Summary
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 ∼ 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 ∼ 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 L0, it was found that ∼60% of the total energy usage was force-independent and the remainder was force-dependent. In the permeabilized extensor digitorum longus treated with 12 mM 2,3-butanedione monoxime, there was a decrease in calcium-activated force production and a decrease in calcium sensitivity. The effects of 2,3-butanedione monoxime were considerably greater in the intact than in the permeabilized mouse extensor digitorum longus. At 2,3-butanedione monoxime concentrations that block force production in the intact muscle, the effects on in vitro motility were small, yet far greater than those on smooth muscle myosin. These results suggest that 2,3-butanedione monoxime has a direct effect on the contractile proteins, but what cannot be ignored is the decrease in myosin light chain phosphorylation in the skeletal muscle, which, like the decreased force output, may result from a reduction in calcium release from the sarcoplasmic reticulum. For these reasons, the use of 2,3-butanedione monoxime to probe the components of energy usage during the contraction of skeletal muscle requires considerable caution and a full definition of its actions.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
ALPERT, N. R., BLANCHARD, E. M. & MULIERI, L. A. (1989) Tensionindependent heat in rabbit papillary muscle. J. Physiol. 414, 433–53.
BAGNI, M. A., CECCHI, G., CoLOMO, F. & GARZELLA, P. (1992) Effects of 2,3-butanedione monoxime on the crossbridge kinetics in frog single muscle fibres. J. Muscle Res. Cell Motil. 13, 516–22.
BARSOTTI, R. J. & BUTLER, T. M. (1984) Chemical energy usage and myosin light chain phosphorylation in mammalian skeletal muscle. J. Muscle Res. Cell Motil. 5, 45–64.
Blanchard, E. M., Mulieri, L. A. & Alpert, N. R. (1984) The effect of 2,3-butanedione monoxime (BDM) on the relation between initial heat and mechanical output of rabbit papillary muscle. Biophys. J. 45, 48a.
BLANCHARD, E. M., SMITH, G. L., ALLEN, D. G. & ALPERT, N. R. (1990) The effects of 2,3-butanedione monoxime on initial heart, tension, and aequorin light output of ferret papillary muscles. Pflügers Arch. 416, 219–21.
BLINKS, J. R., RUDEL, R. & TAYLOR, S. R. (1978) Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. 277, 291–323.
BUTLER, T. M. & SIEGMAN, M. J. (1983) Chemical energy usage and myosin light chain phosphorylation in mammalian smooth muscle. Fed. Proc. 42, 57–61.
BUTLER, T. M., SIEGMAN, M. J., MOOERS, S. U. & DAVIES, R. E. (1978) Chemical energetics of single isometric tetani in mammalian smooth muscle. Am. J. Physiol. 235, C1–7.
BUTLER, T. M., SIEGMAN, M. J., MOOERS, S. U. & NARAYAN, S. R. (1990) Myosin-product complex in the resting state and during relaxation of smooth muscle. Am. J. Physiol. 258, C1092–9.
EGGLETON, P., ELSDEN, S. R. & GOUGH, N. (1943) The estimation of creatine and of diacetyl. Biochem. J. 37, 526–9.
FRANK, J. S. & WINEGRAD, S. (1976) Effect of muscle length on 45Ca efflux in resting and contracting skeletal muscle. Am. J. Physiol. 231, 555–9.
FRYER, M. W., GAGE, P. W., NEERING, I. R., DULHUNTY, A. F. & LAMB, G. D. (1988a) Paralysis of skeletal muscle by butanedione monoxime, a chemical phosphatase. Pflügers Arch. 411, 76–9.
FRYER, M. W., NEERING, I. R. & STEPHENSON, D. G. (1988b) Effects of 2,3-butanedione monoxime on the contractile activation properties of fast and slow twitch rat muscle fibres. J. Physiol. 407, 53–75.
GAGELMANN, M., GUTH, K. & RUEGG, J. C. (1984) Stretch induced tension rise in a molluscan smooth muscle skinned by freeze-drying. J. Comp. Physiol. 154, 187–9.
GIBBS, C. L. & LOISELLE, D. (1978) The energy output of tetanized cardiac muscle: species differences. Pflügers Arch. 373, 31–8.
GORDON, A. R. & SIEGMAN, M. J. (1971) Mechanical properties of smooth muscle. I. Length-tension and force-velocity relations. Am. J. Physiol. 221, 1243–7.
GREEN, A. L. & SaVILLE, B. (1956) The reaction of oxime with isopropyl methylphosphonofluoridate (sarin). J. Chem. Soc. 756, 3887–92.
GUTH, K. & JUNGE, J. (1982) Low Ca2+ impedes crossbridge detachment in chemically skinned taneia coli. Nature 300, 775–6.
