Myosin cross-bridge kinetics and the mechanism of catch

Catch force in molluscan smooth muscle requires little, if any, energy input and is controlled by the phosphorylation state of the thick filament-associated mini-titin, twitchin. The kinetic parameters of myosin cross-bridge turnover in permeabilized catch muscle, and how they are potentially modified by the catch mechanism, were determined by single turnover measurements on myosin-bound ADP. Under isometric conditions, there are fast and slow components of cross-bridge turnover that probably result from kinetic separation of calcium-bound and calcium-free cross-bridge pools. The structure responsible for catch force maintenance at intermediate [Ca+2] does not alter the processes responsible for the fast and slow components under isometric conditions. Also, there is no measurable turnover of myosin-bound ADP during relaxation of catch force by phosphorylation of twitchin at pCa > 8. The only effects of the catch link on myosin-bound ADP turnover are 1), a small, very slow extra turnover when catch force is maintained at very low [Ca+2] (pCa > 8); and 2), attenuation of the shortening-induced increase in turnover at subsaturating [Ca(+2)]. These limited interactions between the catch link and myosin cross-bridge turnover are consistent with the idea that catch force is maintained by a thick and thin filament linkage other than the myosin cross-bridge.     

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.     

Regulation of catch muscle by twitchin phosphorylation: effects on force, ATPase, and shortening.

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.     

The N-terminal region of twitchin binds thick and thin contractile filaments: redundant mechanisms of catch force maintenance

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.     

Twitchin of mollusc smooth muscles can induce “catch”-like properties in human skeletal muscle: support for the assumption that the “catch” state involves twitchin linkages between myofilaments

Molluscan catch muscles can maintain tension with low or even no energy utilization, and therefore, they represent ideal models for studying energy-saving holding states. For many decades it was assumed that catch is due to a simple slowing of the force-generating myosin head cross-bridge cycles. However, recently evidences increased suggesting that catch is rather caused by passive structures linking the myofilaments in a phosphorylation-dependent manner. One possible linkage structure is the titin-like thick filament protein twitchin, which could form bridges to the thin filaments. Twitchin is known to regulate the catch state depending on its phosphorylation state. Here, we found that twitchin induces a catch-like stiffness in skinned human skeletal muscle fibres, when these fibres are exposed to this protein. Subsequent phosphorylation of twitchin reduces the stiffness. These findings support the assumption that catch of molluscan smooth muscle involves twitchin linkages between thick and thin filaments.     

Paramyosin and the catch mechanism

1. Catch is a mechanism found in many molluscan smooth muscles in which tension is maintained at relatively low energy cost. 2. Paramyosin forms the core of thick filaments. In catch muscle paramyosin concentrations are high and the thick filaments are relatively long. 3. The mechanism of catch is not understood, but the consensus is that tension during catch is borne by slowly-cycling cross-bridge attachments to actin. 4. Stimulation by acetylcholine increases intracellular Ca2+ and initiates a contraction characterized by a relatively rapid cross-bridge cycling. Reduction of Ca2+ can lead to relaxation or catch. Relaxation occurs only when a second neurotransmitter, serotonin, is present. 5. The catch state is released by serotonin, via activation of adenylate cyclase, increased levels of cAMP and phosphorylation of one or more contractile proteins, possibly paramyosin. Other targets for phosphorylation are discussed. 6. The contractile cycle of catch muscles, therefore, is controlled by both Ca2+ and cAMP.     

Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.     

Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase

Catch in certain molluscan muscles is released by an increase in cAMP, and it was suggested that the target of cAMP-dependent protein kinase (PKA) is the high molecular weight protein twitchin [Siegman, M. J., Funabara, J., Kinoshita, S., Watabe, S., Hartshorne, D. J., and Butler, T. M. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 5384-5388]. This study was carried out to investigate the phosphorylation of twitchin by PKA. Twitchin was isolated from Mytilus catch muscles and was phosphorylated by PKA to a stoichiometry of about 3 mol of P/mol of twitchin. There was no evidence of twitchin autophosphorylation. Two phosphorylated peptides were isolated and sequenced, termed D1 and D2. Additional cDNA sequence for twitchin was obtained, and the D2 site was located at the C-terminal side of the putative kinase domain in a linker region between two immunoglobulin C2 repeats. Excess PKA substrates, e.g., D1 and D2, blocked the reduction in force on addition of cAMP, confirming the role for PKA in regulating catch. Papain proteolysis of (32)P-labeled twitchin from permeabilized muscles showed that the D1 site represented about 50% of the (32)P labeling. Proteolysis of in-situ twitchin with thermolysin suggested that the D1 and D2 sites were at the N- and C-terminal ends of the molecule, respectively. Thermolysin proteolysis also indicated that D1 and D2 were major sites of phosphorylation by PKA. The direct phosphorylation of twitchin by PKA is consistent with a regulatory role for twitchin in the catch mechanism and probably involves phosphorylation at the D1 and D2 sites.     

Twitchin, a thick-filament protein from molluscan catch muscle, interacts with F-actin in a phosphorylation-dependent way

Twitchin belongs to the titin-like giant proteins family, it is co-localized with thick filaments in molluscan catch muscles and regulates the catch state depending on its level of phosphorylation. The mechanism by which twitchin controls the catch state remains to be established. We report for the first time the ability of twitchin to interact with F-actin. The interaction is observed at low and physiological ionic strengths, irrespective of the presence or absence of Ca(2+). It was demonstrated by viscosity and turbidity measurements, low- and high-speed co-sedimentation, and with the light-scattering particle size analysis revealing the specific twitchin-actin particles. The twitchin-actin interaction is regulated by twitchin phosphorylation: in vitro phosphorylated twitchin does not interact with F-actin. We speculate that the catch muscle twitchin might provide a mechanical link between thin and thick filaments, which contributes to catch force maintenance.     

Regulation of the Ca2+ dependence of smooth muscle contraction.

Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.     

Temporal aspects of excitation-contraction coupling in airway smooth muscle.

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations (5-30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]i to force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]i oscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]i and contraction in intact porcine ASM cells was found to be approximately 450 ms. In beta-escin-permeabilized ASM strips, photolytic release of caged Ca2+ resulted in force generation after approximately 800 ms. When calmodulin (CaM) was added, this delay was shortened to approximately 500 ms. In the presence of exogenous CaM and 100 microM Ca2+, photolytic release of caged ATP led to force generation after approximately 80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20 and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred approximately 50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]i after photolytic release of caged diazo-2 resulted in relaxation after a delay of approximately 1.2 s and 50% reduction in force after approximately 57 s. We conclude that in ASM cells agonist-induced [Ca2+]i oscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.     

