Cell Tiss. Res. 170, 187-201 (1976)
Cell and Tissue Research ',~}by Springer-Verlag 1976
Structural Changes in Smooth Muscle Cells during Isotonic Contraction Giorgio Gabella* Department of Anatomy, University College London, England
Summary. Smooth muscle ceils of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000. m m - 2 to 18,000" m m - 2 . The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted ceils are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells: on the other hand, they run obliquely and transversely around the isotonically contracted cells. Key words: Smooth muscle - Contraction - Taenia coli - Ultrastructure Collagen.
Introduction In spite of the growing interest in smooth muscle morphology in the past few years, few studies on the structural changes in smooth muscle ceils during conSend off~vrint requests to." Dr.
Giorgio Gabella, Department of Anatomy, University College London, Gower Street, London, WC1E 6BT, England * This work is supported by the Medical Research Council. I thank Miss E.M. Franke and Mr S.J. Sarsfield for excellent technical assistance
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traction have been published. Lane (1965) described structural changes in the muscle cells of the m o u s e j e j u n u m in various stages of c o n t r a c t i o n . D u r i n g s h o r t e n i n g the muscle cell becomes ellipsoid a n d its borders show i n v a g i n a t i o n s of the area of a t t a c h m e n t of the myofilaments. Kelly a n d Rice (1969) c o n f i r m e d that some of the isotonically c o n t r a c t e d muscle cells of the guinea-pig t a e n i a coli have n u m e r o u s cytoplasmic projections interdigitating with projections f r o m n e i g h b o u r i n g cells. Recently, studies have been p u b l i s h e d o n single muscle cells isolated from the toad s t o m a c h according to the technique of Bagby et al. (1971). F a y a n d Delise (1973) observed that the isolated c o n t r a c t e d muscle cells have the surface entirely occupied by b u l b o u s a n d m o u n d - l i k e evaginations, whereas the relaxed muscle cells have a fairly s m o o t h surface. In view of the scarsity of i n f o r m a t i o n on the structural changes in muscle cells actively shortened (isotonic contraction), the present investigation was carried out. This paper reports the changes observed at the cell surface a n d in the geometry of muscle cells in the whole muscle d u r i n g isotonic c o n t r a c t i o n . Changes in the a r r a n g e m e n t of filaments, which have received m o r e a t t e n t i o n in previous investigations, will be reported later.
Material and Methods Guinea-pigs of either sex, weighing 480-600 grams, were used. Lengths of the free taenia coli of the caecum, of about 5-20 mm in vivo length, were dissected with part of the underlying circular muscle, loaded with 1 gram weight and incubated in vitro at 24~ in an oxygenated Krebs solution. After 5-20 minutes some taeniae were incubated for 3-6 minutes in a Krebs solution with no calcium, or in a Krebs solution with 1mM atropine; they were then fixed in glutaraldehyde. The taeniae fixed after incubation in the presence of atropine or in the absence of calcium were considered to be mildly stretched. The load of 1 gram was small compared with a maximal tension of between 8 and 11 grams developed by these strips during an isometric contraction. Other taeniae were made to contract isotonically against 1 gram load with a Krebs solution containing 10-5 M carbachol, or with a solution containing 80 mM K+; after 0.5-1 minute they were fixed in glutaraldehyde. In some experiments a long strip of taenia was prepared and mounted in such a way that half of it was made to contract isotonically and the other half made to shorten, as described above. All tissues were fixed with 5 percent glutaraldehyde in 100 mM Na cacodylate at pH 7.4 at room temperature for 2 16 h. After a brief wash in buffer the specimenswere osmicated for 1 2 h in 2 percent osmium tetroxide in cacodylate buffer, then block-stained for 30 minutes in an aqueous saturated solution of uranyl acetate, dehydrated in ethanol and epoxypropane and embedded in Araldite. Sections 1-3 gm thick, cut with glass knives were examined unstained in a phase contrast light microscope. Thin sections cut with glass knives were stained with lead citrate and examined in Philips 300 and 301 electron microscopes. Other technical details were as reported in a previous paper (Gabella, 1976). 'Reticular' fibres were stained with the Wilder's (1935) method, in stretched or in contracted taeniae fixed in formalin and embedded in paraffin.