GUTH, K. & WOJCHIECHOWSKI, R. (1986) Perfusion cuvette for simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibers. Pflügers Arch. 407, 1–6.
GWATHMEY, J. K., HAJJAR, R. J. & SOLARO, R. J. (1991) Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium. Circ. Res. 69, 1280–92.
HAEBERLE, J. R., COOLICAN, S. A., EVAN, A. & HATHAWAY, D. R. (1985) The effects of a calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle. J. Muscle Res. Cell Motil. 6, 347–63.
HERRMANN, C., WRAY, J., TRAVERS, F. & BARMAN, T. (1992) Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPase. An example of an uncompetitive inhbitor. Biochemistry 31, 12227–32.
HIGUCHI, H. & TAKEMORI, S. (1989) Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle. J. Biochem. 105, 638–43.
HOMSHER, E. & KEAN, C. J. (1978) Skeletal muscle energetics and metabolism. Ann. Rev. Physiol. 40, 93–131.
HOMSHER, E., MOMMAERTS, W. F. H. M., RICCHIUTI, N. V. & WALLNER, A. (1972) Activation heat, activation metabolism and tension-related heat in frog semitendinosus muscles. J. Physiol. 220, 601–25.
HORIUTI, K., HIGGUCHI, H., UMAZUME, Y., KONISHI, M., OKAZAKI, O. & KURIHARA, S. (1988) Mechanism of action of 2,3-butanedione 2-monoxime on contraction of frog skeletal muscle fibres. J. Muscle Res. Cell Motil. 9, 156–64.
HUANG, G. J. & MCARDLE, J. J. (1992) Novel suppression of an L-type calcium channel in neurones of murine dorsal root ganglia by 2,3-butanedione monoxime. J. Physiol. 447, 257–74.
Hui, C. S. & Maylie, J. (1988) Effects of BDM on contractile and membrane electrical properties in frog twitch fibres. Biophys. J. 53, 646a.
IKEBE, M., HARTSHORNE, D. J. & ELZINGA, M. (1987a) Phosphorylation of the 20 000 dalton light chain of smooth muscle myosin by the calcium activated, phospholipid dependent protein kinase. J. Biol. Chem. 262, 9569–73.
IKEBE, M., STEPINSKA, M., KEMP, B. E., MEANS, A. R. & HARTSHORNE, D. J. (1987b) Proteolysis of smooth muscle myosin light chain kinase: formation of inactive and calmodulin-independent fragments. J. Biol. Chem. 262, 13828–34.
KAWAI, M., GUTH, K., WINNIKES, K., HAIST, C. & RUEGG, J. C. (1987) The effect of inorganic phosphate on the ATP hydrolysis rate and tension transients in chemically skinned rabbit psoas fibers. Pflügers Arch. 408, 1–9.
KRETZSCHMAR, K. M. & WILKIE, D. R. (1969) A new approach to freezing tissues rapidly. J. Physiol. 202, 66–7P.
KUHN, H., TEWES, A., GAGELMANN, M., GUETH, K., ARNER, A. & RUEGG, J. C. (1990) Temporal relationship between force. ATPase activity, and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle. Pflügers Arch. 416, 512–81.
LANG, R. J. & PAUL, R. J. (1991) Effects of 2,3-butanedione monoxime on whole cell Ca2+ channel currents in single cells of the guinea pig taenia caeci. J. Physiol. 433, 1–24.
Lenart, T. D., Tanner, J. W. & Goldman, Y. E. (1989) 2,3-Butanedione monoxime (BDM) suppresses crossbridge reattachment following laser photolysis of caged ATP. Biophys. J. 55, 260a.
LI, T., SPERELAKIS, N., TENEICK, R. E. & SOLARO, J. (1985) Effects of diacetyl monoxime on cardiac excitation-contraction coupling. J. Pharmacol. Exper. Thera. 232, 688–95.
MANNING, D. R. & STULL, J. T. (1979) Myosin light chain phosphorylation and phosphorylase A activity in rat extensor digitorum longus muscle. Biochem. Biophys. Res. Commun. 90, 164–70.
Maylie J. & Hui C. S. (1988) Effects of BDM on antipyrylazo II calcium signals in frog cut twitch fibers. Biophys. J. 53, 646a.
MORELAND, S., IKEBE, M., HUNT, J. T. & MORELAND, R. S. (1992a) Substrate based inhibitors of smooth muscle myosin light chain kinase. Biochem. Biophys. Res. Comm. 185, 379–85.
MORELAND, S., NISHIMURA, J., van, BREEMEN, C., AHN, H. Y. & MORELAND, R. S. (1992b) Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries. Am. J. Physiol. 263, C540–4.
MRWA, U., GUTH, K., HAIST, C., TROSCHKA, M., HERRMANN, R., Wojciechowski, R. & Gagelmann, M. (1986) Calcium requirement for activation of skinned vascular smooth muscle from spontaneously hypertensive (SHRSP) and normotensive control rats. Life Sciences 38, 191–6.
