An X-Ray Diffraction Study of Contracting Molluscan Smooth Muscle

The living anterior byssus retractor muscle of Mytilus (ABRM), a smooth, “catch” muscle, has been studied by X-ray diffraction while relaxed and while tonically contracted. X-ray reflections were observed from the actin and paramyosin filaments and from the α-helical substructure of the paramyosin filaments. No differences in spacings or relative intensities were observed when the relaxed and contracting muscle patterns were compared. This result is consistent with a sliding filament mechanism involving an interaction between actin and paramyosin filaments.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Actin filament organization and crossbridge decoration in a contracting molluscan smooth muscle

Equatorial intensity distributions of x-ray diffraction patterns from relaxed and contracted states of the anterior byssus retractor muscle, ABRM, are compared with distributions of non-physiological reference states and with calculations based on various packing models of the actin filaments. Relaxed and contracted muscles provide distributions that agree with models, in which actin filaments are packed hexagonally in discrete areas containing 12 to 16 filaments. The crystalline fractions of actin filaments in the relaxed and contracted states are 0.91 and 0.57 respectively. Contracting muscles, however, show deviations from the calculated distributions at small angles of diffraction. This is interpreted as being due to the fact that actin filaments, outside crystalline areas, are decorated by crossbridges as about every 6th actin monomer.     

Cross-bridge arrangements in Limulus muscle

X-ray diffraction patterns show Limulus muscle to have a structure in rigor similar to that of insect flight muscle, except that the thick filaments are staggered. Myosin filaments in relaxed muscle bear a highly ordered helical array of cross-bridges which, however, is very labile. The array undergoes a reversible transition between order and disorder in response to changes in ionic strength.     

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.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Functional states and fine structure of the contractile apparatus of the penis retractor muscle (PRM) of Helix pomatia L.

Summary The ultrastructure of the isolated glycerinated penis retractor muscle (PRM) of Helix pomatia was investigated. The diameter distributions of thick myofilaments from fibre cross sections in the relaxed, phasic contracted, tonic contracted, and in the catch states show that a characteristic filament spectrum is formed in the catch state and its preceding active state. The significant structural differences are discussed in relation to earlier hypotheses related to the catch state.I wish to thank Prof. Dr. Fr.-W. Schlote for helpful discussion. I also thank M. Jenninger and E. Grafahrend for their technical assistance.     

Mechanisms Underlying Nitroglycerin-Induced Suppression of Phenylephrine- and Caffeine-Evoked Contractions of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a strain measurement technique, we studied the mechanisms of the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on contractions of smooth muscles of the main pulmonary artery of the rabbit induced by phenylephrine and caffeine in normal Krebs solution (NKS) or in nominally calcium-free solution (NCFS). Phenylephrine applications caused contractions consisting of an initial fast phasic low-amplitude component followed by a tonic higher-amplitude component. After caffeine-induced monophasic low-amplitude contraction, tension of the smooth muscle strip shifted below the conventional zero. Addition of NG to NKS resulted in a decrease in the smooth muscle tension below the conventional zero. Under the influence of NG, the initial phasic component of phenylephrine-induced contraction was partially suppressed, whereas the next tonic component was suppressed to a greater extent. At the same time, NG exerted nearly no influence on the amplitude of caffeine-induced contractions. Washing out by NKS of phenylephrine dissolved in NCFS resulted in initiation of a fast phasic high-amplitude contraction. Such a contraction did not develop either in the presence of NG or phenylephrine in NCFS or in the case of washing out of caffeine dissolved in NCFS. Our findings allow us to conclude that phenylephrine or caffeine added to the superfusate induce contractions of the smooth muscle cells (SMC) of the main pulmonary artery of the rabbit due to activation of Ca²⁺ release from the respective intracellular calcium stores. In addition, calcium ions entering SMC through the calcium channels of the plasma membrane are also involved in activation of the phenylephrine-induced contraction. The inhibitory effect of NG on the phenylephrine-induced contraction is related to the influence of NO on the release of Ca²⁺ from the inositol trisphosphate-sensitive intracellular calcium store and receptor-operated inflow of Ca²⁺ to SMC. Nitroglycerin did not significantly influence the caffeine-induced contraction and, therefore, Ca²⁺ release from the caffeine-sensitive store.     

Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus (K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.     

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle.

X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al. (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z-disc lattice that correlated very closely with the X-ray results. It is suggested that the Z-disc structure is the major source of this nonindexible reflection.     

Time-Resolved Structural Studies on Insect Flight Muscle after Photolysis of Caged-ATP

The time course of structural changes occurring on ATP-induced relaxation of glycerinated insect flight muscle from the rigor state has been investigated using synchrotron radiation as a source of high intensity x rays and photolysis of caged-ATP to produce a rapid rise in ATP concentration. Temporal resolutions of 1 ms for the strongest equatorial reflections and 5 ms for the 14.5 nm meridional reflection are attainable from single events (i.e., without averaging over several cycles). The equatorial intensity changes completely, the meridional intensity partially, towards their respective relaxed values on a much faster time scale than relaxation of tension. The results suggest that actively cycling bridges present shortly after ATP-release are either too few in number to be detected in the equatorial diffraction pattern or that their structure is different from that of rigor bridges.     

