Cell Tiss. Res. 184, 195-212 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Arrangement of Smooth Muscle Cells and Intramuscular Septa in the Taenia coli Giorgio Gabella* Department of Anatomy, University College London, England
Summary. Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10nm) filaments; about 50 % of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5~ with the septa. In muscles fixed during isotonic contraction the angle increases to about 20-22 ~, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4-6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission. Key words: Taenia coli - Guinea pig - Smooth muscle - Collagen Ultrastructure.
Introduction This paper follows a study on the fine structure of muscle cells of the guinea-pig taenia coli (Gabella, 1976a), and presents further morphological observations Send offprint requests to: Dr. Giorgio Gabella, Department of Anatomy, University College London,
Gower Street, London, WC1E 6BT, England * I thank Mr. S.I. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council
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focussed o n those structures which m a y be involved in the process o f t r a n s m i s s i o n of force. Force is generated by the so-called contractile machinery, i.e. the sets o f myofilaments. While in the case o f striated muscle fibres it is relatively simple to visualize a series o f sarcomeres pulling one against the next a n d the terminal ones pulling the t e n d o n s at each end o f the fibre, it is less clear how a similar effect can be o b t a i n e d in a s m o o t h muscle. It is true that in m a n y s m o o t h muscles the shortening o f the muscle m a y n o t be the principal mechanical effect o f a c o n t r a c t i o n ; for example, the circular m u s c u l a t u r e of the intestine shortens u p o n c o n t r a c t i o n a n d the size of the l u m e n is therefore reduced, b u t some of the force generated is applied in a transverse direction, both radially (towards the l u m e n ) a n d along the length of the intestine (so that e l o n g a t i o n o f the gut ensues). However, the taenia coli is a conspicuous a n d discrete strap o f l o n g i t u d i n a l m u s c u l a t u r e a n d the mechanical b e h a v i o u r of a strip of taenia can be c o n v e n i e n t l y simplified as that o f a muscle s h o r t e n i n g or pulling its two ends towards each other. But how, then, are the forces generated by the individual a n d m i n u t e cells added up to each other to o b t a i n the 'macroscopic' pull o f the tissue?
Material and Methods Strips of taenia coli were obtained from adult guinea-pigs of either sex, weighing400-600 grams. The strips were dissected from the caecum and contained also the myenteric plexus and some circular musculature; usually they were incubated for several minutes in oxygenated Krebs' solution, before fixation.A load (usually 1 gram weight)was attached to one end of the strip. The fixation was carried out at room temperature with 5 % (v/v) glutaraldehyde in 100 millimol/1Na cacodylate buffered at pH 7.4. After 2-16 h the specimenswere washed in buffer alone, then osmicated in a 2 ~o OsO4 solution for 2 h, dehydrated in ethanol and epoxypropane and embedded in Araldite. Block-stainingwas carried out in a 2 % aqueous solution of uranyl acetate. Thick sections were examined unstained in a phase contrast microscope, thin sections(stained with 1% uranyl acetate in 70 % ethanol and lead citrate or with lead alone) were examined in Philips 300 and 301 electron microscopes, equipped with goniometer stage. Some strips were fixed for 1 h in Carnoy solution, and embedded in wax; sections were stained with hematoxylin and eosin. The collagen concentration was estimated from the hydroxyproline content of acid hydrolysates of the followingorgans of guinea-pigs: heart, uterus, central tendon of the diaphragm, caecum, sartorius, thoracic aorta. From the heart, myocardium was obtained from the ventricular wall taking care to exclude all valvesand tendons. For the uterus, measurements were made on the myometrium, dissected in small strips from the rest of the wall, and on the whole uterine wall. The central tendon of the diaphragm was dissectedexcludingthe inferior vena cava and all muscular attachments. For the caecum, the taenia coli was dissected with the myenteric plexus and some of the underlying circular muscle. The thoracic aorta was dissected free from all connectivelinks to the mediastinal organs. The hydroxyproline content of the acid hydrolysates of tissue samples was determined by the colourimetric method of Stegemann (1958) and modified for a Autoanalyser by Grant (1964).
Results
a) Distribution of Dense Patches (Dense Bands) In s m o o t h muscle cells transversely cut at the level of the nucleus dense patches occupy a b o u t 50 % (range 4 0 - 6 0 %) o f the cell perimetre (Fig. 1 A). The percentage
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of the cell profile occupied by dense patches increases in small profiles, i.e. towards the tapering ends of the cell. In some of the smallest profiles the percentage is near to, or reaches, 1 0 0 ~ (Fig. 1 B). In longitudinal section the dense patches are elongated and, in mildly stretched muscles, they are approximately parallel to the major axis of the muscle cell. Their length was not determined, but some measured over 2.5 lam. The term dense bands is therefore preferred to the more widely used ones of dense patches or attachment plaques, At the level of the dense bands the trilaminar appearance of the cell membrane is maintained (Fig. l C). In some cases a layer of electron-dense material appears directly stuck to the inner aspect of the cell membrane (and this arrangement was confirmed by tilting the sections in the microscope). More often a layer of reduced electron density intervenes between cell membrane and electron-dense material and in the latter a row of regularly spaced, circular particles (sectioned filaments?) can be recognized (Fig. 1 C). Thin filaments and sometimes also intermediate (10 nm) filaments are present immediately beneath the dense bands. Some dense bands lie underneath the basal lamina and collagen fibrils; others are matched by another dense band in an adjacent muscle cell, and form the so-called intermediate contacts or desmosome-like attachments (Fig. 2 B).
b) Basal Lamina The basal lamina, present over the entire surface of all muscle cells, is a layer of fuzzy material, of medium electron-density, about 20 nm thick, separated by an electronlucent space from the cell membrane (Fig. 1 B). Where it lies over a dense band, the basal lamina is often slightly thicker than elsewhere and appears less clearly separated from the cell membrane. The basal lamina of blood capillaries of the taenia has a similar electron density but is 2-3 times thicker. The basal lamina was studied enface, in sections grazing the surface of smooth muscle cells, but little was seen of its substructure. In some preparations a dim and ill-defined meshwork ofmicrofibrillar material appears to be a major constituent of the basal lamina (better visible in stereo-pair electron micrographs [Fig. 2 C, D ]); this meshwork also appears to be connected to other microfibrils in the intercellular space and to collagen fibrils.
c) Specializations at the Muscle Cell's End (Terminal Anchoring Apparatuses) Serial sections of strips of taenia fixed in condition of mild stretch were studied in the electron microscope. The muscle cell profiles become progressively smaller as the plane of section approaches the cell's end. However, most muscle cells in the last 10-201am of their length acquire an irregular profile with folds, projections and invaginations; consequently, while the area of the cell profile decreases, its perimetre does not, and at some points it is considerably increased. Some muscle cells acquire prominent laminar projections and appear star-shaped in transverse sections (Fig. 3 A). The basal lamina adhering to these parts of the cell is thicker and merges at several points with that of neighbouring muscle cells.
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At the ends of other muscle cells finger-like invaginations of the cell membrane parallel to the longitudinal axis of the cell are visible (Fig. 3 B). These invaginations are lined by a thickened basal lamina; they may contain collagen fibrils (measuring only about 25 nm in diameter). Some cell profiles are extremely convoluted, surrounded by, or almost embedded into, a much thickened basal lamina, often displaying a granular texture (Fig. 3 C). When these profiles are followed in serial sections they lead to a complete muscle cell in both directions along the series (Figs. 4, 5). These structures are in fact end-to-end junctions between two muscle cells, characterized by a complex interdigitation of laminar and finger-like processes originating from each cell. The laminar projections are often fenestrated. Corresponding images are obtained from longitudinal sections (Fig. 2 A), but they can be correctly interpreted only on the basis of the full reconstructions obtained with serial transverse sections.
d) Other Intercellular Anchoring Apparatuses Muscle cells can be attached to each other side-by-side by intermediate junctions (see a) (Fig. 2 B). In addition, there are anchoring apparatuses, similar to those described in the previous paragraph (end-to-end), where a muscle cell ends against the flank of another cell (end-to-side) (Fig. 6) or, less often, two cell sides are anchored to each other (side-to-side). In the area of an end-to-side contact, which can extend for 10-25 gm, the tapering cell acquires an irregular profile and gives origin to long laminar and finger-like processes parallel to the cell longitudinal axis. The matching muscle cell has grooves often continuing into tunnels, which are parallel to the cell length and are penetrated by thin processes of the tapering cell.