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399–4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K_a of (2.44 ± 0.09) × 10⁵ m⁻¹ with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K_a of (5.83 ± 0.05) × 10⁴ m⁻¹ with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca²⁺ sensitivity in that in the presence of Ca²⁺ complex formation was reduced.Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM)² and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch. Catch muscle develops active tension following an increase of the intracellular [Ca²⁺] induced by secretion of acetylcholine. Myosin is activated by direct binding of Ca²⁺ to the regulatory myosin light chain and initiates a relative sliding between thick and thin filaments (1). After a decrease of intracellular [Ca²⁺] to resting levels, the catch state is formed where tension is maintained over long periods of time with little energy consumption (2, 3). Catch tension is abolished by secretion of serotonin and an increase of intracellular [cAMP] with the resulting activation of cAMP-dependent protein kinase and phosphorylation of twitchin (4, 5). Twitchin phosphorylation is required for relaxation of the muscle from catch. For this cycle to repeat, dephosphorylation of twitchin is necessary (6). Thus, in this scheme, twitchin is a major regulator of the catch state.Molluscan twitchin is known as a myosin-binding protein belonging to the titin/connectin superfamily. It is a single polypeptide of 530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin type 3-like motifs in addition to a single kinase domain homologous to the catalytic domain of myosin light chain kinase of vertebrate smooth muscle (7). There are several possible phosphorylation sites in molluscan twitchin recognized by cAMP-dependent protein kinase, and two, D1 and D2, have been identified. The D1 phosphorylation site (Ser-1075) is in the linker region between the 7th and 8th Ig motifs (numbering from the N terminus). The D2 site (Ser-4316) is in the linker region between the 21st and 22nd Ig motifs. Additional sites are found close to D1, but are thought not to be vital for catch regulation.The molecular mechanisms underlying development and maintenance of the catch state have been controversial for several years. One theory proposes that catch reflected attached frozen or slowly cycling cross-bridges (8, 9). What distinguished the attached cross-bridge from the detached relaxed state is not clear. Also it was suggested that interactions between thick filaments, other than cross-bridges, or between thin and thick filaments are responsible for the catch contraction (10). In either of the latter cases, the cross-bridge (myosin head) was not involved.Recently we found that a twitchin fragment including the D2 phosphorylation site and its flanking Ig motifs (TWD2-S) interacted with myosin and actin in a phosphorylation-sensitive manner, and it was suggested that this trimeric complex contributed to tension maintenance in catch (11). TWD2-S bound to a region of the actin molecule known also to interact with loop 2 of myosin that is involved in the ATP-driven movement of myosin with actin (12). In the present study, we show that the myosin loop 2 binds to TWD2-S using competitive cosedimentation assays and isothermal titration calorimetry (ITC). These techniques were applied to also study in more detail the interactions of the twitchin-binding peptide of actin (identified in the previous study (11)). In addition, the effects of pH and Ca²⁺ on the binding of TWD2-S to myosin and actin were investigated.     

Ca2+]i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

The effects of force inhibition by sodium vanadate on cross-bridge binding, force redevelopment, and Ca2+ activation in cardiac muscle

Strongly bound, force-generating myosin cross-bridges play an important role as allosteric activators of cardiac thin filaments. Sodium vanadate (Vi) is a phosphate analog that inhibits force by preventing cross-bridge transition into force-producing states. This study characterizes the mechanical state of cross-bridges with bound Vi as a tool to examine the contribution of cross-bridges to cardiac contractile activation. The K(i) of force inhibition by Vi was approximately 40 microM. Sinusoidal stiffness was inhibited with Vi, although to a lesser extent than force. We used chord stiffness measurements to monitor Vi-induced changes in cross-bridge attachment/detachment kinetics at saturating [Ca(2+)]. Vi decreased chord stiffness at the fastest rates of stretch, whereas at slow rates chord stiffness actually increased. This suggests a shift in cross-bridge population toward low force states with very slow attachment/detachment kinetics. Low angle x-ray diffraction measurements indicate that with Vi cross-bridge mass shifted away from thin filaments, implying decreased cross-bridge/thin filament interaction. The combined x-ray and mechanical data suggest at least two cross-bridge populations with Vi; one characteristic of normal cycling cross-bridges, and a population of weak-binding cross-bridges with bound Vi and slow attachment/detachment kinetics. The Ca(2+) sensitivity of force (pCa(50)) and force redevelopment kinetics (k(TR)) were measured to study the effects of Vi on contractile activation. When maximal force was inhibited by 40% with Vi pCa(50) decreased, but greater force inhibition at higher [Vi] did not further alter pCa(50). In contrast, the Ca(2+) sensitivity of k(TR) was unaffected by Vi. Interestingly, when force was inhibited by Vi k(TR) increased at submaximal levels of Ca(2+)-activated force. Additionally, k(TR) is faster at saturating Ca(2+) at [Vi] that inhibit force by > approximately 70%. The effects of Vi on k(TR) imply that k(TR) is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca²⁺; and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin–myosin–myorod complex increased the actin-activated Mg²⁺-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.     

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.     

Adenosine triphosphate induces a low [Ca2+]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.