Two elementary models for the regulation of skeletal muscle contraction by calcium

It is shown by use of an extremely simple explicit two-state model that two basic ideas may be sufficient to understand at least qualitatively the sensitive activation of isometric muscle contraction by Ca2+. (a) Ca2+ binds much more strongly on troponin if myosin is already attached to actin. The steady state analogue of this is that the single rate constant (in the two-state model) for myosin attachment plus Pi release is much larger if Ca2+ is bound to troponin. (b) End-to-end tropomyosin interactions are responsible for positive cooperativity. Although these ideas seem to be sufficient, this of course does not mean that they are necessary. These same ingredients were used in two previous, more elaborate models for the cooperative equilibrium binding of myosin subfragment-1 on actin-tropomyosin-troponin, with and without Ca2+, and for a study of the steady state ATPase activity of the same system. Essentially as an appendix, the above-mentioned simple treatment is extended to a somewhat more realistic and complicated model of isometric contraction.     

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.     

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.     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric 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.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxytocin contracts rat uterine smooth muscle in Ca2(+)-free medium without any phosphorylation of myosin light chain

Contraction of rat uterine smooth muscle related to phosphorylation state of myosin light chain under various conditions was investigated. In the Ca2(+)-containing medium, both high K+ and oxytocin induced marked contraction of the muscle accompanied by pronounced phosphorylation of myosin light chain. In the Ca2(+)-free medium, although both vanadate and oxytocin induced slight contraction, phosphorylation of myosin light chain was only evident for vanadate but not for oxytocin. It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2(+)-independent contraction of uterine smooth muscle elicited by oxytocin.     

Effect of phosphorylation of light chain myosin and Ca2+ on the conformation of F-actin during skeletal muscle contraction

The dependence of polarized fluorescence of rhodaminylphalloin specifically bound to F-actin and the tension developed by a fiber upon phosphorylation of myosin (18.5 kD) light chains as well as on the concentration of free Ca2+ was observed during the contraction of glycerinated rabbit skeletal muscle fibers. Still greater changes in the polarized fluorescence and higher values of tension were recorded for fibers with phosphorylated light chains at low (0.6 microM) Ca2+ concentrations as well as for those with dephosphorylated light chains at high (10 microM) Ca2+ concentrations. It is concluded that phosphorylation of myosin light chains modulates skeletal muscle contraction. The mechanisms of modulation involve conformational changes in F-actin.     

Adenosine 5'-triphosphate consumption by smooth muscle as predicted by the coupled four-state crossbridge model.

We have proposed a four-state crossbridge model to explain contraction and the latch state in arterial smooth muscle. Ca(2+)-dependent crossbridge phosphorylation was the only postulated regulatory mechanism and the latchbridge (a dephosphorylated, attached crossbridge) was the only novel element in the model. In this study, we used the model to predict rates of ATP consumption by crossbridge phosphorylation (JPhos) and cycling (JCycle) during isometric and isotonic contractions in arterial smooth muscle; then we compared model predictions with experimental data. The model predicted that JPhos and JCycle were similar in magnitude in isometric contractions, and both increased almost linearly with myosin phosphorylation. The predicted relationship between isometric stress and ATP consumption was quasihyperbolic, but approximately linear when myosin phosphorylation was below 35%, in agreement with most of the available data. Muscle shortening increased the predicted values of JCycle up to 3.7-fold depending on shortening velocity and the level of myosin phosphorylation. The predicted maximum work output per ATP was 7.4-7.8 kJ/mol ATP and was relatively insensitive to changes in myosin phosphorylation. The predicted increase in JCycle with shortening was in agreement with available data, but the model prediction that work output per ATP was insensitive to changes in myosin phosphorylation was unexpected and remains to be tested in future experiments.     

Relationship between length-tension relation and 20,000 dalton myosin light chain phosphorylation in guinea-pig taenia caeci

1. Relationship between length-tension relation and phosphorylation of 20,000 dalton myosin light chain (LC20) in guinea-pig taenia caeci was investigated. 2. At in situ length (Lb), a good linear correlation was obtained between isometric tension and LC20 phosphorylation in high-K+-stimulated muscle. 3. In 100 mM K+-stimulated muscle, the active tension decreased at muscle lengths other than Lb, but no significant decrease in degree of LC20 phosphorylation was observed. 4. These results suggest that in guinea-pig taenia caeci, the major portion of the decrease in active tension at muscle lengths other than Lb is not due to a decrease in degree of activation.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Phosphorylation of the regulatory light chains of myosin affects Ca2+ sensitivity of skeletal muscle contraction.