e) Collagen Fibrils Numerous collagen fibrils are present in the intercellular space (Fig. 1 A). The fibrils measure 30-35 nm in diameter, and in longitudinal section show cross-striation with a 64 nm period. Collagen fibrils often approach the basal lamina of the muscle cells but rarely get nearer to the cell membrane (Fig. 7 A). Elastic fibres, formed by a
Fig. 1. A A smooth muscle cell in transverse section, showing the nucleus and mitochondria. Near the cell surface there are numerous incrustations of electron-dense material (dense bands in transverse section) which alternate fairly regularly with areas mainly occupied by caveolae. Collagen and elastic fibrils are visible in the extracellular space. Marker: 2 I~m.B In the centre a small muscle cell profile whose surface is almost entirely occupied by electron-dense material. At the level of the dense patches in the neighbouring muscle cells the basal lamina appears thicker and denser than elsewhere. Marker: 2 ~tm. C Detail of a smooth muscle cell showing thick (a), intermediate (10nm) (b) and thin (c) filaments, the plasma membrane with caveolae, and sarcoplasmic reticulum (d). The trilaminar appearance of the plasma membrane at the level of a dense band is visible (e); granular material is present in the dense band itself, and underneath there is an array of intermediate filaments. This section was cut at the level of the nucleus, f i s a microtubule. Marker: 0.5 ~tm
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Fig. 2 A-D. Longitudinal sections of taenia coli fixed in condition of moderate stretch. A In the centre two muscle cells join each other end-to-end. This interpretation is based on the study of serial transverse sections of similar regions. Conspicuous basal lamina material is present between the two cells. Marker: 2/am. B An intermediate contact between two muscle cells. Marker: 2 lam. C A stereopair from a section grazing the surface of a muscle cell. Caveolae, with glycogen granules between them, are in transverse section and a sense band is seen enface. In the basal lamina an ill-defined meshwork of microfilaments, some of which link with collagen fibrils, can be seen. Marker: 1 I~m
Fig. 3 A - C . Transverse sections of taenia coli. These muscle cell profiles were studied in serial sections. A In the last few microns of its length the muscle cell in the centre acquires a convoluted profile with laminar projections. Parts of the basal lamina are thickened. Marker: 2 lam. B The profile in the centre is the tapering end of a muscle cell showing finger-like invaginations of the cell membrane parallel to the longitudinal axis. Marker: 2 rtm. C A complex profile which in serial sections turned out to derive from two muscle cells joined end-to-end. A r o u n d the profile there are collagen and elastic fibres; between the laminar projections of the two cells there is mainly basal lamina material and few collagen fibrils. To the left a small nerve with two agranular vesicle containing axons. Marker: 2 g m
Fig. 4 A-I-l, Serial transverse sections of a muscle cell. Eight representative levels from a series through 52 Ilm. The level is expressed as the distance in microns from the beginning of the series. A 6 lain; B 10 lam; C 15 ~tm; D 17 txm; E 19 lam; F 20 ~tm; G 22 ~tm; H 25 ~tm. The complex aspect of this profile in C - F is due to the end-to-end interdigitation of laminar projections from two muscle cells, which appear as plain profiles in A and H respectively. Marker: 2 lain
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19
26
27
29
31
32
33
34
35
36
37
38
Fig. 5. Drawings from serial transverse sections.The levelis indicated by the number beloweach section, which is the distance in microns from the beginning of the series. Two muscle cells (shaded)join each other end-to-end by means of elaborate laminar and finger-like processes
core of amorphous material surrounded by micro filaments about 10nm in diameter, are also present in the intercellular space. They range between 0.1 and 0.7 ~tm in diameter. The small elastic fibres run approximately parallel to the cell length and are sometimes lodged in grooves of the cell surface. Most of the large elastic fibres run approximately orthogonal to the longitudinal axis of the taenia. Microfilaments are also present in small groups among the collagen fibrils and streaks of amorphous material of electron density similar to that of the basal lamina are often observed in the intercellular space between the collagen fibrils. Crossbanded structures with a period of 86-87 nm were also found (Fig. 7B). In taeniae fixed in condition of mild stretch (load 1 gram) most collagen fibrils run parallel to, or at a small angle with, the muscle cells. However, there is also a small number of fibrils which appear to be wound around a muscle cell or to form loops near its surface (Fig. 7 C). Changes in the arrangement of collagen fibrils during an isotonic contraction have been reported in a previous paper (Gabella, 1976 b), where it was shown that around maximally shortened smooth muscle cells the collagen fibrils are mainly wound in spirals of small pitch, and therefore run nearly orthogonal to the longitudinal axis of the muscle cells.
Fig. 6 A-J. Serial transverse sections of two muscle cells. Ten representative levels from a series through 45 ~tm. The level is expressed as the distance in microns from the beginning of the series. A 10p,m; B 22 p,m; C 25 p_m; D 26 p_m; E 27 p,m; F 30 p.m; G 32 l.tm; H 34 p_m; 135 p,m; J 37 lain. The series illustrates an end-to-end attachment between two muscle cells, which appear as plain profiles in A. Marker: 2 lim
Fig. 7 A - C . Longitudinal sections of taenia coli fixed in condition of moderate stretch. A Thin collagen fibrils with the characteristic cross-striation are present between muscle cells and run mainly parallel to them. One fibril (centre) approaches very closely the cell membrane probably piercing through the basal lamina. Marker: 1 lam. B Cross-banded structures with a period of about 86 n m are present a m o n g the collagen fibrils in the intercellular space. Marker: 1 pm. C A fairly thick section grazing the surface of a muscle cell. Caveolae, with cisternae of sarcoplasmic reticulum between them, are in transverse section. Most of the collagen fibrils are longitudinally arranged, but those closer to the cell surface seem to form loops and spirals around the cell. Marker: 2 p.m
Fig. 8 A-C. Longitudinal sections of taenia coli. Light microscopic micrograph. The sections were tangential to the serosal surface of the taenia. A Taenia fixed in condition of moderate stretch. Unstained section photographed in a phase contrast microscope. The septa of connective tissue (light areas) extend only for a limited distance along the length of the taenia. Few septa span the length of this microscope field (520~tm). Marker: 100~tm. B Same as A. It shows that a small angle is formed between the longitudinal axes of muscle cells and connective tissue septa, Marker: 20 ~tm. C Taenia fixed during isotonic contraction obtained with 10-5 M carbachol against a load of 1 gram. Section stained with hematoxylin and eosin. The angle between the longitudinal axes of muscle cells and connective tissue septa is much greater than in A, and gives an overall impression of a herring-bone pattern. Marker: 1 O0 lam
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f) Connective Tissue Septa When strips oftaenia are cut in longitudinal section on a plane parallel to the serosa (tangential longitudinal sections) the connective tissue septa appear sectioned along their tength (Fig. 8 A). It is clear (as already gathered from serial transverse sections) that the septa extend only for a limited distance along the muscle. The thick septa, more conspicuous near the inner surface of the taenia, are up to 1-2 mm long; the thin ones, more numerous towards the outer surface of the taenia, extend for only 100-400 microns. In taeniae fixed in condition of mild stretch, the muscle ceils and the septa of connective tissue are not parallel but form an angle with each other of about 5~(Fig. 8 B). In taeniae fixed during isotonic contraction against a light load (e.g. 1 gram weight) and therefore with extensive shortening (to 25-40 ~o the resting in vitro length) the angle between muscle cells and septa increases; values of 22-25 ~ were measured. With an increase of the angle it becomes also apparent that muscle cells are not only obliquely arranged but also alternate their direction in adjacent cell groups; this arrangement gives an overall impression of a herring-bone pattern (Fig.
8C).
g) Collagen Content The concentration of collagen in taenia coli has been estimated from the content of hydroxyproline. For comparison, estimations were carried out in parallel in the uterus (whole uterine wall and myometrium alone), a skeletal muscle (diaphragm), the heart, the thoracic aorta and the central tendon of the diaphragm (Table 1). Taenia coli and uterus contain larger concentrations of collagen than skeletal and cardiac muscles. The collagen concentration in these smooth muscles is intermediate between that of tendon and that of striated muscles.
