Glycerol-treated Muscle Fibers Research Articles

Factors affecting the transient tension change after applying stepwise length change to glycerol-treated muscle fibers. Effects of temperature, divalent cations, and modification with p-chloromercuribenzoate.

The dynamic properties of cross-bridge movement were investigated in glycerol-treated muscle fibers under various conditions by analyzing tension responses to two types of length change. First, the fiber bundles were stretched linearly with time for 0.3 s from the rest length (L0) by 2.5% of L0, suddenly released, then fixed at L0 (sudden release of the slow stretch). Second, they were stretched for 0.01 s by 2.5% of L0, then held at the plateau length (a quick stretch). 1. The transient tension responses following both length changes were divided into three phases: (i) very quick recovery of tension (0 approximately 0.05 s), (ii) quick recovery (0.05 approximately 0.3-0.4 s), and (iii) gradual recovery (0.3-0.4 s approximately several seconds). 2. The effects of activating conditions on the rates of the quick phases (0 approximately 0.3-0.4 s) were not associated with those on the nucleoside triphosphatase [EC 3.6.1.3] rates: the rates of the quick phases increased with increase in temperature and Mg2+-ATP concentration, with decrease in Ca2+ concentration, and also on replacement of Mg2+-ATP by Mg2+-ITP or Mn2+-ATP. Only a small amount of ADP, 0.07 mol per mol of myosin (Fig. 24 in the preceding paper), was liberated during the quick recovery phases. 3. The remaining slow tension recovery was concluded to be associated with one cycle of ATP splitting, and progressed very smoothly. This suggests that most of the cross-bridges do not exist in a synchronously dissociated state during one cycle of ATP splitting.

Coupling of Movement of Cross-Bridges with ATP Splitting Studied in Terms of the Acceleration of the ATPase Activity of Glycerol-Treated Muscle Fibers on Applying Various Types of Repetitive Stretch-Release Cycles1

Glycerol-treated muscle fiber bundles were subjected to three types of repetitive stretch-release cycle, and the Mg2+-ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was determined from the rate of decrease in NADH absorbance by linking the ATPase reaction with the pyruvate kinase [EC 2.7.1.40] and lactate dehydrogenase [EC 1.1.1.27] system. The fiber bundles were stretched linearly with time for 0.3 s by 2.5% of the rest length (Lo), released suddenly, then fixed at Lo for a seconds. The length change cycle was repeated (repetitive jagged stretch-release followed by return to rest length). The activity of the oscillated fibers increased with increase in the value of α, and at α=2 s, it reached a maximal level of up to 1.9 times that of static fibers. It decreased gradually with further increase in α to a value slightly larger than that of static fibers. The calculated time course of ADP liberation after the sudden release of slow stretch showed a sigmoidal increase, a decrease in the rate to almost zero, then a return to the rate of static fibers. The amount of ADP liberated at the end of the change was about 0.7 mol per mol of myosin. The fiber bundles were stretched linearly with time for β seconds by 2.5 % of Lo, then restretched immediately after a sudden release to Lo. The cycle was repeated (simple repetitive jagged stretch-release). The ATPase activity increased with increase in β and at β≈0.06 s, it reached a maximal level of up to 1.75 times that of static fibers. It decreased gradually with further increase in β to a value slightly larger than that of static fibers. The fiber bundles were stretched by 2.5% of Lo for 0.01 s, held at the plateau length, then re-stretched immediately after a sudden release to Lo. The cycle was repeated (intermittent stretch-release). The ATPase activity increased with increase in the length of the cycle (β) and at β≈ 0.05 s, it reached a maximal level of up to 1.8 times that of static fibers (the first acceleration). Then it decreased gradually with increase in β4, becoming nearly equal to that of static fibers, and increased again with further increase in β to reach a maximal level of up to 1.5 times that of static fibers at β≈1 s (the second acceleration). Finally, it decreased with further increase in β to a value slightly larger than that of static fibers. The acceleration of ATPase activity on applying a repetitive jagged stretch-release followed by return to rest length and the second acceleration on applying an intermittent stretch-release had the following common properties. Both were saturated at ΔL/Lo≈2%, showed substrate inhibition, and markedly decreased when ATP was replaced with ITP or Mg2+with Mn2+. They were suppressed by modification of the muscle fibers with PCMB. The value of α or β for the maximum acceleration decreased to one-fourth when the temperature was raised from 3−4°C to 20°C, and increased to 4–5 s when the Ca2+concentration was lowered from 10 to 0.1 μM. All these results, together with those mentioned in point 1, clearly indicate that one cycle of ATP splitting of cross-bridges is synchronously initiated by the dissociation of cross-bridges by both types of length change. On the other hand, the acceleration of ATPase activity on applying a simple repetitive jagged stretch-release and the first acceleration on applying an intermittent stretch-release had the following common properties. Both showed a bell-shaped dependence on the amplitude of the length change (ΔL/Lo) with the maximum at ΔL/Lo of 2–3%. They were almost unaffected by raising the temperature or lowering the Ca2+concentration. Furthermore, the first acceleration on intermittent stretch-release was only slightly affected by change in ATP concentration, and by replacement of ATP with ITP or Mg2+with Mn2+, while it was markedly enhanced by modification of the muscle fibers with PCMB. These results strongly suggest that the ATPase rate of one cross-bridge increases markedly at an angle greater than 90°.