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.     

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation

Chemically skinned anterior byssus retractor muscle (ABRM) preparations were prepared by treatment with the nonionic detergents saponin and Triton X-100. Both maximum peak tension and rate of contraction were found to be greater in saponin-treated ABRM than in ABRM treated with Triton X-100. Active tension was initiated at a concentration of free Ca2+ above 0.1 microM, and maximum tension development was found at a [Ca2+] = approximately 32 microM. During exposure of the muscle preparation to optimal Ca2+ concentration, a high and almost constant tension level was sustained. The force recovery was high after a quick release during this period indicating the presence of an "active" state rather than a "catch" state. Actually, a state equivalent to the catch state in the living ABRM could not be induced, if the Ca2+ concentration was above 0.1 microM. Variations in the ionic strength in the range of 0.07--0.28 M had no influence on active state and only slightly affected the maximum tension developed. The influence of Mg2+ on the Ca2+-activated tension was examined by studying the tension-pCa relation at two concentrations of free Mg2+ (0.43 and 4.0 mM). The tension-pCa relation was found to be S-shaped with tension increasing steeply over approximately 1 pCa unit, indicating the existence of cooperativity between Ca2+ sites. Increasing the free concentration of Mg2+ shifted the tension-pCa relation to lower pCa as in striated muscles, demonstrating a decreasing Ca2+ sensitivity with increasing Mg2+. At [Mg2+] = 4.0 mM the half-maximum tension was found at [Ca2+] = 0.43 microM, decreasing to 0.20 microM at [Mg2+] = 0.43 mM. At both Mg2+ concentrations studied, plots of log Prel/(1--Prel) vs. log [Ca2+] were nonlinear with a shape indicating a rather complicated model for cooperativity, probably involving four sites for Ca2+. These Ca2+--Mg2+ interactions are most probably taking place at the myosin head itself because troponin is absent in this myosin-regulated muscle.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

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.     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.     

Regulation of smooth muscle contractile proteins by calmodulin and cyclic AMP

The various protein components of a reversible phosphorylating system regulating smooth muscle actomyosin Mg-ATPase activity have been purified. The enzyme catalyzing phosphorylation of smooth muscle myosin, myosin-kinase, requires Ca2+ and the Ca2+-binding protein calmodulin for activity and binds calmodulin in a ratio of 1 mol calmodulin to 1 mol of myosin kinase. Myosin kinase can be phosphorylated by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, and phosphorylation of myosin kinase that does not have calmodulin bound results in a marked decrease in the affinity of this enzyme for Ca2+-calmodulin. This effect is reversed when myosin kinase is dephosphorylated by a phosphatase purified from smooth muscle. When the various components of the smooth muscle myosin phosphorylating-dephosphorylating system are reconstituted, a positive correlation is found between the state of myosin phosphorylation and the actin-activated Mg-ATPase activity of myosin. Unphosphorylated and dephosphorylated myosin cannot be activated by actin, but the phosphorylated and rephosphorylated myosin can be activated by actin. The same relationship between phosphorylation and enzymatic activity was found for a chymotryptic peptide of myosin, smooth muscle heavy meromyosin. The findings reported here suggest one mechanism by which Ca2+ and calmodulin may act to regulate smooth muscle contraction and how cAMP may modulate smooth muscle contractile activity.     

Structural changes of cross-bridges on transition from isometric to shortening state in frog skeletal muscle

Structural changes in the myosin cross-bridges were studied by small-angle x-ray diffraction at a time resolution of 0.53 ms. A frog sartorius muscle, which was electrically stimulated to induce isometric contraction, was released by approximately 1% in 1 ms, and then its length was decreased to allow steady shortening with tension of approximately 30% of the isometric level. Intensity of all reflections reached a constant level in 5-8 ms. Intensity of the 7.2-nm meridional reflection and the (1,0) sampling spot of the 14.5-nm layer line increased after the initial release but returned to the isometric level during steady shortening. The 21.5-nm meridional reflection showed fast and slow components of intensity increase. The intensity of the 10.3-nm layer line, which arises from myosin heads attached to actin, decreased to a steady level in 2 ms, whereas other reflections took longer, 5-20 ms. The results show that myosin heads adapt quickly to an altered level of tension, and that there is a distinct structural state just after a quick release.     

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.