TaMe 1. Hydroxyproline concentration (in micrograms per milligram of wet weight) in various tissues of the guinea-pig. The values in each row are from the same animal. Bottom row: mean -- standard error. The weight of collagen in the samples can be calculated, on the assumption that all the hydroxyproline is collagenous, by multiplying the weight of hydroxyproline by 7,5. The principal source of error is elastim but even in the aorta, which m a y have as m u c h elastin as collagen (Harkness et at., 1957), the error will be less than 10 ~, which in this context can be ignored Aorta 1 2 3 4 5 •
Myocardium
9.47 8.14 11.06 14.84 8.75
0.49 0,74 0.67 0.61 .
10.45
0.63 •
.
Sartorius 1.21 1.28 -1.25 . 1.25 +0.02
Taenia
Uterus
4.36 3.83 4.07 4.72
7.58 3.26 4.87 4.36
.
Myometrium
Tendon
-3.32 5.69 5.51
-18.98 26.96 28,10 31.16
. 4.24 •
5.02 +0.92
4.81 •
26.3 •
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Discussion
The morphological evidence obtained in this investigation can be summed up into the following points: 1. Dense bands (which appear as dense patches in transversely sectioned muscle cells) are present over the entire length of the cell. Thin filaments can be clearly seen merging in the electron-dense material beneath the cell membrane, and during contraction the dense bands appeared pulled towards the axis of the cell whereas areas of the cell membrane rich in caveolae appear pushed outwards. Both observations indicate that dense bands are attachment sites for the thin filaments which run near the cell surface, while the rest of the thin filaments of the cell are attached at one end to dense bodies, as suggested by many authors (Pease and Molinari, 1960; Prosser et al., 1960; Rosenbluth, 1965; Lane, 1965; Bois, 1973; Ashton et al., 1975). The number of thin filaments which are attached to the cell membrane must be substantial, since up to 50 % of the cell membrane is occupied by dense bands at the level of the middle parts of the cell, and more towards the cell's ends. In other words, a large number of thin filaments are attached to the 'lateral' walls of the muscle cell, along its entire length, as opposed to the attachment to the cell's ends. Elsewhere it has been suggested that this arrangement may account for the remarkably large tension developed by this muscle per unit of transverse sectional area (Gabella, 1976 c). Part of the tension produced by the myofilaments would be distributed to the cell surface at all levels along the cell length, so that the functional transverse sectional area (indicative of the number of contractile elements acting in parallel) is greater than the anatomical transverse sectional area. In the model of smooth muscle contraction proposed by Pease and Molinari (1960) it was already suggested that the force generated by the myofilaments is directly applied to the cell surface. According to Cooke (1976) the tension is transmitted to the cell surface by means of a framework of intermediate (10nm) filaments; the latter, however, are mainly attached to the cell surface at or near the ends of the muscle cells, the 'lateral' attachments along the length of the cell being relatively infrequent (Cooke, 1975). Similar ideas have been discussed by Ashton et al. (1975) and by Murphy (1976), but recently Driska and Murphy (1977) have argued that the large force output of arterial smooth muscle is accounted for by the properties of the contractile material. If the large tension per unit sectional area developed by the taenia coli is to be explained partly by means of the presence of numerous 'lateral' attachments of myofilaments, it is also required that the dense bands be capable of transmitting forward (i.e. along the length of the taenia) the tension generated inside the cell by the myofilaments. This is achieved where the dense bands match similar structures in a neighbouring muscle cell, i.e. at the intermediate junctions, or at the level of more complex anchoring apparatuses (discussed below, para 2.). Since, however, many dense bands do not match each other and appear to be facing a wide intercellular space, it is tentatively suggested that the sarcolemma itself can bear and transmit some of the muscular tension. Direct evidence of a mechanical link between cell membrane and basal lamina on one side and the collagen fibril network on the other is lacking, except for a few collagen fibrils which are seen to get very close to the basal lamina and for the occurrence of a microfilamentous feltwork
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between collagen fibrils and basal lamina. Indirect evidence was obtained with isotonical[y contracted smooth muscle cells: in this condition most dense bands are situated in deep grooves at the cell surface and run obliquely to the cell long axis, but the collagen fibrils remain in close proximity of the dense bands, being found within the grooves and parallel to them. However, the physical properties of the sarcolemma in smooth muscle and its role in the transmission of force are only poorly understood. By contrast, in skeletal muscle fibres the sarcomeres act in series and the tension develops between the ends of the fibre as a simple, linear pull. However, the sarcolemma (plasma membrane, basal lamina, collagen and other fibrillar material) of skeletal fibres seems also adequate to bear and transmit maximum tension developed during stimulation, at least in injured fibres (Street and Ramsey, 1965). Moreover, Fields (1970) has found that the sarcolemma of the frog semitendinosus muscle is anisotropic, being much stiffer in the longitudinal direction, and Schmalbruch (1974) has observed that the orientation of collagen fibrils of the sarcolemma with respect to the fibre axis changes at different sarcomere lengths. It is therefore possible that even in skeletal muscle fibres (where the tension is mainly transmitted from one end of the fibre to the other through a long series of sarcomeres) the sarcolemma at the 'lateral' sides of the fibre plays some role in the transmission of force, particularly during isotonic contraction. Attachment of thin filaments to the plasma membrane in regions far from the cell's ends is not a unique characteristic of smooth muscle. It is known to occur to a limited extent in cardiac muscle (e.g. Bogusch, 1974; McNutt, 1975), particularly in embryonic cardiac cells (Hagopian and Spiro, 1970), and it is a common occurrence in transversely and obliquely striated muscles of invertebrates (e.g. Smith et al., 1966; Mill and Knapp, 1970; Rosenbluth, 1972; Dewey et al., 1973; and many others). Further evidence of mechanical links between muscle cells and material in the intercellular space is provided by finger-like invaginations of the cell membrane at the cell's end. Similar structures have been described before in smooth muscle cells of the guinea-pig vas deferens (Merrillees et al., 1963) and are a prominent feature of the muscle-tendon junctions in skeletal fibres (Gelber et al., 1960; Schwarzacher, 1960; Ishikawa, 1965; Mackay et al., 1969; Hanak and B6ck, 1971). 2. Various types of structural specialization join together adjacent muscle cells. In addition to the well known intermediate junctions (see Henderson, 1975), there are more elaborate apparatuses linking two cells together end-to-end or side-to-side or end-to-side. In each case the contact is characterized by an increase of the surface of both cells, by means of folds, invaginations, projections. The area of contacts is, therefore, much greater than if the two cells were facing each other with smooth surfaces, and also the ratio of surface to volume is increased at that particular level of the cell. Moreover, the basal lamina appears thickened and may fill a gap of 100 nm or more between the apposed cell surfaces. These observations strongly suggest that the apparatuses described anchor two muscle cells to each other and allow the mechanical pull of one to be transmitted to the other. Considerable mechanical stability of the intermediate junctions or desmosome-like attachments is shown by the observations that, in the dog duodenum, these junctions are not pulled apart by osmotic cell shrinkage (Henderson et al., 1971).