Results
a) Cell Size and Packing Density I n phase contrast microscopy the muscle cells of taeniae fixed d u r i n g isotonic c o n t r a c t i o n were m u c h larger (in transverse section) t h a n those of taeniae fixed
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when mildly stretched (Fig. 1 a, b). Most cell profiles of the shortened muscle had a rough surface with numerous spines and extrusions (Fig. 1 b). The extrusions from adjacent cells often interdigitated (indeed, on the sole light microscopic evidence it would be hard to exclude that they were intercellular bridges). In view of the evidence reported in (d.) a transverse section of an isotonically contracted taenia does not cut the muscle cells exactly transversely. The cells do, however, appear transverselysectioned in light micrographs, since the angles formed by the longitudinal axes of the cells are small. It has previously been shown that muscle cells cut at 30~ from the transverse plane still appear as if they were transversely cut (Gabella, 1976). In one experiment two contiguous portions of the same taenia were fixed, respectively during a moderate stretch and during isotonic contraction against 1 gram load: the latter muscle shortened from 80 to 19 mm, i.e. to about a quarter its resting length. The transverse sectional area of the two muscles was about 0.19mm 2 and 0.85mm 2, respectively. The number of muscle cell profiles on a full transverse section, counted on phase contrast micrographs, was 17,700 and 15,400 in the stretched and the isotonically contracted taenia, respectively. The packing density of the muscle cells was 94,000.ram 2 and 18,000 2, respectively. In another similar experiment, the contracting taenia shortened to about 40 percent its resting length. The packing density of muscle cells was in this case 105,000.mm -2 in the stretched taenia and 37,000-mm -2 in the isotonically contracted taenia.
b) Nuclei and Nucleated Cell Profiles The nuclei of the shortened muscle cells usually occupied the centre of the cell profile, and showed deep indentations and a characteristic convoluted surface which has been described by other authors in contracted muscle cells (Lane, 1965; Kelly and Rice, 1969). The shortening of the nuclei during isotonic contraction was roughly proportional to the overall shortening of the muscle strip. Nucleated muscle cell profiles, counted on low-power electron micrographs, were 5.9 percent of all muscle cell profiles in a mildly stretched taenia and 8.5 percent in an adjacent portion of the same taenia, isotonically contracted (2,000 cell profiles were counted). The transverse sectional area of the nucleated profiles averaged 65.6 ~tm2 in isotonically contracted muscle cells as opposed to 10.8 ~tm2 in stretched muscle cells, a difference of about six times. Since in the contracted taeniae the muscle cells are not exactly transversely sectioned, the nucleated cell profiles must be larger than expected from the total area of the taenia. However, the difference observed was too big to be accounted for entirely by the direction of the plane of section (see Discussion, d). Whereas in the mildly stretched taenia the nucleus constituted about 33.4 percent of the corresponding cell profile (in terms of transverse sectional area), in the isotonically contracted taenia the nucleus represented about 25.1 percent of the muscle cell profile (50 muscle cell profiles were measured in both cases; the standerd error was 1.05 and 1.27, respectively).
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c) Longitudinal Arrangement of Muscle Cells Longitudinal sections o f mildly stretched taeniae showed in phase contrast microscopy a very regular arrangement. The muscle cells were almost perfectly parallel to each other over long distances and t h r o u g h the full thickness o f the taenia (Fig. 1 c). By contrast, in longitudinal sections o f isotonically contracted taeniae, the muscle cells were no longer parallel to each other (Fig. 1 d). Even when the plane o f section coincided with the longitudinal axis of the taenia, all muscle cells were obliquely sectioned; the axis o f each cell made a small angle with both the plane of section and the neighbouring cells. They appeared twisted and m a n y seemed to entwine a r o u n d each other. The outline of the cells was very rough, studded with n u m e r o u s processes and folds covering most o f the cell surface. On rare occasions, in isotonically contracted taeniae single muscle cells were found whose surface showed a regular ondulation (Fig. 1e). Other authors have based their description of contracted muscle cells on cells of this appearance (e.g. Figs. 11 and 12 in Kelly and Rice, 1969). These cells, however, were different from the majority of the shortened muscle cells (and in the few preparations in which they were found they were less than 1 percent of all the muscle cells), and it was not clear whether they were genuinely contracted muscle cells. They might have been cells which had been shortened and deformed by the contraction of the neighbouring cells.
d) Striation of Muscle Cells In stretched taeniae the muscle cell surface appeared s m o o t h in longitudinal sections examined in phase contrast, but a very faint and ill-defined longitudinal striation was visible in a n u m b e r o f cells (Fig. 2 a). On the other hand, the cells f r o m taeniae fixed after shortening showed prominent striations of two types.