Acceleration of the ATPase activity of glycerol-treated muscle fibers by repeated stretch-release cycles.

Glycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KC1, 2 mM MgC1(2), and 10 micron CaC1(2) at pH 7.8 and 0 degrees C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 s at a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (alpha seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that: 1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1--2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, alpha, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (alpha=0). 2. The ATPase activity of oscillated muscle fibers increased with increase in the value of alpha, reached a maximal level, then decreased gradually with further increase of alpha to a value slightly larger than that of static fibers. At 0 degrees C, the value of alpha for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20 degrees C, the alpha value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers. 3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, alpha, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of alpha of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0 degrees C and 20 degrees C during one cycle of the changes in the rate of ADP liberation.

Double sucrose-gap method applied to single muscle fiber of Xenopus laevis.

Passive electrical properties (internal conductance, membrane conductance, low frequency capacity, and high frequency capacity obtained from the foot of the action potential) of normal and glycerol-treated muscle of Xenopus were determined with the intracellular microelectrode technique. The results show that the electrical properties of Xenopus muscle are essentially the same as those of frog muscle. Characteristics of the action potential of Xenopus muscle were also similar to those of frog muscle. Twitch tension of glycerol-treated muscle fibers of Xenopus recovered partially when left in normal Ringer for a long time (more than 6 h). Along with the twitch recovery, the membrane capacity increased. Single isolated muscle fibers of Xenopus were subjected to the double sucrose-gap technique. Action potentials under the sucrose gap were not very different from those obtained with the intracellular electrode, except for the sucrose-gap hyperpolarization and a slight tendency toward prolongation of the shape of action potential. Twitch contraction of the artificial node was recorded as a change of force from one end of the fiber under the sucrose gap. From the time-course of the recorded force and the sinusoidal stress-strain relationship at varying frequencies of the resting muscle fiber, the time-course of isotonic shortening of the node was recovered by using Fourier analysis. It was revealed that the recorded twitch force can approximately be regarded as isotonic shortening of the node.

Action of a serum protein on muscular contraction.

The mechanism of action of a “serum protein” isolated from human serum was assessed in several experimental preparations including glycerol-treated muscle fibers, rat heart papillary muscle and isolatedin vitro perfused rat heart. The action of the “serum protein” was studied also on canine and human heart papillary muscles which were made to respond to electrical stimulation with “ultrasonication modified epinephrine”. In addition the action of the protein on adenosine 5′ triphosphate generated precipitation of purified human actomyosin was investigated. The “serum protein” enhanced and intensified the generation of ATP induced tension in glycerol-extracted muscle fibers. It intensified the developed tension (DT) and increased the rate of development of tension (dT/dt) without influencing the time peak tension (TPT) of capillary muscles from rat, canine and human hearts in response to electrical stimulation. The “serum protein” increased the force of contraction of the isolatedin vitro perfused rat heart, and accelerated the adenosine 5′ triphosphate generated precipitation of purified human heart actomyosin.