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3. The present estimations of hydroxyproline content, performed in parallel in a number of muscles of the guinea-pig, show that the taenia coli contains a larger concentration of collagen than skeletal and cardiac muscles (the values for cardiac and skeletal muscles are within the range of those previously found in the rat by Lowry et al., 1942). The collagen concentration in the guinea-pig uterus is similar to that in the taenia, and it is slightly higher than those previously reported for the uterus of the mouse (Finn et al., 1963) or the rat (Grant, 1965). Much higher values of collagen concentration were found in the tendon examined. Therefore, the collagen concentration in taenia coli (and uterus) is intermediate between that of tendon and that of striated muscles. Characterization of the collagen types present in the taenia coli has not yet been carried out. It should be noted that in addition to collagen fibrils, the basal laminae are a major component probably containing collagen. On the basis of muscle cell size and packing density in the taenia coli (Gabella, 1976 a) it can be calculated that there are nearly 10 cm 2 of muscle cell membrane (and approximately the same amount of basal lamina) per mm 3 of muscle. The amount of type IV collagen (specific of basal lamina, Kefalides, 1971) present in the taenia should be substantial. It is also worth noting that intramuscular collagen fibrils measure about 30-35 nm in diameter and never reach the diameter of 50 nm and over typical of the fibrils of tendons and other connective tissues. Fibrils of the large type are found in other parts of the wall of the caecum. Similarly, in large arteries of the guinea-pig, such as thoracic aorta and external iliac artery, the collagen fibrils of the intima and media measure about 35 nm in diameter, whereas those of the outer part of the adventitia measure over 50 nm (unpublsished observations). It seems, therefore, that the collagen fibrils present within vascular and intestinal muscles are of small diameter, like those which constitute the 'reticular' fibres. Elastic fibres, microfibrils and other ill-defined material is present between the muscle cells of the taenia coli. Microfibrils similar in appearance to those which are part of the elastic fibres are known to be associated with the basal lamina of smooth muscle cells (Haust, 1965). The cross-banded structures occasionally found in the intercellular space are similar to those described in other connective tissues (Phillai, 1964; Cravioto and Lockwood, 1968; Sun and White, 1975); their significance is still obscure. On the basis of the present measurements of the collagen concentration in various types of muscle, it can be suggested that the taenia coli, although it lacks discrete tendons, has in fact a kind of diffuse intramuscular tendon, made up of a dense collagen network. The light microscopic observations show that a large part of this collagen constitute septa, different in thickness and rather limited in length, onto which many muscle cells about. 4. Septa of connective tissue are present among the muscle cells and appear to subdivide these into more or less clear-cut groups. Similar observations are at the basis of the current notion that smooth muscles (and the taenia coli in particular) are composed of bundles and that the latter, more than the individual muscle cells, are the functional units of the muscle. In a study of serial transverse sections of taenia coli doubts were cast on this notion, since the groups of'bundles' of smooth muscle cells were found to loose their individuality within tens of microns of their length, splitting into or merging with other 'bundles' (Gabella, 1976a). In the present investigation the attention was focussed on the septa themselves and it has
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been found that their length along the longitudinal axis of the taenia is rather limited, usually in the order of hundreds of microns. From the analysis of serial sections the septa can be visualized as laminae or plaques of connective tissue, slightly longer than broader and of various thicknesses, with little or no continuity with each other. Only the thicker septa are anchored at one side to the connective tissue which lies between taenia and circular musculature of the caecum. The septa are nearly parallel to each other, but the longitudinal axis of the muscle cells is not exactly parallel to the plane of the septa on which they abut. In taeniae fixed in condition of mild stretch this structural detail is barely visible, an angle of about 5 ~ being formed between muscle cells and septa, but in taeniae fixed during isotonic contraction the angle is up to 22-25 ~. In tangential section the taenia show a herring-bone pattern, with the muscle cells directed from one septum to another. This pattern is reminiscent of a multipennate skeletal muscle except that instead of proper tendons the taenia has septa which are much more numerous and are entirely intramuscular. Each connective tissue septum is tentatively visualized as giving attachment to several arrays of muscle cells pulling in opposite directions.
References Ashton, F.T., Somlyo, A.V., Somlyo, A.P.: The contractile apparatus of vascular smooth muscle: intermediate high voltage stereo electron microscopy. J. molec. Biol. 98, 17-29 (1975) Bogusch, G.: Investigations on the fine structure of Purkinje fibres in the atrium of the avian heart. Cell Tiss. Res. 150, 43-56 (1974) Bois, R.M.: The organization of the contractile apparatus of vertebrate smooth muscle. Anat. Rec. 177, 61-78 (1973) Cooke, P.: A filamentous cytoskeleton in vertebrate smooth muscle fibers. J. Cell Biol. 68, 539-556 (1976) Cravioto, H., Lockwood, R.: Long-spacing fibrous collagen in human acoustic nerve tumors, in vivo and in vitro observations. J. Ultrastruct. Res. 24, 70-85 (1968) Dewey, M.M., Levine, R.J.C., Colflesh, D.E.: Structure of Limulus striated muscle. J. Cell Biol. 58, 574593 (1973) Driska, S.P., Murphy, R.A.: Force generation by vascular smooth muscle cells. Biophys. J. 17, 266a (1977) Fields, R.W.: Mechanical properties of the frog sarcolemma. Biophys. J. 10, 462-479 (1970) Finn, C.A., Fitch, S.M., Harkness, R.D.: Collagen content of barren and previously pregnant uterine horns in old mice. J. Reprod. Fertil. 6, 405-407 (1963) Gabella, G.: Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli. Cell Tiss. Res. 170, 161-186 (1976a) Gabella, G.: Structural changes in smooth muscle cells during isotonic contraction. Cell Tiss. Res. 170, 187-201 (1976 b) Gabella, G.: The force generated by a visceral smooth muscle. J. Physiol. (Lond.) 263, 199-213 (1976 c) Gelber, D., Moore, D.H., Ruska, H.: Observations on the myo-tendon junction in mammalian skeletal muscle. Z. Zellforsch. 52, 396-400 (1960) Grant, R.A.: Estimation of hydroxyproline by the autoanalyzer. J. clin. Path. 17, 685-686 (1964) Grant, R.A.: Chemical changes in the uterus of the rat during late pregnancy and postpartum involution. The effects of lactation and hormone treatment. J. Reprod. Fertil. 9, 285-299 (1965) Hanak, H., B6ck, P.: Die Feinstruktur der Muskel-Sehnenverbindung von Skelett- und Herzmuskel. J. Ultrastruct. Res. 36, 68-85 (1971) Harkness, M.L.R., Harkness, R.D., McDonald, D.A.: Collagen and elastin content of the arterial wall of the dog. Proc. roy. Soc. B 146, 541-551 (1957) Haust, M.D. : Fine fibrils of extracellular space (microfibrils). Amer. J. Path. 37, 1113-1137 (1965)
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Henderson, R.M.: Cell-to-cell contacts. In: Methods in pharmacology, Vol. 3, pp. 47-77 (E.E. Daniel and D.M. Paton, eds.). New York: Plenum Press 1975 Henderson, R.M., Duchon, G., Daniel, E.E.: Cell contacts in duodenal smooth muscle layers. Amer. J. Physiol. 221, 564-574 (1971) Hagopian, M., Spiro, D.: Derivation of the Z line in the embryonic chick heart. J. Cell Biol. 44, 683-687 (1970) Ishikawa, H.: The fine structure of myo-tendon junction in some mammalian skeletal muscles. Arch. histol, jap. 25, 275-296 (1965) Kefalides, N.A.: Chemical properties of basement membranes. Int. Rev. exp. Path. 10, 1-39 (1971) Lane, B.P.: Alterations in the cytologic detail of intestinal smooth muscle cells in various stages of contraction. J. Cell Biol. 27, 199-213 (1965) Lowry, O.H., Hastings, A.B., Hull, T.Z. : Histochemical changes associe.ted with aging. II. Skeletal and cardiac muscle in rat. J. biol. Chem. 143, 271-280 (1942) Mackay, B., Harrop, T.J., Muir, A.R.: The fine structure of the muscle tendon junction in the rat. Acta anat. (Basel) 73, 588-604 (1969) McNutt, N.S. : Ultrastructure of myocardial sarcolemma. Circulat. Res. 37, 1-13 (1975) Merrillees, N.C.R., Burnstock, G., Holman, M.E.: Correlation of fine structure and physiology of the innervation of smooth muscle of the guinea pig vas deferens. J. Cell Biol. 19, 529-550 (1963) Mill, P.J., Knapp, M.F.: The fine structure of obliquely striated body wall muscles in the earthworm, Lumbricus terrestris Linn. J. Cell Sci. 7, 233-261 (1970) Murphy, R.A.: Contractile system in mammalian smooth muscle. Blood Vess. 13, 1-23 (1976) Pease, D.C., Molinari, S.: Electron microscopy of muscular arteries: pial vessels of the cat and monkey. J. Ultrastruct. Res. 3, 447-468 (1960) Phillai, P.A.: A banded structure in the connective tissue of nerve. J. Ultrastruct. Res. 11, 455-468 (1964) Prosser, C.L., Burnstock, G., Kahn, J.: Conduction in smooth muscle: comparative structural properties. Amer. J. Physiol. 199, 545-552 (1960) Rosenbluth, J.: Smooth muscle: an ultrastructural basis for the dynamics of its contraction. Science 148, 1337-1339 (1965) Rosenbluth, J. : Obliquely striated muscle. In: Structure and function of striated muscle. Vol. 1, pp. 389420 (G.E. Bourne, ed.). New York and London: Academic Press 1972 Schmalbruch, H.: The sarcolemma of skeletal muscle fibres as demonstrated by a replica technique. Cell Tiss. Res. 150, 377-387 (1974) Schwarzacher, H.G.: Untersuchungen fiber den Feinbau der Muskelfaser-Sehnenverbindungen. Acta anat. (Basel) 40, 59-86 (1960) Smith, D.S., Gupt, B.L., Smith, U.: The organization and myofilament array of insect visceral muscles. J. Cell Sci. 1, 49-57 (1966) Stegemann, H.: Mikrobestimmung von Hydroxyprolin mit Chloramin-T und pDimethylaminebenaldehyd. Hoppe-Seylers Z. physiol. Chem. 311, 41-45 (1958) Street, S.F., Ramsey, R.W.: Sarcolemma: transmitter of active tension in frog skeletal muscle. Science 149, 1379-1380 (1965) Sun, C.N., White, H.J.: Extracellular cross-striated banded structures in human connective tissue. Tissue and Cell 7, 419-432 (1975)
Accepted June 29, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Arrangement of Smooth Muscle Cells and Intramuscular Septa in the Taenia coli Giorgio Gabella* Department of Anatomy, University College London, England
Summary. Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10nm) filaments; about 50 % of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5~ with the septa. In muscles fixed during isotonic contraction the angle increases to about 20-22 ~, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4-6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission. Key words: Taenia coli - Guinea pig - Smooth muscle - Collagen Ultrastructure.