Effect of inflation on trachealis muscle tone in canine tracheal segments in vitro

Isolated tracheal segments were studied in vitro to determine how inflation affects the length and tension of the contracted and relaxed trachealis muscle. Circumferential trachealis muscle lengths were measured from cross-sectional radiographs taken during stepwise inflation of intact 20-cm-long tracheal segments to an inflation pressure of 25 cmH2O. A tracheal length spanning two cartilage rings was then cut out and mounted in a tissue bath using clips attached at the points of muscle insertion into the cartilage. The ring was stretched open along the axis of the muscle, and the resulting forces of the relaxed and contracted muscle and the cartilage were measured. Muscle lengths and tensions during inflation of the trachea were determined by comparing pressure vs. length and force vs. length measurements. During inflation from 0 to 25 cmH2O, the circumferential length of the trachealis muscle contracted with 10(-5) M acetylcholine increased from 48 to 70% of its length of maximal active tension (Lmax), while the relaxed muscle increased from 80 to 93% Lmax. The length of the contracted muscle was maintained at a nearly constant proportion of its relaxed length at each pressure.     

Some properties of glycerinated skeletal muscle fibers containing phosphorylated myosin

The structural changes of phalloidin-rhodamin labelled F-actin at relaxed and contracted skeletal muscle fibre containing phosphorylated myosin and at contracted state after dephosphorylation were investigated by measuring of polarized fluorescence of the fluorophore. The mechanical properties (isometric tension development) of fibre were studied in parallel. At submaximal concentration of Ca ions (0.6 mumol/l) the isometric tension was decreased after dephosphorylation of fibre myosin. The changes in polarization of fluorophore bound to actin filament were correlated with isometric tension developed by the muscle fibre. The angles between the actin filament long axis and the absorption and emission dipoles for contracted and relaxed fibre were different, suggesting changes in the organization of the actin monomers in thin filament, dependent on the physiological state of the fibre. The flexibility of the thin filaments during transition of the fibre from relaxed to "contracted" state increases as indicated by greater average angle between the F-actin long axis and the fibre axis.     

Effect of activation by calcium on the x-ray diffraction pattern from insect flight muscle.

A low-angle X-ray diffraction pattern of calcium-activated Lethocerus flight muscle was formed and the intensities of various parts of the pattern observed by means of a proportional counter. The muscle was sinusoidally oscillated in length to produce mechanical work. The resultant changes in diffraction intensity were related to the state of the muscle and to the phase of the mechanical oscillatory cycle. The measurements were interpreted in terms of a movement of the heads of the myosin molecules into contact with the actin filaments. In these terms the results showed that between 10 and 20% of the myosin heads attached to actin during work-producing oscillation of the muscle. The time-course of this attachment followed that of tension generation with a small delay. Calculation suggests that not all of the myosin molecules attached to actin at any one moment were generating tension.     

Activation of two types of fibres in ferret,Mustela putorius furo,cremaster muscle

Summary Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small(<400 m²) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and-59.1 mV) and intracellular sodium activity (8.3 mmol·l^-1 and 14.1 mmol·l^-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15–200 mmol·l^-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5–1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca²⁺ withdrawal and inhibited by Ni²⁺, Cd²⁺, Co²⁺, Gd³⁺ and gallopamil (D600). In Ca²⁺-free medium the sustained tension was restored by adding Sr²⁺. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca²⁺ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.Abbreviations a _i ^Na intracellular sodium activity - ATPase myosin adenosine triphosphatase - D600 gallopamil - E _m membrane potential - E _r resting potential - EDL muscle, extensor digitorum longus muscle - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - e.c. excitation-contraction - SDHase succinate dehydrogenase - NADHase nicotinamide adenine, dinucleotide hydrogen-diaphorase - SOL muscle, soleus muscle - T time constant of relaxation - TEACI tetraethylammonium chloride - [Ca]_o, [K]_o, [Na]_o extracellular calcium, potassium, sodium concentration     

Changes in the X-ray diffraction pattern from rigor muscles by application of external length changes

The effect of external force on the X-ray pattern from frog muscles in rigor was studied by a time-resolved diffraction technique. When sinusoidal length changes (1.5–3% of the muscle length, 5Hz) were applied to the muscle, the 14.3 nm intensity decreased during the releasing phase and increased during the stretching phase. The intensity ratio of the equatorial 1,0 and 1,1 reflections did not change, nor were there any appreciable intensity changes in the 5.9 nm and 5.1 nm reflections during the length change. Experiments were also done with the relaxed muscles and no change was seen in any reflection, indicating that the rigor linkages are needed to produce the 14.3 nm intensity change. Thus the distinct effect of the length change was detected only on the 14.3 nm reflection. These results suggest no large conformational changes are induced in both the distal part of the myosin head attached to actin and the actin filament during the oscillation. It is therefore most probable that the proximal portion of myosin heads including S-2 contributes to the intensity change in response to the length change (see, also ref.21). When the muscle was stretched beyond the filament overlap, the 14.3 nm intensity change was suppressed to less than 50% of that of the slack length. It was also found that the tension change delayed the intensity change during the length oscillation. However, this delay of the tension change as observed in the muscle at the slack length was lacking in the overstretched muscle, indicating that the 14.3 nm intensity change may arise partly from a portion other than the crossbridges.     

Time-resolved synchrotron X-ray diffraction studies of a single frog skeletal muscle fiber. Time courses of intensity changes of the equatorial reflections and intracellular Ca2+ transients.

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.     

Thick-filament strain and interfilament spacing in passive muscle: effect of titin-based passive tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d_1,0) and Z-disk (d_Z) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I_1,1/I_1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I_1,1/I_1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.     

Laser Raman scattering. A molecular probe of the contractile state of intact single muscle fibers.

The 500 to 1,800-cm-1 region of the Raman spectra of intact single muscle fibers from the giant barnacle are dominated by bands caused by the protein component of the fibers. The frequency and the intensity of the conformationally sensitive bands indicate that the contractile proteins adopt a predominantly alpha-helical structure and are not affected when the contractile state of the fibers is changed from relaxed to contracted by addition of ATP and Ca. However, the contraction induces a decrease of the scattering intensity of some of the Raman bands caused by the acidic and tryptophan side chains, showing that these amino acids are involved during the generation of tension.     

Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.