Structural changes in myosin during contraction and the state of ATP in the intact frog muscle

The reactivity of myosin to [¹⁴C]-labeled N-ethylmaleimide ([¹⁴C] NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [¹⁴C] NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [¹⁴C] NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [¹⁴C] NEM into myosin and that upon the irreversible loss of ATP the rate increases. ³¹P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.     

Intracellular Na+ and K+ shifts induced by contractile activities of rat skeletal muscles

The effects of direct and indirect electrical stimulation on intracellular potassium and sodium contents ([K]_i and [Na]_i, respectively) in rat soleus muscle (SOL) and extensor digitorum longus muscle (EDL) were investigated under in vivo conditions. The changes of [K]_i and [Na]_i contents in both muscles which were stimulated indirectly reached respective values at 30 min or 1 hr after the beginning of stimulation, whereas those of EDL stimulated with 60 Hz changed gradually through 2 hr stimulation. The shifts of [K]_i and [Na]_i in EDL occurred during the twitch contraction at considerably lower frequency stimulation (0.5–10 Hz), whereas those in SOL were observed during the tetanus contraction at high frequency stimulation (10–40 Hz). The difference of change in cationic shifts between EDL and SOL under low frequency stimulation was reduced by ouabain treatment, though the difference was still significant. When the muscles were indirectly stimulated 6000 times at 1,5,10 and 20 Hz, the cationic shifts in EDL were greater than those in SOL, extending over all frequencies. It was concluded that such a difference in ionic shift between contracting EDL and SOL may be primarily due to the difference in unidirectional ionic fluxes per stimulation and, secondly, to the difference in Na⁺-K⁺ pump activity.     

Gender modulates the energy cost of muscle contraction in untrained healthy subjects. A P magnetic resonance spectroscopy analysis

The forearm flexor muscles of 56 untrained volunteers (26 women and 30 men) were examined by ³¹P magnetic resonance spectroscopy, during a rest-exercise-recovery protocol, in order to document the impact of gender on muscle energetics. Absolute concentrations of high-energy phosphate compounds, intracellular pH and rates of aerobic and anaerobic ATP production were calculated. An inverse correlation was found between body mass index (BMI) and power output in women but not in men. After correcting for power output and BMI, the measured energy cost of contraction was twice larger for women than for men. This increase was also reflected in larger ATP production from aerobic and anaerobic pathways. This higher energy cost might be explained in part by differences in local muscle mass, a higher impact of fatness, but also by a reduced metabolic efficiency of muscle fibers in untrained women.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and On 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insullin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661–668). Brief exposure (1 min) to N-ethylmaleimide (0.3?10 nM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-¹⁴C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H₂O₂ (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin was paralleled by the inhibition of ¹²⁵I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and ¹²⁵I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockae on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20–30%. Conversely, when muscles were first allowed to bind ¹²⁵I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effet of sulphydryl blockade but no protective effect was observed with H₂O₂. None of the oxidants protected against the inhibitory effect of 3 nM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

Lack of phosphate incorporation into TN-I in live frog muscle

The phosphorylation of TN-I was investigated in muscles of live frogs injected with [³²P]orthophosphate. Isolation of TN-I was carried out by the rapid and specific technique of affinity chromatography developed by Syska, Perry, and Trayer [FEBS Lett.40, 253–257 (1974)] followed by gel electrophoresis in the presence of sodium dodecyl sulfate. No significant labeling of TN-I was found even in frogs which were exposed to the ³²P-treatment for several days. A comparison of the specific radioactivity of TN-I from resting and contracting frog muscle showed no change in the ³²P content of TN-I during muscle contraction.     