Introduction This paper follows a study on the fine structure of muscle cells of the guinea-pig taenia coli (Gabella, 1976a), and presents further morphological observations Send offprint requests to: Dr. Giorgio Gabella, Department of Anatomy, University College London,
Gower Street, London, WC1E 6BT, England * I thank Mr. S.I. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council
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focussed o n those structures which m a y be involved in the process o f t r a n s m i s s i o n of force. Force is generated by the so-called contractile machinery, i.e. the sets o f myofilaments. While in the case o f striated muscle fibres it is relatively simple to visualize a series o f sarcomeres pulling one against the next a n d the terminal ones pulling the t e n d o n s at each end o f the fibre, it is less clear how a similar effect can be o b t a i n e d in a s m o o t h muscle. It is true that in m a n y s m o o t h muscles the shortening o f the muscle m a y n o t be the principal mechanical effect o f a c o n t r a c t i o n ; for example, the circular m u s c u l a t u r e of the intestine shortens u p o n c o n t r a c t i o n a n d the size of the l u m e n is therefore reduced, b u t some of the force generated is applied in a transverse direction, both radially (towards the l u m e n ) a n d along the length of the intestine (so that e l o n g a t i o n o f the gut ensues). However, the taenia coli is a conspicuous a n d discrete strap o f l o n g i t u d i n a l m u s c u l a t u r e a n d the mechanical b e h a v i o u r of a strip of taenia can be c o n v e n i e n t l y simplified as that o f a muscle s h o r t e n i n g or pulling its two ends towards each other. But how, then, are the forces generated by the individual a n d m i n u t e cells added up to each other to o b t a i n the 'macroscopic' pull o f the tissue?
Material and Methods Strips of taenia coli were obtained from adult guinea-pigs of either sex, weighing400-600 grams. The strips were dissected from the caecum and contained also the myenteric plexus and some circular musculature; usually they were incubated for several minutes in oxygenated Krebs' solution, before fixation.A load (usually 1 gram weight)was attached to one end of the strip. The fixation was carried out at room temperature with 5 % (v/v) glutaraldehyde in 100 millimol/1Na cacodylate buffered at pH 7.4. After 2-16 h the specimenswere washed in buffer alone, then osmicated in a 2 ~o OsO4 solution for 2 h, dehydrated in ethanol and epoxypropane and embedded in Araldite. Block-stainingwas carried out in a 2 % aqueous solution of uranyl acetate. Thick sections were examined unstained in a phase contrast microscope, thin sections(stained with 1% uranyl acetate in 70 % ethanol and lead citrate or with lead alone) were examined in Philips 300 and 301 electron microscopes, equipped with goniometer stage. Some strips were fixed for 1 h in Carnoy solution, and embedded in wax; sections were stained with hematoxylin and eosin. The collagen concentration was estimated from the hydroxyproline content of acid hydrolysates of the followingorgans of guinea-pigs: heart, uterus, central tendon of the diaphragm, caecum, sartorius, thoracic aorta. From the heart, myocardium was obtained from the ventricular wall taking care to exclude all valvesand tendons. For the uterus, measurements were made on the myometrium, dissected in small strips from the rest of the wall, and on the whole uterine wall. The central tendon of the diaphragm was dissectedexcludingthe inferior vena cava and all muscular attachments. For the caecum, the taenia coli was dissected with the myenteric plexus and some of the underlying circular muscle. The thoracic aorta was dissected free from all connectivelinks to the mediastinal organs. The hydroxyproline content of the acid hydrolysates of tissue samples was determined by the colourimetric method of Stegemann (1958) and modified for a Autoanalyser by Grant (1964).
Results
a) Distribution of Dense Patches (Dense Bands) In s m o o t h muscle cells transversely cut at the level of the nucleus dense patches occupy a b o u t 50 % (range 4 0 - 6 0 %) o f the cell perimetre (Fig. 1 A). The percentage
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of the cell profile occupied by dense patches increases in small profiles, i.e. towards the tapering ends of the cell. In some of the smallest profiles the percentage is near to, or reaches, 1 0 0 ~ (Fig. 1 B). In longitudinal section the dense patches are elongated and, in mildly stretched muscles, they are approximately parallel to the major axis of the muscle cell. Their length was not determined, but some measured over 2.5 lam. The term dense bands is therefore preferred to the more widely used ones of dense patches or attachment plaques, At the level of the dense bands the trilaminar appearance of the cell membrane is maintained (Fig. l C). In some cases a layer of electron-dense material appears directly stuck to the inner aspect of the cell membrane (and this arrangement was confirmed by tilting the sections in the microscope). More often a layer of reduced electron density intervenes between cell membrane and electron-dense material and in the latter a row of regularly spaced, circular particles (sectioned filaments?) can be recognized (Fig. 1 C). Thin filaments and sometimes also intermediate (10 nm) filaments are present immediately beneath the dense bands. Some dense bands lie underneath the basal lamina and collagen fibrils; others are matched by another dense band in an adjacent muscle cell, and form the so-called intermediate contacts or desmosome-like attachments (Fig. 2 B).
b) Basal Lamina The basal lamina, present over the entire surface of all muscle cells, is a layer of fuzzy material, of medium electron-density, about 20 nm thick, separated by an electronlucent space from the cell membrane (Fig. 1 B). Where it lies over a dense band, the basal lamina is often slightly thicker than elsewhere and appears less clearly separated from the cell membrane. The basal lamina of blood capillaries of the taenia has a similar electron density but is 2-3 times thicker. The basal lamina was studied enface, in sections grazing the surface of smooth muscle cells, but little was seen of its substructure. In some preparations a dim and ill-defined meshwork ofmicrofibrillar material appears to be a major constituent of the basal lamina (better visible in stereo-pair electron micrographs [Fig. 2 C, D ]); this meshwork also appears to be connected to other microfibrils in the intercellular space and to collagen fibrils.
c) Specializations at the Muscle Cell's End (Terminal Anchoring Apparatuses) Serial sections of strips of taenia fixed in condition of mild stretch were studied in the electron microscope. The muscle cell profiles become progressively smaller as the plane of section approaches the cell's end. However, most muscle cells in the last 10-201am of their length acquire an irregular profile with folds, projections and invaginations; consequently, while the area of the cell profile decreases, its perimetre does not, and at some points it is considerably increased. Some muscle cells acquire prominent laminar projections and appear star-shaped in transverse sections (Fig. 3 A). The basal lamina adhering to these parts of the cell is thicker and merges at several points with that of neighbouring muscle cells.
