Transverse bands in smooth muscle cells

Summary In several preparations of intestinal musculature of mammals and other vertebrates, the smooth muscle cells showed distinct transverse striations. Their distribution was irregular, but they were often in register in adjacent cells. They were studied by light and electron microscopy, and it was concluded that they represent areas where the muscle cells had been crumpled, probably during or after fixation, especially in muscles that are curved and arranged in concentric arrays. A characteristic disruption in the arrangement of myofilaments was observed. These striations were not seen in control preparations of the guinea-pig taenia coli; however, when the taenia was wound around a rod after partial or complete fixation, transverse striations became apparent in the concave part of the coiled-up strip of muscle. The longitudinal compression of the muscle cells, which were made somewhat rigid by the fixation, is the most likely explanation of the transverse bands.     

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.     

Light and dark smooth muscle cells in estrogen-stimulated rat myometrium.

Smooth muscle cells of different densities to transmission of electrons (termed light and dark cells) were found in rat myometrium examined in the electron microscope following fixation by immersion in glutaraldehyde. Light cells accounted for about 4% of the total population of cells. No light cells were found in tissues fixed in situ by intraarterial perfusion with glutaraldehyde. In addition to staining differences, light cells were distinguished from most dark cells by differences in nuclear, mitochondrial, endoplasmic reticular, and surface structures. The relative number of light and dark cells after in vitro fixation was not changed in tissues relaxed with adrenaline or contracted with oxytocin. Mechanical injury resulted in increased numbers of light cells. Similarly, chemical injury with metabolic inhibitors resulted in ATP depletion, followed by increased numbers of light cells and gain in water content. We concluded that light cells were produced by mechanical or metabolic damage, leading to loss of volume control mechanisms, swelling, and leakage of protein. Light cells found after fixation in vitro in numerous prior studies represent cells damaged during isolation, and not a physiological variant among smooth muscle cells.     

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.     

Antibodies probe for folded monomeric myosin in relaxed and contracted smooth muscle

Regulatory light chain phosphorylation is required for assembly of smooth and non-muscle myosins in vitro, but its effect on polymerization within the cell is not understood. Relaxed smooth muscle cells contain dephosphorylated thick filaments, but this does not exclude the presence of a pool of folded myosin monomers which could be recruited to assemble when phosphorylated, thus forming part of smooth muscle's activation pathway. To test this hypothesis, relaxed and contracted avian gizzard cryosections were labeled with a fluorescently conjugated monoclonal antibody specific for the folded monomeric conformation, or with an antibody against the tip of the tail whose epitope is accessible in the monomeric but not the filamentous state. Fluorescence intensity observed in the two physiological states was quantitated by digital imaging microscopy. Only trace amounts of folded monomeric myosin were detected in both the relaxed and contracted states. The amount of monomer also did not increase when alpha-toxin permeabilized gizzard was equilibrated in a solvent that disassembles filaments in vitro. Assembly/disassembly is therefore unlikely to play a major role in regulating the contraction/relaxation cycle in smooth muscle cells.     

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.     

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.     

Organization, ultrastructure, and development of midgut visceral muscle in larval Aedes aegypti

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.     

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).     

The effects of some natural prostaglandins on isolated human circular bronchial muscle.

PGE1 relaxed isolated human circular bronchial muscle over a wide concentration range as did isoprenaline. Surprisingly isoprenaline was more potent than PGE1. PGF2alpha weakly contracted this muscle preparation whereas histamine was more potent. PGE2, however, produced paradoxical results, relaxing some tissues and contracting others, always in a concentration-related manner irrespective of tissue tone. In preparations that contracted to PGE2, tachyphylaxis induced to PGF2alpha also applied to PGE2, but did not affect PGE1 relaxations of histamine contractions. These findings suggest that pge2 can stimulate either PGF2alpha or PGE1 receptors of isolated human bronchial muscle.     

ULTRASTRUCTURAL STUDIES ON THE CONTRACTILE MECHANISM OF SMOOTH MUSCLE

Fresh taenia coli and chicken gizzard smooth muscle were studied in the contracted and relaxed states. Thick and thin filaments were observed in certain (but not all) cells fixed in contraction. Relaxed smooth muscle contained only thin filaments. Several other morphological differences were observed between contracted and relaxed smooth muscle. The nuclear chromatin is clumped in contraction and evenly dispersed in the relaxed state. The sarcolemma is more highly vesiculated in contraction than in relaxation. In contraction, the sarcoplasm also appears more electron opaque. Over-all morphological differences between cells fixed in isometric and in unloaded contraction were also noticeable. The results suggest a sliding filament mechanism of smooth muscle contraction; however, in smooth muscle, unlike striated muscle, the thick filaments appear to be in a highly labile condition in the contractile process. The relation between contraction and a possible change in pH is also discussed.     

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.     