Longitudinal striation. Most muscle cells showed along part o f their length longitudinal, parallel bands, alternately light and dark (Fig. 2 b, c, d). In some cells this striation formed a small angle (up to 20 ~ with the cell longitudinal axis (Fig. 2 b, c). The dense bands were ill-defined, o f variable density and often showing discontinuities along their length. The centre-to-centre distance between adjacent dark bands or stripes was about 1.1-1.4 lam, their length up to 25 tam. In m a n y instances the stripes clearly appeared associated with the superficial parts of the muscle cells; this occurred, for example, where muscle cells were seen 'entering' or 'leaving' the section or where the knife had 'grazed' the surface o f a cell (Fig. 2 d). In obliquely sectioned cells, light and dark bands (similar to those o f the striation, but o f shorter length) clearly appeared related to the cell surface (Fig. 2e). By cutting a taenia at various degrees o f tilt in the microtome, all the transition aspects between a regular longitudinal striation (in longitudinally sectioned cells) and a regular arrangement of extrusions or processes at the cell Fig. I a-e. Phase contrast micrographs of plastic embedded taeniae coli. a Transverse section of a
mildly stretched taenia, x 1,500. b Transverse section of an isotonically contracted taenia. • 1,500. c Longitudinal section of a mildly stretched taenia. • 600. d Longitudinal section of an isotonically contracted taenia. • 600. e Longitudinal section of an isotonically contracted taenia; the arrowpoints to a cell of unusual appearance (see text for description). • 1,500
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surface (in transversely sectioned cells) were obtained. The longitudinal striation was therefore related to structures present at the cell surface (see, below, the evidence in electron microscopy). The 'striated' parts of a cell were those where the cell surface was included in the section, the smooth parts were those were the section passed well within the cell. The dark bands would seem to correspond to evaginated parts of the cell surface, the light bands to invaginated parts. Occasionally, a criss-cross appearance was observed (Fig. 2 b), each set of bands on a slightly different plane of focus, which was interpreted as due to extrusions on the surface of two adjacent muscle cells (or on opposed surfaces of one, very slender cell) both included for a short length within one section. Transverse Striation. Other, more irregular stripes run across longitudinally sectioned muscle cells (from taeniae fixed after shortening) or at an angle between 90 ~ and 45 ~ to their longitudinal axis (Fig. 2b, f). The centre-to-centre distance between successive dark bands was about 1.0-1.2~tm, but in many cases the pattern was too irregular for the bands to be measured. This transverse or oblique striation was related to laminar cell processes running in that direction and clearly seen in electron microscopy (Fig. 5a). Here again the striation was better visible where the microtome knife had 'grazed' the surface of a muscle cell. The transverse or oblique striation was often superimposed on the longitudinal striation described above. The dark bands of the latter were interrupted by thinner light bands running across or obliquely (Fig. 2 f).
e) Fine Structure
In the electron microscope muscle cells of stretched taeniae cut in longitudinal section lay parallel to each other and had a fairly smooth surface (Fig. 3a). In transverse section the cells partly moulded their shapes upon one another, but at the level of the nucleus they tended to have a circular profile. Various types of intercellular contacts were observed, as described in a previous paper (Gabella, 1976). In isotonically contracted taeniae, the muscle cells were much larger in transverse section (Fig. 4). Their surfaces were very rough and irregular with
Fig. 2 a - L Phase contrast micrographs of plastic embedded taeniae coli. All x 1,500. a Longitudinal section of a mildly stretched taenia. Some muscle cells show an ill-defined longitudinal striation (arrow). h - d Longitudinal sections of isotonically contracted taeniae. Many muscle cells have a crenated profile and show a striation. Longitudinal stripes are visible in d (arrow), transverse stripes in b (arrow), oblique stripes in c (arrow), and a criss-cross pattern in b (arrowhead). e Obliquely sectioned muscle cells, isotonically contracted, to show an intermediate appearance between the longitudinal striation (e.g. in d) of longitudinally sectioned muscle cells and the crenations (Fig. 1 b) of transversely sectioned muscle cells, f Longitudinal section of an isotonically contracted taenia. The cell in the centre has longitudinal stripes superimposed on an oblique striation. To the left a capillary Fig. 3. a Longitudinal section of a mildly stretched taenia, showing muscle cells with a fairly smooth surface, x6,300, h Longitudinal section of an isotonically contracted taenia, showing numerous processes, of various sizes and shapes, covering most of the cell surface. Nuclei have deep indentations. x 2,300
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Fig. 4. Transverse section of an isotonically contracted taenia. Muscle cells have a rough surface with numerous extrusions and interdigitations. There are also many desmosome-like attachments between muscle cells (e.g. at arrows), n Nerve bundles: c capillary, x 2,300