On the Inhibition of Muscle Contraction Caused by Exposure to Hypertonic Solutions

The evidence supporting a site of inhibition of excitation contraction (E-C) coupling near the plasma membrane (the "glycerol effect," the K⁺-potentiating effect) for muscle in hypertonic solution was reinvestigated. It was found, using whole frog sartorii, that there was a rehydration of muscle soaked in glycerol Ringer after 30 min and a large swelling (to 140% after 1 hr soaking) upon return of the muscle to normal Ringer, suggesting that significant amounts of glycerol enter the fibers during this time. While contrary to the original report of the glycerol effect, this finding was consistent with other studies involving the use of single fibers. Also reexamined was the potentiating effect of K⁺ on the hypertonic inhibition of muscle contraction. It was found that muscles exposed to this KCl pretreatment swell so that they are less dehydrated in hypertonic solutions, thus accounting for the observed potentiation. After being treated instead with a K₂-tartrate Ringer solution, muscles did not swell and, as determined with twitch recordings, did not display any potentiation in hypertonic solutions—even though the [K₊] was higher than an osmotically equivalent KCl solution. The evidence was thus consistent with alternative hypotheses in which inhibition of contraction occurs at a later stage in E-C coupling or involves the contractile process itself.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [³H]glucose released from a wide range of [³H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na⁺-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2–2.0 mM phlorizin, the [³H]glucose uptake was a constant 11–12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [³H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [¹⁴C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [¹⁴C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na⁺-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medium by virtue of the position of the site where it is formed, i.e. inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.     

Impedance of the chest wall during sustained respiratory muscle contraction

We measured total chest wall impedance (Zw), "pathway impedances" of the rib cage (Zrcpath), and diaphragm-abdomen (Zd-apath), and impedance of the belly wall including abdominal contents (Zbw+) in five subjects during sustained expiratory (change in average pleural pressure [Ppl] from relaxation = 10 and 20 cmH2O) and inspiratory (change in Ppl = -10 and -20 cmH2O) muscle contraction, using forced oscillatory techniques (0.5-4 Hz) we have previously reported for relaxation (J. Appl. Physiol. 66: 350-359, 1989). Chest wall configuration and mean lung volume were kept constant. Zw, Zrcpath, Zd-apath, and Zbw+ all increased greatly at each frequency during expiratory muscle contraction; increases were proportional to effort. Zw, Zrcpath, and Zd-apath increased greatly during inspiratory muscle contraction, but Zbw+ did not. Resistances and elastances calculated from each of the impedances showed the same changes during muscle contraction as the corresponding impedances. Each of the resistances decreased as frequency increased, independent of effort; elastances generally increased with frequency. These frequency dependencies were similar to those measured in relaxed or tetanized isolated muscle during sinusoidal stretching (P.M. Rack, J. Physiol. Lond. 183: 1-14, 1966). We conclude that during respiratory muscle contraction 1) chest wall impedance increases, 2) changes in regional chest wall impedances can be somewhat independent, depending on which muscles contract, and 3) increases in chest wall impedance are due, at least in part, to changes in the passive properties of the muscles themselves.     

Different motor learning effects on excitability changes of motor cortex in muscle contraction state

Abstract

We aimed to investigate whether motor learning induces different excitability changes in the human motor cortex (M1) between two different muscle contraction states (before voluntary contraction [static] or during voluntary contraction [dynamic]). For the same, using motor evoked potentials (MEPs) obtained by transcranial magnetic stimulation (TMS), we compared excitability changes during these two states after pinch-grip motor skill learning. The participants performed a force output tracking task by pinch grip on a computer screen. TMS was applied prior to the pinch grip (static) and after initiation of voluntary contraction (dynamic). MEPs of the following muscles were recorded: first dorsal interosseous (FDI), thenar muscle (Thenar), flexor carpi radialis (FCR), and extensor carpi radialis (ECR) muscles. During both the states, motor skill training led to significant improvement of motor performance. During the static state, MEPs of the FDI muscle were significantly facilitated after motor learning; however, during the dynamic state, MEPs of the FDI, Thenar, and FCR muscles were significantly decreased. Based on the results of this study, we concluded that excitability changes in the human M1 are differentially influenced during different voluntary contraction states (static and dynamic) after motor learning.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Ultrastructure of a molluscan smooth muscle during phasic contractions and in the relaxed state