Capacitance of the surface and transverse tubular membrane of frog sartorius muscle fibers.

The passive electrical properties of glycerol-treated muscle fibers, which have virtually no transverse tubules, were determined. Current was passed through one intracellular microelectrode and the time course and spatial distribution of the resulting potential displacement measured with another. The results were analyzed by using conventional cable equations. The membrane resistance of fibers without tubules was 3759 +/- 331 ohm-cm(2) and the internal resistivity 192 ohm-cm. Both these figures are essentially the same as those found in normal muscle fibers. The capacitance of the fibers without tubules is strikingly smaller than normal, being 2.24 +/- 0.14 microF/cm(2). Measurements were also made of the passive electrical properties of fibers in a Ringer solution containing 400 mM glycerol (which is used in the preparation of glycerol-treated fibers). The membrane resistance and capacitance are essentially normal, but the internal resistivity is somewhat reduced. These results show that glycerol in this concentration does not directly affect the membrane capacitance. Thus, the figure for the capacitance of glycerol-treated fibers, which agrees well with previous estimates made by different techniques, represents the capacitance of the outer membrane of the fiber. Estimates of the capacitance per unit area of the tubular membrane are made and the significance of the difference between the figures for the capacitance of the surface and tubular membrane is discussed.

Selective disruption of the sarcotubular system in frog sartorius muscle. A quantitative study with exogenous peroxidase as a marker.

Skeletal muscles which have been soaked for 1 hr in a glycerol-Ringer solution and then returned to normal Ringer solution have a disrupted sarcotubular system. The effect is associated with the return to Ringer's since muscles have normal fine structure while still in glycerol-Ringer's. Karnovsky's peroxidase method was found to be a very reliable marker of extracellular space, filling 98.5% of the tubules in normal muscle. It was interesting to note that only 84.1% of the sarcomeres in normal muscle have transverse tubules. The sarcotubular system was essentially absent from glycerol-treated muscle fibers, only 2 % of the tubular system remaining connected to the extracellular space; the intact remnants were stumps extending only a few micra into the fiber. Thus, glycerol-treated muscle fibers provide a preparation of skeletal muscle with little sarcotubular system. Since the sarcoplasmic reticulum is not destroyed and the sarcolemma and myofilaments are intact in this preparation, of the properties of the sarcolemma may thus be separated from those of the tubular system.

Chemistry, microscopy, and performance of methylated glycerol-treated muscle fibers.

Chemistry, microscopy, and performance of methylated glycerol-treated muscle fibers.

Effects of pH, buffers, carnosine, histidine, and β-alanine on the shortening of glycerol-treated muscle fibers

It has been shown that carnosine and histidine are markedly more effective than K + in accelerating ATP-induced shortening of glycerol-treated rabbit psoas muscle fiber. This holds with or without Mg ++ being present in the solutions. All tests were carried out at pH 8 after determining that pH 8–9 is an optimal range for ATP-induced contraction of this muscular model. Some buffers, particularly tris and maleate, are also effective potentiators for ATP-induced contraction.

Carnosine and tetramethylammonium ion and ATP-induced shortening of glycerol-treated muscle fibers

Recently, it has been reported that tetramethylammonium chloride causes contraction of acetylated glycerol-treated muscle fibers while small metallic cations cause little or no contraction ( Bowen and Martin, 1964). This led to speculation concerning the effect of large cations on the ATP-induced contraction of normal glycerinated muscle fibers. Several large cations have been tested and will be discussed in forthcoming publications. The present report concerns the effect of tetramethylammonium ion (TMA) and carnosine on the ATP-induced contraction of glycerinated muscle fibers. Carnosine is a constituent of vertebrate muscle and exists in rabbit to the extent of about 0.6 percent ( Severin, 1964).

The binding of ions and the shortening of glycerol-treated muscle fibers

Since 1948 when Riseman and Kirkwood (6) originated it and 1952 when Morales and Botts (5) modified it, the so-called “binding theory” to account for the ATP-induced contraction of biological models of muscular contraction has been in and out of popularity. In the past, the milieux into which the models of contraction were put were modified and, except for differences of physical form, models have not been altered. This communication reports experiments in which the models of contraction (glycerol-treated rabbit psoas muscle fibers) were methylated and acetylated before use as an enzyme or as a model of contraction.

