontracting muscle compared with relaxed muscle. In the taeniacoli we find no change. These results are in qualitativeagreement with earlier findings. They provide evidence forpolymerization of myosin in contracting rat anococcygeus, andsuggest that this process is subtle and occurs only in somesmooth muscles     

Regulation of the smooth muscle contractile phenotype by nonmuscle myosin

The contractile phenotype of a smooth muscle can broadly be classified as phasic or tonic. Following activation, phasic smooth muscle exhibits an initial period of rapid force activation, following which force falls to a lower steady state level. In contrast, force generated by tonic smooth muscle rises slowly to a sustained steady state. The differences in contractile patterns cannot be explained by the time course of either the Ca(2+) transient or phosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)). Therefore, a molecular marker that defines tonic and phasic smooth muscle contractile properties remains elusive. Further, smooth muscle can maintain force at low levels of MLC(20) phosphorylation; often referred to as the latch state. The mechanism for the latch state is unknown and has been hypothesized to be due to a number of mechanisms including the formation of slowly cycling dephosphorylated or latch cross-bridges (Hai and Murphy, Am J Physiol 253:H1365-H1371, 1988). This review will focus evidence suggesting that nonmuscle myosin IIB (NMIIB) are the latch cross-bridges in smooth muscle and NMIIB content could define the tonic contractile phenotype.     

Vectorial phosphorylation of filamentous smooth muscle myosin by calmodulin and myosin light chain kinase complex

Vertebrate smooth muscle myosin extracted from myofibrils and isolated via filament assembly was co-purified with calmodulin (CaM) and myosin light chain kinase (MLCK) which are tightly associated with the filament architecture and, therefore, it may be considered as a native-like preparation. These endogenous contaminates also co-precipitated with a native-like actomyosin, for both cases, at levels sufficient to fully phosphorylate myosin within 10–20 s after addition of ATP and calcium, although their molar ratio to myosin was only about 1 to 100. Phosphorylation progress curves obtained from mixtures of the native-like, and CaM- and MLCK-free filaments indicated that the CaM/MLCK complex preferentially phosphorylated its parent filaments and, as result, the whole myosin present was not maximally phosphorylated. Solubilization of the filaments' mixtures at high ionic strength resulted in slower phosphorylation rates but with maximal phosphorylation levels being attainable. Similar observations were made on the filamentous myosin system reconstituted from the kinase- and CaM-free myosin with added purified MLCK and CaM as well as on the native-like myosin from which only one of these endogenous contaminates was removed by affinity chromatography. These data indicated that not only the MLCK but also CaM was necessary for the observed preferential phosphorylation kinetics. Thus, the native-like filamentous myosin appeared to be phosphorylated by some kind of vectorial mechanism. Similar experiments were carried out on the native-like actomyosin where these vectorial effects were even more pronounced.     

Characterization of smooth muscle organs by determination of cellular myosin content

The mean relative content of myosin of cells from smooth muscular organs was estimated using immunocytochemical methods after enzymatical tissue disintegration. The cells were classified according to myosin content. Aortas and urinary bladders of newborn rats and chicken amnions were used as models. Cells of a permanent cell strain, being free of smooth muscular myosin served as negative controls. It could be shown, that the content of myosin, estimated by immunocytochemical methods correlates with the state of development of smooth muscular organs.     

Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca²⁺ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of smooth muscle myosin in myoid cells of the testes

Smooth muscle myosin was found by the Coons' immunomorphological test in the outer wall of the seminiferous tubules of man, rats, and mice. The results of the investigation confirm the smooth-muscle nature the myoid cells.Laboratory of General Pathological Anatomy, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Strukov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsniny, Vol. 82, No. 12, pp. 1499–1501, December, 1976.     

Heterogeneity of myosin antigenic expression in vascular smooth muscle in vivo

Rabbit antisera elicited against purified human nonmuscle (platelet) and smooth muscle (uterine myometrium) myosins identified distinct species of myosin when frozen sections of a variety of mammalian tissues were examined by immunofluorescence microscopy. Antiplatelet myosin antiserum specifically stained several nonmuscle cell types including epithelial, some connective tissue, and all vascular endothelial (arterial, venous, capillary) cells. Antismooth muscle myosin antiserum stained only smooth muscle and no other cell types. Neither antiserum reacted with rat cardiac (ventricular) or skeletal muscle cells. Antismooth muscle myosin antiserum staining was detectable in medial vascular smooth muscle in all vessels examined from rat, bovine, human, and guinea pig sources (including elastic and muscular arteries, arterioles, venules, and veins). Although antiplatelet myosin antiserum did not stain nonvascular smooth muscle or vascular smooth muscle in muscular arteries, arterioles, venules, or veins, it did uniformly and specifically stain medial vascular smooth muscle in elastic arteries. This staining of elastic arteries was abolished by absorption of antiplatelet myosin antiserum with purified platelet myosin but not uterine myosin. Similarly, the reactivity of antismooth muscle myosin antiserum was abolished by incubation with uterine but not platelet myosin. The differences in staining patterns observed with antiplatelet myosin antiserum and antismooth muscle myosin antiserum in elastic arteries versus other blood vessels suggests a heterogeneity of antigenic expression in vascular smooth muscle myosin. The most likely explanations for this heterogeneity are the presence of different gene products (myosin isozymes) or a posttranslational alteration (possibly conformational) of a single myosin species. Heterogeneity in this important component of the contractile apparatus of vascular smooth muscle may have significant implications for the physiology and pathophysiology of the vessel wall.