THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

CYTOPLASMIC FILAMENTS IN DEVELOPING AND ADULT VERTEBRATE SMOOTH MUSCLE

An extensive study of adult and developing smooth muscle has revealed the widespread occurrence of a distinct filament with an average diameter of about 100 A (termed the 100 A filament). Unlike that of myofilaments, their appearance in longitudinal section is uniform, but in transverse section they have a round profile, occasionally exhibiting a less electron-opaque core. The 100 A filaments are almost invariably preserved under a variety of fixation procedures, whereas myofilaments, particularly the thicker filaments, are preserved inconsistently. The 100 A filaments appear to be randomly oriented throughout the cytoplasm, either singly or in small groups, although they are sometimes concentrated in the juxtanuclear region of the smooth muscle cells. The intimate association of 100 A filaments with dark bodies, in both developing and adult smooth muscle cells, may indicate that these filaments either play a role in dark body formation or, at least, constitute a part of the dark body. The 100 A filaments are conspicuous in developing smooth muscle cells and occasionally form networks or clusters; they appear to decrease in relative number as maturation proceeds, but considerable numbers are still present in adult tissue.     

LOCUS AND STATE OF AGGREGATION OF MYOSIN IN TISSUE SECTIONS OF VERTEBRATE SMOOTH MUSCLE

Structures with the characteristics of molecular myosin were identified by electron microscopy in tissue sections of vertebrate smooth muscle. No thick filaments of myosin were found regardless of preparative procedures, which included fixation at rest and in contraction, glycerine extraction, and storage at low pH prior to fixation. Absence of thick myosin filaments and presence of what appear to be myosin molecules is in accord with conclusions based on X-ray diffraction (3, 12) and birefringence data (4) from living smooth muscles at rest and in contraction. Explanations are provided for appearances thought by others (6, 20, 21) to represent thick myosin filaments. Our present observations are in accord with the model for smooth muscle contraction which we have previously proposed (1).     

THE COAGULATION OF MYOSIN IN MUSCLE

1. Muscle can be prepared in the form of a dry powder in which myosin exists in a state similar to that in intact muscle. As in intact muscle, myosin in powdered muscle is soluble and can be caused to rapidly coagulate. 2. Restoring to powdered muscle the quantity of water previously removed causes coagulation of myosin. The rate of coagulation is considerably slower at 0° than at 20°. 3. Adding the powder to a large volume of dilute salt solution also results in coagulation. 4. The water soluble constituents of muscle can be removed from the powder without thereby causing coagulation. Coagulation occurs in water extracted muscle when it is suspended in a dilute salt solution. 5. Coagulation of myosin in muscle resembles the coagulation of myosin caused by dehydration. 6. Myosin coagulates readily only when it is imbedded in the structure of muscle. The significance for coagulation of the arrangement of myosin particles in muscle has been indicated.     

大麦细胞核及染色体肌球蛋白免疫细胞化学定位

对去除DNA、组蛋白和大部分非组蛋白的大麦(Hordeum vulgare)细胞核和染色体间接免疫荧光标记实验结果表明:抗肌球蛋白抗体的荧光标记弥散分布在整个细胞核和染色体上;进一步应用免疫胶体金技术分析肌球蛋白在细胞核和染色体的分布情况,发现在染色体中散布着大量的胶体金颗粒;间期细胞核中胶体金颗粒主要分布在核仁和染色质中。上述实验结果表明:肌球蛋白是细胞核及染色体非组蛋白组成成分。本文还对肌球蛋白在细胞核和染色体中的分布规律进行了讨论。     

THE DOUBLE ARRAY OF FILAMENTS IN CROSS-STRIATED MUSCLE

The conditions under which one might expect to see the secondary filaments (if they exist) in longitudinal sections of striated muscle, are discussed. It is shown that these conditions were not satisfied in previously published works for the sections were too thick. When suitably thin sections are examined, the secondary filaments can be seen perfectly easily. It is also possible to see clearly other details of the structure, notably the cross-bridges between primary and secondary filaments, and the tapering of the primary filaments at their ends. The arrangement of the filaments and the changes associated with contraction and with stretch are identical to those already deduced from previous observations and described in terms of the interdigitating filament model in previous papers. There are therefore excellent grounds for believing that this model is correct. The alternative models which have been proposed appear to be incompatible both with the present observations and with much of the other available evidence.     

REVERSIBLE DISAGGREGATION OF MYOFILAMENTS IN VERTEBRATE SMOOTH MUSCLE

Strips of taenia coli from guinea pigs were incubated under isometric conditions in Krebs-Ringer bicarbonate saline (MKR) containing various concentrations of Ca⁺² and/or Mg⁺². Spontaneous or chemically induced contractile activity was abolished within 15 min of exposure to MKR containing Ca⁺² at concentrations below 10^-6 M; contractile activity was restored by reincubation in normal MKR after 1–2 h. Exposure of taenia coli to MKR containing Ca⁺² at concentrations below 10^-6 M for 1 h or more led to loss of thick and thin myofilaments from the sarcoplasm as observed with the electron microscope. Except for the loss of these two filament types, the cells contained all other structural features observed in preparations incubated in MKR containing Ca⁺² at its normal level (1.3 x 10^-3 M). The loss of thick and thin myofilaments in strips exposed to a Ca⁺² concentration below 10^-6 M was reversed by reincubation for 30 min in MKR containing normal Ca⁺² levels. The observed loss of thick and thin myofilaments in response to low Ca⁺² is interpreted as resulting from the disaggregation of some or all of the molecular components of these two filament types.     