A study of diffusion of ATP through glycerol-treated muscle

One and two m M ATP will penetrate bundles of glycerol-treated muscle fibers of about 100 μ thickness. This conclusion has been reached from studies by a physical method which involves recording the tension developed during ATP-induced isometric contraction. The method cannot be used to determine the precise diffusion coefficient through rabbit psoas muscle because the fibers are not thick enough to provide sufficiently long pathways for complete absence of substrate in the central core at substrate concentrations high enough to get measurable tension. It can be used, however, to compute the diffusion coefficient of ATP through this muscular material necessary to maintain a concentration of ATP in the center of fibers equal to 1 10 that of the concentration in the bath. The value obtained, 1.4–2.0 × 10 −6 sq. cm./sec., is about 100 times larger than that in the literature and one-half the free diffusion of ATP through aqueous solution. This large difference may be explained by the results of an earlier study in this laboratory of the interactions of ATP and glycerol-treated muscle which showed that the splitting of ATP by myosin was affected more by diffusion than was the change in length of bundles of muscle during isotonic ATP-induced contractions.

The ability of glycerol-treated muscle fibers to do work during ATP-induced contractions and the free energy of ATP

The free energy available from adenosine triphosphate (ATP) was varied by adding adenosine diphosphate and phosphate. Such additions had no influence on the ability of glycerol-treated muscle fibers to do work until Δ F was decreased below −9000 cal./ mole ATP. At energies below −9000 cal., the ability to work decreased until it reached zero at −6900 cal. at 0.01 M ATP and −5210 and −5640 cal. at 0.005 M and 0.0025 M ATP. At energy levels of −7329, −8160, and −14,360 cal./mole of 0.01 M ATP, the energy derived from the ATP was about ten times excess over that required by the working fiber bundles during ATP-induced contraction.

Relaxation of ATP-induced tension of glycerol-treated muscle fiber by polyphosphate

Relaxation of ATP-induced contraction of glycerol-treated muscle fibers by polyphosphate [(NaPO 3) 14–20 and (NaPO 3) 30] is described. Several parameters of the reaction were investigated.

EDTA relaxation of glycerol-treated muscle fibers, and the effects of magnesium, calcium and manganese ions

The relaxing effects of EDTA on glycerol-treated muscle fibers were studied quantitatively in the presence of Mg ++, Ca ++, and Mn ++ in various combinations. These conclusions were drawn: 1. 1. All of the three substances EDTA, Mg ++, and ATP must be present to produce relaxation of well-washed fibers. 2. 2. The effective concentration of EDTA depends upon the concentration of Mg ++ present. 3. 3. Enough Mg ++ and EDTA remain on an EDTA-relaxed fiber after it has been washed up to 15 min. in 160 m M KCl to exert a measurable effect. 4. 4. In the initial contraction, Mn ++ can take the place of Mg ++, but Ca ++ cannot. 5. 5. EDTA relaxation can be reversed by both Ca ++ and Mn ++, but more Mn ++ is required. 6. 6. Neither Mn ++ nor Ca ++ can take the place of Mg ++ in the EDTA relaxation process. 7. 7. ITP cannot replace ATP in the relaxation process though it can in contraction. 8. 8. PDTA is as effective as EDTA in relaxing fibers. 9. 9. The relaxing effect of EDTA decreases as pH increases, and has nearly vanished at pH 8. 10. 10. An explanation of some of the above results in terms of a “four species” complex of ATP, Mg ++, EDTA, and the contractile protein is discussed.

Effect of concentration of KI and of temperature on shortening of glycerol-treated muscle fibers in KI solutions.

Experiments reported here further support the previous conclusion that the shortening of glycerol-treated muscle caused by neutral Nessler's reagent (HgI2 in KI solution) is essentially due to the KI component. Analysis of the data obtained indicates that the apparent binding constant of KI to the contractile protein is 5 or 6 m/l. The same value for the binding ATP is about 10,000. The extent and speed of shortening in HgI2-KI solution are temperature dependent. The molecular rearrangement which results in the shortening of glycerol-treated fibers is considered to be directly related to the binding KI to the protein.