Mulieri, L. A. & Alpert, N. R. (1984) Differential effects of 2,3-butanedione monoxime (BDM) on activation and contraction. Biophys. J. 45, 47a.
OSTERMAN, A., ARNER, A. & MALMQVIST, U. (1993) Effects of 2,3-butanedione monoxime on activaton of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli. J. Muscle Res. Cell Motil. 14, 186–94.
OTUN, H., GILLESPIE, J. I., GREENWELL, J. R. & DUNLOP, W. (1993) The actions of caffeine and 2,3-butanedione monoxime on calcium transients in human vascular smooth muscle. Exper. Physiol. 78, 255–8.
PACKER, S., KAGAN, J. F., ROBERTSON, S. A. & STEPHENS, N. L. (1988) The effect of 2,3-butanedione monoxime (BDM) on smooth muscle mechanical properties. Pflügers Arch. 412, 659–64.
PATO, M. D. & KERC, E. (1985) Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. J. Biol. Chem. 260, 12359–66.
PETERSON, J. W. & PAUL, R. J. (1974) Effects of initial lengh and active shortening on vascular smooth muscle contractility. Am. J. Physiol. 227, 1019–25.
SADA, H., SADA, S. & SPERELAKIS, N. (1985) The calcium channel agonist. Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues. Can. J. Physiol. Pharmacol. 63, 1267–70.
SIEGMAN, M. J., BUTLER, T. M., MOOERS, S. U. & DAVIES, R. E. (1980) Chemical energetics of force development force maintenance and relaxation in mammalian smooth muscle. J. Gen. Physiol. 76, 609–29.
SIEGMAN, M. J., BUTLER, T. M., MOOERS, S. U. & MICHALEK, A. (1984) Ca2+ can affect V max without changes in myosin light chain phosphorylation in smooth muscle. Pflügers Arch. 401, 385–90.
STEPHENSON, D. G. & WILLIAMS, D. A. (1981) Calcium-activated force responses in fast and slow-twitch skinned muscle fibres of the rat at different temperatures. J. Physiol. 317, 281–302.
SWEENEY, H. L. & STULL, J. T. (1986) Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells. Am. J. Physiol. 250, C657–60.
WALSH, M. P., HINKINS, S., DABROWSKA, R. & HARTSHORNE, D. J. (1983) Smooth muscle myosin light chain kinase. Methods Enzymol. 99, 279–88.
Warren, T. B., Butler, T. M., Siegman, J. M. & Mooers, S. U. (1985) Effects of 2,3-butanedione monoxime (BDM) on force production and myosin light chain phosphorylation (MyLCP) in mammalian smooth and skeletal muscle. Biophys. J. 47, 295a.
WARSHAW, D. M., DESROSIERS, J. M., WORK, S. S. & TRYBUS, K. M. (1990) Smooth muscle myosin crossbridge interactions modulate actin filament sliding in vitro. J. Cell Biol. 111, 453–63.
WARSHAW, D. M., DESROSIERS, J. M., WORK, S. S. & TRYBUS, K. M. (1991) Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin. J. Biol. Chem. 266, 24339–43.
WATANABE, M. (1993) Effects of 2,3-butanedione monoxime on smooth-muscle contraction of guinea-pig portal vein. Pflügers Arch. 425, 462–8.
Wendt, I. R. & Lang, R. J. (1987) Effects of 2,3-butanedione monoxime on smooth muscle contraction. Proc. Australian Physiol. Pharmacol. Soc. 18, 33P.
WIGGINS, J. R., REISER, J., FITZPATRICK, D. F. & BERGEY, J. L. (1980) Inotropic actions of diacetyl monoxime in cat ventricular muscle. J. Pharmacol. Exper. Thera. 212, 217–24.
WILSON, I. B. & GINSBERG, S. (1955) A powerful reactivator of alkyl-phosphate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 18, 168–75.
WOLEDGE, R. C. (1971) Heat production and chemical change in muscle. Prog. Biophys. 22, 37–74.
WORK, S. S. & WARSHAW, D. M. (1992) Computer-assisted tracking of actin filament motility. Anal. Biochem. 202, 275–85.
YAGI, N., TAKEMORI, S., WATANABE, M., HORIUTI, K. & AMEMIYA, Y. (1992) Effects of 2,3-butanedione monoxime on contraction of frog skeletal muscles: An X-ray diffraction study. J. Muscle Res. Cell Motil. 13, 153–60.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Siegman, M.J., Mooers, S.U., Warren, T.B. et al. Comparison of the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and chemical energy usage in intact and permeabilized smooth and skeletal muscles. J Muscle Res Cell Motil 15, 457–472 (1994). https://doi.org/10.1007/BF00122119
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00122119