The mechanism of the inhibitory effect of 2,3-butanedione 2-monoxime (BDM) on contraction of frog skeletal muscles was studied using skinned fibres and aequorin-injected intact fibres. The tension development of skinned fibres directly activated with calcium was strongly inhibited by BDM. This agent also had effects on the sarcoplasmic reticulum in the skinned preparations, suppressing the calcium pump function and enhancing the activity of the 'calcium-induced calcium release' mechanism. In electrically stimulated intact fibres, although BDM slightly suppressed the elevation of the intracellular calcium ion concentration, this effect was so weak that it would not explain the strong inhibitory effect of the agent on the tension development by the intact fibres. It was concluded that the tension reducing effect of BDM on intact fibres was due mainly to its direct action on the contractile system. The mode of this action of BDM was further examined with skinned fibres in view of its effects on the maximum shortening speed and isometric tension in low MgATP environments.

SUMMARY1. In order to clarify the mechanism of contracture on cooling of caffeine-treated intact muscle fibres, the temperature dependence of a calcium (Ca2+) release mechanism, 'Ca2+-induced Ca2+ release', of the sarcoplasmic reticulum (SR) was examined in skinned frog muscle fibres.2. Skinned fibres in a solution containing 1-2 mM-caffeine and 07 mM-EGTA (Mg2+, 1-5 mm, Mg-ATP, 3-5 mm, pH 7), contracted on cooling (from 22 to 2°C) due to Ca2+ release from the SR.3. The rate of Ca2+ release from skinned fibre SR in a medium which contained Ca2+ ions (with 10 mM-EGTA) and no ATP salts, was determined under various conditions using the 'caffeine method'.4. In the absence of Mg2+ ions, adenine nucleotides and caffeine, the rates at room temperature (21-22°C) were 3-4 times greater than those at a lower temperature (1-5-3°C), at any concentrations of Ca2+ ions external to the SR.5. In the presence of Mg2+ ions (1-5 mM) and fl,y-methylene ATP (1 mM), the effect of temperature on the rates disappeared in Ca2+-containing media, although the effect remained in Ca2+-free medium.6. When caffeine (1-2 mM), which is a potentiator of the Ca2+-induced Ca2+ release, was added to the test medium with Mg2+ and fi,y-methylene ATP, the resulting potentiating effect was several times greater than that at lower temperature. 7. In order to examine the temperature dependence of the Ca2+ pump activity of the SR, the initial rate of Ca2+ uptake by the empty SR was determined under various conditions in the presence of Mg2+ ions (1-5 mM) and Mg-ATP (3 5 mM). The Q10 of the pump activity was around 2-0 at the Ca2+ ion concentrations examined (< 10-6 M).8. A numerical model based on the results obtained, together with some reasonable assumptions, suggested that both suppression of the Ca2+ pump and enhancement of the Ca2+ release contribute to the cooling contracture of caffeinized fibres.

Properties of both the contractile system and the sarcoplasmic reticulum (s.r.) of slow fibres from the iliofibularis muscle of Xenopus laevis were examined by using 'skinned' preparations mainly at 4 degrees C and pH 7.0. The results were compared with those of skinned fast fibres. The contractile system was activated with various concentrations of alkaline earth metal ions and it was found that the sensitivity of the contractile system of slow fibres to Sr2+ and Ba2+ was much higher than that of fast fibres while their Ca2+ sensitivity was similar. Caffeine (20-50 mM) reversibly induced appreciable active steady contraction of the slow fibre with one-third to one-half maximal isometric tension in the practical absence of Ca2+ (in the presence of 40 mM-EGTA) at relatively high temperatures (10-20 degrees C). The speed of unloaded shortening of caffeine-activated skinned slow fibres in the absence of Ca2+ was not slow but even faster than fibres activated to the same isometric tension level with Ca2+. When the s.r. of a skinned slow fibre was loaded with Ca2+ or Sr2+, 25 mM-caffeine caused transient contraction of the fibre due to a release of Ca2+ or Sr2+ from the s.r. The magnitude of such caffeine contracture was used as a quantitative indicator of the amount of Ca2+ or Sr2+ in the s.r. The dependence on [Ca2+] or [Sr2+] of the initial rate of Ca2+ or Sr2+ uptake by the empty s.r. of slow fibres was almost the same as that of fast fibres. In both types of fibres, a much higher concentration of Sr2+ than Ca2+ was required to activate the pump. When [Ca2+] outside the s.r. was sufficiently high (e.g. 10(-5) M), the maximum level to which the slow fibre s.r. could take up Ca2+ decreased, suggesting that the Ca2+-induced Ca2+ release mechanism also exists in slow fibre s.r. The rate of the Ca2+ leakage from slow fibre s.r. into the media without Ca2+ was higher than that from fast fibre s.r. In the absence of ATP, the enhancing effect of caffeine on the Ca2+-induced Ca2+ release mechanism was much weaker in slow fibres than in fast fibres, although adenosine-5'-monophosphate (AMP) enhanced it to a similar extent in both slow and fast fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca ~+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guineapig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20~ with half-time (t1#2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadalc acid (an inhibitor of myosin lightchain phosphatase) or thiophosphorylated with ATP3,S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the tl/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of ~ 10% of the tl/2 value. Some possible chemical and mechanical origins of the delay are discussed.

Changes in skeletal muscle of a patient with malignant hyperthermia (MH) were examined in skinned fibers. In the patient's muscle, the Ca-induced Ca release mechanism showed a significantly higher sensitivity to Ca than that in normal muscles and the maximum rate of Ca release at a sufficiently high concentration of Ca was also significantly higher. Halothane accelerated Ca-induced Ca release to a similar extent both in the patient's and normal muscles. No difference was observed in the properties of Ca uptake by the sarcoplasmic reticulum (SR) and of the contractile protein system between the patient's and normal muscles. The changes observed in the Ca-induced Ca release mechanism were considered to be sufficient to explain the disease.