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At the ends of other muscle cells finger-like invaginations of the cell membrane parallel to the longitudinal axis of the cell are visible (Fig. 3 B). These invaginations are lined by a thickened basal lamina; they may contain collagen fibrils (measuring only about 25 nm in diameter). Some cell profiles are extremely convoluted, surrounded by, or almost embedded into, a much thickened basal lamina, often displaying a granular texture (Fig. 3 C). When these profiles are followed in serial sections they lead to a complete muscle cell in both directions along the series (Figs. 4, 5). These structures are in fact end-to-end junctions between two muscle cells, characterized by a complex interdigitation of laminar and finger-like processes originating from each cell. The laminar projections are often fenestrated. Corresponding images are obtained from longitudinal sections (Fig. 2 A), but they can be correctly interpreted only on the basis of the full reconstructions obtained with serial transverse sections.
d) Other Intercellular Anchoring Apparatuses Muscle cells can be attached to each other side-by-side by intermediate junctions (see a) (Fig. 2 B). In addition, there are anchoring apparatuses, similar to those described in the previous paragraph (end-to-end), where a muscle cell ends against the flank of another cell (end-to-side) (Fig. 6) or, less often, two cell sides are anchored to each other (side-to-side). In the area of an end-to-side contact, which can extend for 10-25 gm, the tapering cell acquires an irregular profile and gives origin to long laminar and finger-like processes parallel to the cell longitudinal axis. The matching muscle cell has grooves often continuing into tunnels, which are parallel to the cell length and are penetrated by thin processes of the tapering cell.
e) Collagen Fibrils Numerous collagen fibrils are present in the intercellular space (Fig. 1 A). The fibrils measure 30-35 nm in diameter, and in longitudinal section show cross-striation with a 64 nm period. Collagen fibrils often approach the basal lamina of the muscle cells but rarely get nearer to the cell membrane (Fig. 7 A). Elastic fibres, formed by a
Fig. 1. A A smooth muscle cell in transverse section, showing the nucleus and mitochondria. Near the cell surface there are numerous incrustations of electron-dense material (dense bands in transverse section) which alternate fairly regularly with areas mainly occupied by caveolae. Collagen and elastic fibrils are visible in the extracellular space. Marker: 2 I~m.B In the centre a small muscle cell profile whose surface is almost entirely occupied by electron-dense material. At the level of the dense patches in the neighbouring muscle cells the basal lamina appears thicker and denser than elsewhere. Marker: 2 ~tm. C Detail of a smooth muscle cell showing thick (a), intermediate (10nm) (b) and thin (c) filaments, the plasma membrane with caveolae, and sarcoplasmic reticulum (d). The trilaminar appearance of the plasma membrane at the level of a dense band is visible (e); granular material is present in the dense band itself, and underneath there is an array of intermediate filaments. This section was cut at the level of the nucleus, f i s a microtubule. Marker: 0.5 ~tm
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199
Fig. 2 A-D. Longitudinal sections of taenia coli fixed in condition of moderate stretch. A In the centre two muscle cells join each other end-to-end. This interpretation is based on the study of serial transverse sections of similar regions. Conspicuous basal lamina material is present between the two cells. Marker: 2/am. B An intermediate contact between two muscle cells. Marker: 2 lam. C A stereopair from a section grazing the surface of a muscle cell. Caveolae, with glycogen granules between them, are in transverse section and a sense band is seen enface. In the basal lamina an ill-defined meshwork of microfilaments, some of which link with collagen fibrils, can be seen. Marker: 1 I~m
Fig. 3 A - C . Transverse sections of taenia coli. These muscle cell profiles were studied in serial sections. A In the last few microns of its length the muscle cell in the centre acquires a convoluted profile with laminar projections. Parts of the basal lamina are thickened. Marker: 2 lam. B The profile in the centre is the tapering end of a muscle cell showing finger-like invaginations of the cell membrane parallel to the longitudinal axis. Marker: 2 rtm. C A complex profile which in serial sections turned out to derive from two muscle cells joined end-to-end. A r o u n d the profile there are collagen and elastic fibres; between the laminar projections of the two cells there is mainly basal lamina material and few collagen fibrils. To the left a small nerve with two agranular vesicle containing axons. Marker: 2 g m
Fig. 4 A-I-l, Serial transverse sections of a muscle cell. Eight representative levels from a series through 52 Ilm. The level is expressed as the distance in microns from the beginning of the series. A 6 lain; B 10 lam; C 15 ~tm; D 17 txm; E 19 lam; F 20 ~tm; G 22 ~tm; H 25 ~tm. The complex aspect of this profile in C - F is due to the end-to-end interdigitation of laminar projections from two muscle cells, which appear as plain profiles in A and H respectively. Marker: 2 lain
Arrangement of Muscle Cells in Taenia coli
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19
26
27
29
31
32
33
34
35
36
37
38
Fig. 5. Drawings from serial transverse sections.The levelis indicated by the number beloweach section, which is the distance in microns from the beginning of the series. Two muscle cells (shaded)join each other end-to-end by means of elaborate laminar and finger-like processes
core of amorphous material surrounded by micro filaments about 10nm in diameter, are also present in the intercellular space. They range between 0.1 and 0.7 ~tm in diameter. The small elastic fibres run approximately parallel to the cell length and are sometimes lodged in grooves of the cell surface. Most of the large elastic fibres run approximately orthogonal to the longitudinal axis of the taenia. Microfilaments are also present in small groups among the collagen fibrils and streaks of amorphous material of electron density similar to that of the basal lamina are often observed in the intercellular space between the collagen fibrils. Crossbanded structures with a period of 86-87 nm were also found (Fig. 7B). In taeniae fixed in condition of mild stretch (load 1 gram) most collagen fibrils run parallel to, or at a small angle with, the muscle cells. However, there is also a small number of fibrils which appear to be wound around a muscle cell or to form loops near its surface (Fig. 7 C). Changes in the arrangement of collagen fibrils during an isotonic contraction have been reported in a previous paper (Gabella, 1976 b), where it was shown that around maximally shortened smooth muscle cells the collagen fibrils are mainly wound in spirals of small pitch, and therefore run nearly orthogonal to the longitudinal axis of the muscle cells.
Fig. 6 A-J. Serial transverse sections of two muscle cells. Ten representative levels from a series through 45 ~tm. The level is expressed as the distance in microns from the beginning of the series. A 10p,m; B 22 p,m; C 25 p_m; D 26 p_m; E 27 p,m; F 30 p.m; G 32 l.tm; H 34 p_m; 135 p,m; J 37 lain. The series illustrates an end-to-end attachment between two muscle cells, which appear as plain profiles in A. Marker: 2 lim
Fig. 7 A - C . Longitudinal sections of taenia coli fixed in condition of moderate stretch. A Thin collagen fibrils with the characteristic cross-striation are present between muscle cells and run mainly parallel to them. One fibril (centre) approaches very closely the cell membrane probably piercing through the basal lamina. Marker: 1 lam. B Cross-banded structures with a period of about 86 n m are present a m o n g the collagen fibrils in the intercellular space. Marker: 1 pm. C A fairly thick section grazing the surface of a muscle cell. Caveolae, with cisternae of sarcoplasmic reticulum between them, are in transverse section. Most of the collagen fibrils are longitudinally arranged, but those closer to the cell surface seem to form loops and spirals around the cell. Marker: 2 p.m
Fig. 8 A-C. Longitudinal sections of taenia coli. Light microscopic micrograph. The sections were tangential to the serosal surface of the taenia. A Taenia fixed in condition of moderate stretch. Unstained section photographed in a phase contrast microscope. The septa of connective tissue (light areas) extend only for a limited distance along the length of the taenia. Few septa span the length of this microscope field (520~tm). Marker: 100~tm. B Same as A. It shows that a small angle is formed between the longitudinal axes of muscle cells and connective tissue septa, Marker: 20 ~tm. C Taenia fixed during isotonic contraction obtained with 10-5 M carbachol against a load of 1 gram. Section stained with hematoxylin and eosin. The angle between the longitudinal axes of muscle cells and connective tissue septa is much greater than in A, and gives an overall impression of a herring-bone pattern. Marker: 1 O0 lam
Arrangement of Muscle Cells in Taenia coli
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f) Connective Tissue Septa When strips oftaenia are cut in longitudinal section on a plane parallel to the serosa (tangential longitudinal sections) the connective tissue septa appear sectioned along their tength (Fig. 8 A). It is clear (as already gathered from serial transverse sections) that the septa extend only for a limited distance along the muscle. The thick septa, more conspicuous near the inner surface of the taenia, are up to 1-2 mm long; the thin ones, more numerous towards the outer surface of the taenia, extend for only 100-400 microns. In taeniae fixed in condition of mild stretch, the muscle ceils and the septa of connective tissue are not parallel but form an angle with each other of about 5~(Fig. 8 B). In taeniae fixed during isotonic contraction against a light load (e.g. 1 gram weight) and therefore with extensive shortening (to 25-40 ~o the resting in vitro length) the angle between muscle cells and septa increases; values of 22-25 ~ were measured. With an increase of the angle it becomes also apparent that muscle cells are not only obliquely arranged but also alternate their direction in adjacent cell groups; this arrangement gives an overall impression of a herring-bone pattern (Fig.
