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We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919-3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg(2+)-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met1-Gly773) and the C-terminal long alpha-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly773-Ser1104) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.

1. The effects of inorganic phosphate (P(i)) on force, Ca(2+)-force relationship, ATPase activity, maximal shortening velocity (Vmax) and rate of tension development were investigated in chemically skinned preparations of smooth muscle from the guinea-pig taenia coli. 2. In maximally thiophosphorylated fibres, P(i) in the range 1-40 mM inhibited isometric force, with a reduction of 20% at 20 mM P(i). 3. The relative force was similar at all [Ca2+], i.e. the Ca(2+)-force relationship was not affected, when 20 mM P(i) was present. 4. After photolytic release of ATP from caged ATP in maximally thiophosphorylated fibres in the presence of 20 mM P(i), tension rose to a lower level but with a higher rate constant than in the absence of P(i). 5. Inorganic phosphate (20 mM) did not affect the ATP hydrolysis in fibres activated at intermediate [Ca2+] or by maximal thiophosphorylation. 6. Inorganic phosphate (20 mM) decreased force but did not influence Vmax in maximally activated fibres. At lower levels of activation by Ca2+, P(i) increased the Vmax and decreased force slightly without affecting the degree of myosin light chain phosphorylation. 7. We conclude that P(i) influences cross-bridge reactions associated with force generation in smooth muscle. These reactions are not rate limiting for cross-bridge turnover under isotonic or isometric conditions in maximally activated smooth muscle fibres, since P(i) did not influence Vmax or the rate of ATP turnover. 8. Since P(i) increased Vmax in submaximally activated muscles, we propose that, under these conditions, shortening velocity is rate limited by cross-bridge states, reached early after attachment, which impose a mechanical resistance to shortening.

The primary goal of this study was to determine the utility of 2,3-butanedione monoxime as a tool for determining and separating the chemical energy usage associated with force production from that of force-independent, or 'activation' processes in smooth and skeletal muscles. We determined the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and high energy phosphate usage in intact and permeabilized smooth (rabbit taenia coli) and skeletal (mouse extensor digitorum longus) muscles. In the intact taenia coli, 2,3-butanedione monoxime depressed the tonic phase of the tetanus, contractures evoked by high potassium (90 mM) and by carbachol (10(-5) M) and the small force response evoked by these agonists after treatment with D-600 (10(-5) M). In the electrically stimulated intact taenia coli 2,3-butanedione monoxime (0-20 mM) caused a proportional inhibition of tetanic force output, myosin light chain phosphorylation and high energy phosphate usage (ED50 approximately 7 mM for all these parameters). At 20 mM 2,3-butanedione monoxime, force and energy usage fell to near zero and the degree of myosin light chain phosphorylation decreased to resting values, indicating a shut-down of both force-dependent and force-independent energy usage at high concentrations of 2,3-butanedione monoxime. In permeabilized taenia coli, 2,3-butanedione monoxime had little or no depressant effects on force production, ATPase activity or calcium sensitivity. 2,3-butanedione monoxime had a very modest inhibitory effect on the in vitro motility of unregulated actin filaments interacting with thiophosphorylated myosin. In solution, 2,3-butanedione monoxime inhibited myosin light chain kinase, but not the phosphatase (SMP-IV). These results suggest that the major effect of 2,3-butanedione monoxime is not on the contractile proteins themselves, but rather on calcium delivery during excitation, thereby reducing the degree of activation of myosin light chain kinase and subsequent activation of myosin by light chain phosphorylation. Thus, 2,3-butanedione monoxime is not useful for the determination of the energetics of activation processes in smooth muscle because of its inhibition of both force-dependent and force-independent processes. In contrast, in the intact mouse extensor digitorum longus, 2,3-butanedione monoxime inhibits tetanic force production (ED50 approximately 2 mM) to a much greater extent than myosin light chain phosphorylation. When 2,3-butanedione monoxime was used to manipulate force production in muscles at L(o), it was found that approximately 60% of the total energy usage was force-independent and the remainder was force-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

A full-length cDNA of smooth muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system. The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy [Morita, J., Takashi, R., & Ikebe, M. (1991) Biochemistry 30, 9539-9545]. The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity. To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains. Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19. Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation. The Asp/Asp mutant further activated actomyosin ATPase activity. On the other hand, the Ala mutant did not affect the ATPase activity. Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin. The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin. These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position. Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied. Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18.(ABSTRACT TRUNCATED AT 250 WORDS)

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.

The direct effect of okadaic acid (OA) on the ATP-dependent interaction between actin and myosin of smooth muscle was examined not only by the conventional measurement of ATPase activity but also by application of in vitro motility assay developed recently. The motility was effectively enhanced by microM levels of OA. Measurements of the activities of myosin confirmed that the myosin mediated this effect. The result of this study, which was carried out in the absence of protein phosphatase, are not compatible with the recent reports that the stimulatory effect of OA on smooth muscle contraction is attributable to its inhibitory effect on the activity of the protein phosphatase.

The effects of cadmium (Cd) on smooth muscle myosin phosphorylation have been investigated using an in vitro system comprising myosin filaments containing endogenous calmodulin (CM) and myosin light chain kinase (MLCKase). In the absence of calcium (Ca), Cd as well as some other divalent cations caused no activation of phosphorylation. However, when at least one (or possibly two) Ca2+ were bound per CM, the addition of 10 microM to 40 microM Cd2+ resulted in a 2 to 3 fold acceleration of the phosphorylation rate. Higher Cd concentrations caused inhibition of the system independent of Ca2+ concentration through the formation of Cd-ATP complexes. These results explain some previously controversial data on the complex effects of Cd in intact smooth muscles.