A STUDY OF NEXUSES IN VISCERAL SMOOTH MUSCLE

Nexuses are described between the smooth muscle cells of the gizzard of the chick and the pigeon, the vas deferens of the mouse and the guinea pig, and the taenia coli of the guinea pig. The nexuses in the gizzard were examined after osmium tetroxide and potassium permanganate had been used as fixatives. Although differences in the dimensions of the unit membranes and the nexuses were noted, the results with the two fixation techniques were complementary. The distribution of nexuses within the smooth muscle tissues examined was uneven. Nexuses were still present in both small and large pieces of tissue incubated in hypertonic solutions at varying temperatures. Other experiments showed that the degree of contraction at the time of fixation did not affect the presence of nexuses in the tissue. These results indicate that nexuses between smooth muscle cells are stable under a variety of conditions.     

LOCALIZATION AND PROPERTIES OF THE CHOLINESTERASE IN CRUSTACEAN MUSCLE

Cholinesterase (ChE) activity is present in crustacean muscle extracts. However, since acetylcholine (ACh) is not a neuromuscular transmitter in these animals, the role and exact localization of ChE was unknown. The histochemical localization of the enzyme was studied in whole muscle and in the sarcoplasmic reticulum fraction of the extract, 50-µm frozen sections of glutaraldehyde-fixed crayfish tail flexor muscle were incubated with acetylthiocholine (ATC) as substrate, and examined under the electron microscope. After some modifications in published techniques, dense deposits were found associated with the sarcolemma, sarcolemmal invaginations, and transverse tubules. No deposits were found in 10^-4 M eserine, or if butyrylthiocholine (BTC) was substituted for ATC. The vesicles in the sarcoplasmic reticulum fraction which demonstrate the activity must represent minced bits of these membranes. Using a spectrophotometric method, the kinetics of the crustacean muscle enzyme was compared to the acetylcholinesterase (AChE) on mammalian red blood cells and in the lobster ventral nerve cord. Surprisingly, and contrary to previous reports, the crustacean muscle enzyme did not demonstrate substrate inhibition. While a number of similarities to AChE were found, this lack of substrate inhibition makes questionable an unequivocal similarity with classical AChE.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (~ 10.0 µ) and intermediate (~ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–~6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.     

CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.     

FINE STRUCTURE OF SMOOTH MUSCLE CELLS GROWN IN TISSUE CULTURE

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150–250 A in diameter) and thin (30–80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80–110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.     

AUTORADIOGRAPHIC LOCALIZATION OF NEW RNA SYNTHESIS IN HYPERTROPHYING SKELETAL MUSCLE

Work-induced growth of rat soleus muscle is accompanied by an early increase in new RNA synthesis. To determine the cell type(s) responsible for the increased RNA synthesis, we compared light autoradiographs of control and hypertrophying muscles from rats injected with tritiated uridine 12, 24, and 48 h after inducing hypertrophy. There was an increased number of silver grains over autoradiographs of hypertrophied muscle. This increase occurred over connective tissue cells; there was no increase in the number of silver grains over the muscle fibers. Quantitative studies demonstrated that between 70 and 80% of the radioactivity in the muscle that survived fixation and washing was in RNA. Pretreatment of the animals with actinomycin D reduced in parallel both the radioactivity in RNA and the number of silver grains over autoradiographs. Proliferation of the connective tissue in hypertrophying muscle was evident in light micrographs, and electron micrographs identified the proliferating cells as enlarged fibroblasts and macrophages; the connective tissue cells remained after hypertrophy was completed. Thus, proliferating connective tissue cells are the major site of the increase in new RNA synthesis during acute work-induced growth of skeletal muscle. It is suggested that in the analysis of physiological adaptations of muscle, the connective tissue cells deserve consideration as a site of significant molecular activity.     

FINE STRUCTURAL LOCALIZATION OF ADENOSINETRIPHOSPHATASE ACTIVITY IN HEART MUSCLE MYOFIBRILS

Activity of myofibrillar adenosinetriphosphatase was demonstrated histochemically at a fine structural level in isolated, unfixed or hydroxyadipaldehyde-fixed cardiac myofibrils in the rat, using a lead precipitation technique and either Ca⁺⁺ or Mg⁺⁺ as activating ion. Activity in relaxed myofibrils was found in the A band, but not the H, I, or Z bands. Deposits of final product frequently exhibited an axial periodicity of near 365 A, and bore a close relationship to filaments within the A band. Several patterns of distribution occurred in contracted myofibrils. In myofibrils which had shortened to the point of disappearance of the I band, final product was distributed throughout the sarcomere, except for the unreactive Z band. A second type of distribution occurred in strongly contracted fibers in which there was intensification of activity in the center of the sarcomere. These findings are discussed in the light of the recent morphological evidence and it is suggested that the distribution of final product is consistent with localization of enzyme activity to the cross-bridges between the thick and thin filaments.