8C).
g) Collagen Content The concentration of collagen in taenia coli has been estimated from the content of hydroxyproline. For comparison, estimations were carried out in parallel in the uterus (whole uterine wall and myometrium alone), a skeletal muscle (diaphragm), the heart, the thoracic aorta and the central tendon of the diaphragm (Table 1). Taenia coli and uterus contain larger concentrations of collagen than skeletal and cardiac muscles. The collagen concentration in these smooth muscles is intermediate between that of tendon and that of striated muscles.
TaMe 1. Hydroxyproline concentration (in micrograms per milligram of wet weight) in various tissues of the guinea-pig. The values in each row are from the same animal. Bottom row: mean -- standard error. The weight of collagen in the samples can be calculated, on the assumption that all the hydroxyproline is collagenous, by multiplying the weight of hydroxyproline by 7,5. The principal source of error is elastim but even in the aorta, which m a y have as m u c h elastin as collagen (Harkness et at., 1957), the error will be less than 10 ~, which in this context can be ignored Aorta 1 2 3 4 5 •
Myocardium
9.47 8.14 11.06 14.84 8.75
0.49 0,74 0.67 0.61 .
10.45
0.63 •
.
Sartorius 1.21 1.28 -1.25 . 1.25 +0.02
Taenia
Uterus
4.36 3.83 4.07 4.72
7.58 3.26 4.87 4.36
.
Myometrium
Tendon
-3.32 5.69 5.51
-18.98 26.96 28,10 31.16
. 4.24 •
5.02 +0.92
4.81 •
26.3 •
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Discussion
The morphological evidence obtained in this investigation can be summed up into the following points: 1. Dense bands (which appear as dense patches in transversely sectioned muscle cells) are present over the entire length of the cell. Thin filaments can be clearly seen merging in the electron-dense material beneath the cell membrane, and during contraction the dense bands appeared pulled towards the axis of the cell whereas areas of the cell membrane rich in caveolae appear pushed outwards. Both observations indicate that dense bands are attachment sites for the thin filaments which run near the cell surface, while the rest of the thin filaments of the cell are attached at one end to dense bodies, as suggested by many authors (Pease and Molinari, 1960; Prosser et al., 1960; Rosenbluth, 1965; Lane, 1965; Bois, 1973; Ashton et al., 1975). The number of thin filaments which are attached to the cell membrane must be substantial, since up to 50 % of the cell membrane is occupied by dense bands at the level of the middle parts of the cell, and more towards the cell's ends. In other words, a large number of thin filaments are attached to the 'lateral' walls of the muscle cell, along its entire length, as opposed to the attachment to the cell's ends. Elsewhere it has been suggested that this arrangement may account for the remarkably large tension developed by this muscle per unit of transverse sectional area (Gabella, 1976 c). Part of the tension produced by the myofilaments would be distributed to the cell surface at all levels along the cell length, so that the functional transverse sectional area (indicative of the number of contractile elements acting in parallel) is greater than the anatomical transverse sectional area. In the model of smooth muscle contraction proposed by Pease and Molinari (1960) it was already suggested that the force generated by the myofilaments is directly applied to the cell surface. According to Cooke (1976) the tension is transmitted to the cell surface by means of a framework of intermediate (10nm) filaments; the latter, however, are mainly attached to the cell surface at or near the ends of the muscle cells, the 'lateral' attachments along the length of the cell being relatively infrequent (Cooke, 1975). Similar ideas have been discussed by Ashton et al. (1975) and by Murphy (1976), but recently Driska and Murphy (1977) have argued that the large force output of arterial smooth muscle is accounted for by the properties of the contractile material. If the large tension per unit sectional area developed by the taenia coli is to be explained partly by means of the presence of numerous 'lateral' attachments of myofilaments, it is also required that the dense bands be capable of transmitting forward (i.e. along the length of the taenia) the tension generated inside the cell by the myofilaments. This is achieved where the dense bands match similar structures in a neighbouring muscle cell, i.e. at the intermediate junctions, or at the level of more complex anchoring apparatuses (discussed below, para 2.). Since, however, many dense bands do not match each other and appear to be facing a wide intercellular space, it is tentatively suggested that the sarcolemma itself can bear and transmit some of the muscular tension. Direct evidence of a mechanical link between cell membrane and basal lamina on one side and the collagen fibril network on the other is lacking, except for a few collagen fibrils which are seen to get very close to the basal lamina and for the occurrence of a microfilamentous feltwork
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between collagen fibrils and basal lamina. Indirect evidence was obtained with isotonical[y contracted smooth muscle cells: in this condition most dense bands are situated in deep grooves at the cell surface and run obliquely to the cell long axis, but the collagen fibrils remain in close proximity of the dense bands, being found within the grooves and parallel to them. However, the physical properties of the sarcolemma in smooth muscle and its role in the transmission of force are only poorly understood. By contrast, in skeletal muscle fibres the sarcomeres act in series and the tension develops between the ends of the fibre as a simple, linear pull. However, the sarcolemma (plasma membrane, basal lamina, collagen and other fibrillar material) of skeletal fibres seems also adequate to bear and transmit maximum tension developed during stimulation, at least in injured fibres (Street and Ramsey, 1965). Moreover, Fields (1970) has found that the sarcolemma of the frog semitendinosus muscle is anisotropic, being much stiffer in the longitudinal direction, and Schmalbruch (1974) has observed that the orientation of collagen fibrils of the sarcolemma with respect to the fibre axis changes at different sarcomere lengths. It is therefore possible that even in skeletal muscle fibres (where the tension is mainly transmitted from one end of the fibre to the other through a long series of sarcomeres) the sarcolemma at the 'lateral' sides of the fibre plays some role in the transmission of force, particularly during isotonic contraction. Attachment of thin filaments to the plasma membrane in regions far from the cell's ends is not a unique characteristic of smooth muscle. It is known to occur to a limited extent in cardiac muscle (e.g. Bogusch, 1974; McNutt, 1975), particularly in embryonic cardiac cells (Hagopian and Spiro, 1970), and it is a common occurrence in transversely and obliquely striated muscles of invertebrates (e.g. Smith et al., 1966; Mill and Knapp, 1970; Rosenbluth, 1972; Dewey et al., 1973; and many others). Further evidence of mechanical links between muscle cells and material in the intercellular space is provided by finger-like invaginations of the cell membrane at the cell's end. Similar structures have been described before in smooth muscle cells of the guinea-pig vas deferens (Merrillees et al., 1963) and are a prominent feature of the muscle-tendon junctions in skeletal fibres (Gelber et al., 1960; Schwarzacher, 1960; Ishikawa, 1965; Mackay et al., 1969; Hanak and B6ck, 1971). 2. Various types of structural specialization join together adjacent muscle cells. In addition to the well known intermediate junctions (see Henderson, 1975), there are more elaborate apparatuses linking two cells together end-to-end or side-to-side or end-to-side. In each case the contact is characterized by an increase of the surface of both cells, by means of folds, invaginations, projections. The area of contacts is, therefore, much greater than if the two cells were facing each other with smooth surfaces, and also the ratio of surface to volume is increased at that particular level of the cell. Moreover, the basal lamina appears thickened and may fill a gap of 100 nm or more between the apposed cell surfaces. These observations strongly suggest that the apparatuses described anchor two muscle cells to each other and allow the mechanical pull of one to be transmitted to the other. Considerable mechanical stability of the intermediate junctions or desmosome-like attachments is shown by the observations that, in the dog duodenum, these junctions are not pulled apart by osmotic cell shrinkage (Henderson et al., 1971).