Tension and X-ray diffraction patterns are not always correlated in the smooth anterior retractor muscle (ABRM) of Mytilus edulis. The muscle produces equatorial intensity profiles of X-ray diffraction patterns corresponding to either a relaxed or a contracted structure. During phasic contractions, comprising a contracted as well a a relaxed phase, the diffracted intensity on the equator at 0.003 A^?1 changes within the first 10s after onset of stimulation. The tension reaches a maximum after about the same time. The time dependence of this intensity change during phasic contraction has been measured. It shows that the tension decays within 10s, but the relaxed structure needs 30–40 s to reestablish. There is no difference between the observed intensities from the tonic and phasic contracted states. Inactivated muscles with minimum tension, normally termed relaxed, can have either a “contracted” or a relaxed structure.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Palmitate incorporation into lipids pools of contracting red and white muscles

We compared the incorporation of the blood-borne [14C]-palmitate into selected lipid and phospholipid pools in rat muscles (soleus, red and white gastrocnemius), at rest and during contractions (15 and 60 tetani/min) in one leg (5 min) while the contralateral leg served as a control. [1-14C]-palmitate (20 µCi/rat) was administered into the carotid artery (t = 1 min). [14C]-palmitate deposition was greatest in soleus (100%) and lower in red (82%) and white gastrocnemius muscles (63%), respectively (p < 0.05). [14C] was deposited primarily into the tri-acylglycerol (50%) and phospholipid pools (30%) of soleus and red gastrocnemius muscles, and into the di-acylglycerol (30%), tri-acylglycerol (30%) and phospholipid pools (30%) in white gastrocnemius muscle. During contraction the concentrations of tri-acylglycerol were not changed. But, contraction increased [14C]-palmitate incorporation into soleus and red gastrocnemius muscles (600-700%) and into white gastrocnemius muscles (200%). Slightly more [14C] was directed from the phospholipids into the tri-acylglycerol pool during contraction. [14C]-palmitate deposition was also increased in the subclasses of phospholipids during contraction in red and white gastrocnemius. In conclusion, the deposition of [14C]palmitate into different lipid and phospholipid pools is quite rapid, and is dependent on contraction and the muscle fiber type. (Mol Cell Biochem 166: 73-83, 1997)     

X-ray diffraction studies of the thick filament in permeabilized myocardium from rabbit

Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25 degrees C to 5 degrees C at 200 mM and 50 mM ionic strengths (mu), respectively. Effects of temperature and mu on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I(1,1)/I(1,0), and the spacing of the filament lattice are similar in both muscles. At 25 degrees C, particularly at mu = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I(1,1)/I(1,0). Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P(i)]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P(i) state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I(1,1)/I(1,0) ratio tends to be higher, and the lattice spacing D(10), larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.     

Requirement of cell proliferation for the induction of presumptive preneoplastic lesions in rat liver by a single dose of 1,2-dimethylhydrazine

Octanol (1 mM) or octanoate (10 mM) almost totally depress the contraction amplitude of directly stimulated muscles in a few minutes. Octanoate in a concentration of 2 mM/l decreases the contraction amplitude by 20% and retards the caffeine contracture. The ratio between twitch and tetanus is affected by octanol only. The results suggest that octanol and octanoate alter binding or releasing properties for Ca²⁺ of skeletal muscle cells.     

Cross-bridge kinetics, cooperativity, and negatively strained cross- bridges in vertebrate smooth muscle. A laser-flash photolysis study

The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig protal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an approximately 45 degree angle. In the absence of Ca2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some cross-bridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM Pi, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca2+, was estimated to be 0.1-2.5 X 10(5) M-1s-1, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca2+, force development occurred at a rate (0.4 s-1) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP gamma S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca2+, consistent with cooperative attachment of cross-bridges. Cooperative reattachment of dephosphorylated cross-bridges may contribute to force maintenance at low energy cost and low cross-bridge cycling rates in smooth muscle.