The effects of some natural prostaglandins on isolated human circular bronchial muscle

PGE₁ relaxed isolated human circular bronchial muscle over a wide concentration range as did isoprenaline. Surprisingly isoprenaline was more potent than PGE₁. PGF₂ weakly contracted this muscle preparation whereas histamine was more potent. PGE₂, however, produced paradoxical results, relaxing some tissues and contracting others, always in a concentration-related manner irrespective of tissue tone. In preparations that contracted to PGE₂, tachyphylaxis induced to PGF₂ also applied to PGE₂, but did not affect PGE₁ relaxations or histamine contractions. These findings suggest that PGE₂ can stimulate either PGF₂ or PGE₁ receptors of isolated human bronchial muscle.     

Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones

We have purified a polyclonal antibody by affinity chromatography which binds specifically to the phosphorylated form of the regulatory light chain (Mr = 20,000) of smooth muscle myosin. This antibody does not stain relaxed, permeabilized smooth muscle cells isolated from guinea pig taenia coli. However, when these cells were stimulated to contract with CaCl2 (100 microM) and ATP (1 mM), the immunofluorescence staining was localized in a series of transverse bands. This distribution of activated myosin appears to reflect an underlying structural organization of the smooth muscle cell cytoskeleton into mechanically coupled contractile zones.     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Troponin and its components from ascidian smooth muscle

Troponin was isolated from the thin filaments of ascidian smooth muscle and separated into three components by ion-exchange chromatography, the molecular weights of which were 33,000, 24,000, and 18,000, respectively. The three components were designated as troponin t (TN-T), troponin I (TN-I), and troponin C (TN-C) in order of molecular weight, since each component had properties similar to those of the respective components of vertebrate skeletal-muscle troponin. The ascidian troponin or the mixture of the three components conferred Ca2+-sensitivity on reconstituted rabbit actomyosin in the presence of tropomyosin. One of the characteristics of the ascidian troponin was Ca2+-dependent activation of actin-myosin interaction in collaboration with tropomyosin, whereas its inhibitory action on the actomyosin ATPase in the absence of Ca2+ was less remarkable. From this, it is concluded that in the ascidian smooth muscle actin-myosin interaction is regulated by an actin-linked troponin-tropomyosin system, but the ascidian troponin acts as a Ca2+-dependent activator of an actomyosin system.     

Subcellular localization of dystrophin and vinculin in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle

The subcellular localization of dystrophin and vinculin was investigated in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle by immunofluorescence confocal microscopy. In ventricular cardiac muscle fibers, strong staining with antibody against dystrophin appeared as regularly arranged transverse striations at the sarcolemmal surface, and faint but uniform staining was seen in narrow strips between these striations. In fibers of the ventricular conduction system, the sarcolemma was stained uniformly with this antibody, but strong staining was found as regular striations in many areas and as scattered patches in other areas of the sarcolemma. These intensely stained striations and scattered patches of dystrophin were colocalized with those of vinculin. Because dystrophin striations were located at the level of Z bands of the underlying myofibrils, they were regarded as the concentration of this protein at costameres together with vinculin. In fibers of the conduction system, myofibrils were close to the sarcolemma where dystrophin and vinculin assumed a striated pattern, at some distance from the cell membrane where these proteins exhibited a patchy distribution, and distant from the sarcolemma where dystrophin was uniformly distributed. These data suggest that the distribution patterns of dystrophin reflect the degree of association between the sarcolemma and underlying myofibrils.     

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.     

The influence of muscle activation on impact dynamics during lateral falls on the hip

Muscle activation has been demonstrated to influence impact dynamics during scenarios including running, automotive impacts, and head impacts. This study investigated the effects of targeted muscle activation magnitude on impact dynamics during low energy falls on the hip with human volunteers. Fifteen university-aged participants (eight females, seven males) underwent 12 lateral pelvis release trials. Half of the trials were muscle-‘relaxed’; in the remaining ‘contracted’ trials participants isometrically contracted their gluteus medius to 20–30% of maximal voluntary contraction before the drop was initiated onto a force plate. Peak force applied to the femur-pelvis complex averaged 9.3% higher in contracted compared to relaxed trials (F = 6.798, p = .022). Muscle activation effects were greater for females, resulting in (on average) an 18.5% increase in effective pelvic stiffness (F = 5.838, p = .046) and a 23.4% decrease in time-to-peak-force (F = 5.109, p = .042). In the relaxed trials, muscle activation naturally increased during the impact event, reaching levels of 12.8, 7.5, 11.1, and 19.1% MVC at the time of peak force for the gluteus medias, vastus lateralis, erector spinae, and external oblique, respectively. These findings demonstrated that contraction of trunk and hip musculature increased peak impact force across sexes. In females, increases in the magnitude and rate of loading were accompanied (and likely driven) by increases in system stiffness. Accordingly, incorporating muscle activation contributions into biomechanical models that investigate loading dynamics in the femur and/or pelvis during lateral impacts may improve estimate accuracy.