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3. The present estimations of hydroxyproline content, performed in parallel in a number of muscles of the guinea-pig, show that the taenia coli contains a larger concentration of collagen than skeletal and cardiac muscles (the values for cardiac and skeletal muscles are within the range of those previously found in the rat by Lowry et al., 1942). The collagen concentration in the guinea-pig uterus is similar to that in the taenia, and it is slightly higher than those previously reported for the uterus of the mouse (Finn et al., 1963) or the rat (Grant, 1965). Much higher values of collagen concentration were found in the tendon examined. Therefore, the collagen concentration in taenia coli (and uterus) is intermediate between that of tendon and that of striated muscles. Characterization of the collagen types present in the taenia coli has not yet been carried out. It should be noted that in addition to collagen fibrils, the basal laminae are a major component probably containing collagen. On the basis of muscle cell size and packing density in the taenia coli (Gabella, 1976 a) it can be calculated that there are nearly 10 cm 2 of muscle cell membrane (and approximately the same amount of basal lamina) per mm 3 of muscle. The amount of type IV collagen (specific of basal lamina, Kefalides, 1971) present in the taenia should be substantial. It is also worth noting that intramuscular collagen fibrils measure about 30-35 nm in diameter and never reach the diameter of 50 nm and over typical of the fibrils of tendons and other connective tissues. Fibrils of the large type are found in other parts of the wall of the caecum. Similarly, in large arteries of the guinea-pig, such as thoracic aorta and external iliac artery, the collagen fibrils of the intima and media measure about 35 nm in diameter, whereas those of the outer part of the adventitia measure over 50 nm (unpublsished observations). It seems, therefore, that the collagen fibrils present within vascular and intestinal muscles are of small diameter, like those which constitute the 'reticular' fibres. Elastic fibres, microfibrils and other ill-defined material is present between the muscle cells of the taenia coli. Microfibrils similar in appearance to those which are part of the elastic fibres are known to be associated with the basal lamina of smooth muscle cells (Haust, 1965). The cross-banded structures occasionally found in the intercellular space are similar to those described in other connective tissues (Phillai, 1964; Cravioto and Lockwood, 1968; Sun and White, 1975); their significance is still obscure. On the basis of the present measurements of the collagen concentration in various types of muscle, it can be suggested that the taenia coli, although it lacks discrete tendons, has in fact a kind of diffuse intramuscular tendon, made up of a dense collagen network. The light microscopic observations show that a large part of this collagen constitute septa, different in thickness and rather limited in length, onto which many muscle cells about. 4. Septa of connective tissue are present among the muscle cells and appear to subdivide these into more or less clear-cut groups. Similar observations are at the basis of the current notion that smooth muscles (and the taenia coli in particular) are composed of bundles and that the latter, more than the individual muscle cells, are the functional units of the muscle. In a study of serial transverse sections of taenia coli doubts were cast on this notion, since the groups of'bundles' of smooth muscle cells were found to loose their individuality within tens of microns of their length, splitting into or merging with other 'bundles' (Gabella, 1976a). In the present investigation the attention was focussed on the septa themselves and it has
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been found that their length along the longitudinal axis of the taenia is rather limited, usually in the order of hundreds of microns. From the analysis of serial sections the septa can be visualized as laminae or plaques of connective tissue, slightly longer than broader and of various thicknesses, with little or no continuity with each other. Only the thicker septa are anchored at one side to the connective tissue which lies between taenia and circular musculature of the caecum. The septa are nearly parallel to each other, but the longitudinal axis of the muscle cells is not exactly parallel to the plane of the septa on which they abut. In taeniae fixed in condition of mild stretch this structural detail is barely visible, an angle of about 5 ~ being formed between muscle cells and septa, but in taeniae fixed during isotonic contraction the angle is up to 22-25 ~. In tangential section the taenia show a herring-bone pattern, with the muscle cells directed from one septum to another. This pattern is reminiscent of a multipennate skeletal muscle except that instead of proper tendons the taenia has septa which are much more numerous and are entirely intramuscular. Each connective tissue septum is tentatively visualized as giving attachment to several arrays of muscle cells pulling in opposite directions.
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Henderson, R.M.: Cell-to-cell contacts. In: Methods in pharmacology, Vol. 3, pp. 47-77 (E.E. Daniel and D.M. Paton, eds.). New York: Plenum Press 1975 Henderson, R.M., Duchon, G., Daniel, E.E.: Cell contacts in duodenal smooth muscle layers. Amer. J. Physiol. 221, 564-574 (1971) Hagopian, M., Spiro, D.: Derivation of the Z line in the embryonic chick heart. J. Cell Biol. 44, 683-687 (1970) Ishikawa, H.: The fine structure of myo-tendon junction in some mammalian skeletal muscles. Arch. histol, jap. 25, 275-296 (1965) Kefalides, N.A.: Chemical properties of basement membranes. Int. Rev. exp. Path. 10, 1-39 (1971) Lane, B.P.: Alterations in the cytologic detail of intestinal smooth muscle cells in various stages of contraction. J. Cell Biol. 27, 199-213 (1965) Lowry, O.H., Hastings, A.B., Hull, T.Z. : Histochemical changes associe.ted with aging. II. Skeletal and cardiac muscle in rat. J. biol. Chem. 143, 271-280 (1942) Mackay, B., Harrop, T.J., Muir, A.R.: The fine structure of the muscle tendon junction in the rat. Acta anat. (Basel) 73, 588-604 (1969) McNutt, N.S. : Ultrastructure of myocardial sarcolemma. Circulat. Res. 37, 1-13 (1975) Merrillees, N.C.R., Burnstock, G., Holman, M.E.: Correlation of fine structure and physiology of the innervation of smooth muscle of the guinea pig vas deferens. J. Cell Biol. 19, 529-550 (1963) Mill, P.J., Knapp, M.F.: The fine structure of obliquely striated body wall muscles in the earthworm, Lumbricus terrestris Linn. J. Cell Sci. 7, 233-261 (1970) Murphy, R.A.: Contractile system in mammalian smooth muscle. Blood Vess. 13, 1-23 (1976) Pease, D.C., Molinari, S.: Electron microscopy of muscular arteries: pial vessels of the cat and monkey. J. Ultrastruct. Res. 3, 447-468 (1960) Phillai, P.A.: A banded structure in the connective tissue of nerve. J. Ultrastruct. Res. 11, 455-468 (1964) Prosser, C.L., Burnstock, G., Kahn, J.: Conduction in smooth muscle: comparative structural properties. Amer. J. Physiol. 199, 545-552 (1960) Rosenbluth, J.: Smooth muscle: an ultrastructural basis for the dynamics of its contraction. Science 148, 1337-1339 (1965) Rosenbluth, J. : Obliquely striated muscle. In: Structure and function of striated muscle. Vol. 1, pp. 389420 (G.E. Bourne, ed.). New York and London: Academic Press 1972 Schmalbruch, H.: The sarcolemma of skeletal muscle fibres as demonstrated by a replica technique. Cell Tiss. Res. 150, 377-387 (1974) Schwarzacher, H.G.: Untersuchungen fiber den Feinbau der Muskelfaser-Sehnenverbindungen. Acta anat. (Basel) 40, 59-86 (1960) Smith, D.S., Gupt, B.L., Smith, U.: The organization and myofilament array of insect visceral muscles. J. Cell Sci. 1, 49-57 (1966) Stegemann, H.: Mikrobestimmung von Hydroxyprolin mit Chloramin-T und pDimethylaminebenaldehyd. Hoppe-Seylers Z. physiol. Chem. 311, 41-45 (1958) Street, S.F., Ramsey, R.W.: Sarcolemma: transmitter of active tension in frog skeletal muscle. Science 149, 1379-1380 (1965) Sun, C.N., White, H.J.: Extracellular cross-striated banded structures in human connective tissue. Tissue and Cell 7, 419-432 (1975)
Accepted June 29, 1977