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some finger-like and bulbous projections and many, short extrusions. Some processes from adjacent cells interdigitated, some penetrated deep into a neighbouring cell. Intermediate or desmosome-like contacts between shortened muscle cells were often found. In longitudinal section the contracted muscle cells showed myriad processes, over most of their surface (Fig. 3 b). These varied greatly in shape and size (Fig. 5 b, c, d, f); slender processes only 0.15 gm thick and 1 ~tm long; stout, triangular or square processes, 0.5-1 gm at their base and 1-1.5 gm long; Y-shaped processes studded with caveolae; small extrusions of the cell surface only a few tenth of a micron in size; and all intermediate appearances. In the areas where the microtome knife had 'grazed' the surface of a muscle cell, it appeared that many cell processes were not cylindrical or conical projections of the cell surface but laminar projections arranged at an angle of 90 ~ to 45 ~ to the longitudinal axis of the cell (Fig. 5 a). As in stretched muscle cells, the two major structural specializations associated to the cell membrane were caveolae (plasmalemmal vesicles) and dense patches. Both were obvious in isotonically contracted muscle cells. Quantitative studies on these structures, such as those carried out on stretched muscle cells (Gabella, 1976), were not feasible because of the irregular surface of the cells. From qualitative observations there was no indication of large changes in the number of caveolae or dense patches during cell shortening. Whether more subtle changes occurred, such as small changes in the caveolar volume or in the size of the neck or in the relation to the sarcoplasmic reticulum, remains to be seen. Caveolae were mainly found on the extrusions of the cell surface; dense patches were mainly found on the invaginated parts of the cell surface. However, it was not uncommon to observe dense patches on cell evaginations (Fig. 5 e). The slimmest membrane folds could lodge only one set of caveolae, originating from both surfaces (Fig. 5d). The large cell extrusions and all those which had dense patches contained myofilaments. Cisternae of sarcoplasmic reticulum were often present within the folds, closely associated to caveolae (Fig. 5c, d). In the stretched muscle cells the cisternae intervened between caveolae and myofilaments; those present in the thin folds of shortened cells were situated at a greater distance from the contractile structures. Occasionally, cisternae of sarcoplasmic reticulum were stacked at the base of an extrusion of the cell surface (Fig. 5 b).
f) Arrangementof Collagen In taeniae mildly stretched, collagen fibrils run parallel or at a small angle to the longitudinal axis of the muscle cells (Fig. 5 e). On the other hand, in taeniae which Fig. 5. a Longitudinal section of an isotonically contracted taenia. Sets of parallel membrane folds from different muscle cells interdigitate, x4,200, b Membrane evaginations of various sizes and shapes in isotonically contracted cells. The a r r o w points to a stack of sarcoplasmic reticulum cisternae located at the base of a cell process, x 19,000. c A cell process, covered with caveolae and with cisternae of sarcoplasmic reticulum in the centre, x 46,000. d A thin process with caveolae arising from both its surfaces and a cisterna of sarcoplasmic reticulum, x 37,000. e Two isotonically contracted muscle cells in longitudinal section. Between them there are collagen fibrils mainly transversely cut. x 27,000. f Two mildly stretched muscle cells in longitudinal section. The collagen fibrils mainly run parallel to the cell axis. x 50,000
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Fig. 6a and b. Longitudinal sections of taeniae coli impregnated with a silver method for 'reticular'
fibres. Both • a Mildly stretched muscle, with the fine fibres mainly running longitudinally. b Isotonically contracted muscle, with fine fibres running across muscle cell, occasionally giving a ladder-like appearance
were fixed after shortening the collagen fibrils had a more complicated arrangement. In transversely sectioned muscles many fibrils were longitudinally cut; in muscles in longitudinal section m a n y fibrils appeared transversely sectioned (Fig. 5 f). Collagen fibrils were present between the folds and processes of the cell surface, a basal lamina always intervening between cell m e m b r a n e and collagen. After silver impregnation for 'reticular' fibres, the stretched taenia showed fine fibres longitudinally arranged (Fig. 6 a). On the other hand, in the shortened taenia m a n y 'reticular' fibres were arranged transversely to the longitudinal axis of the muscle cells. Around most muscle cells the 'reticular' fibres appeared arranged in a ladder-like fashion (Fig. 6 b).
Discussion
Smooth muscle contraction is a complex process effected by a number of mechanisms at sub-cellular, cellular and tissue level. The present experiments have cast some light on the following aspects:
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a) During isotonic contraction against a moderate load (1 gram) the taenia undergoes a remarkable reduction in length. Shortening to less than a quarter the initial length can be observed in vitro. The shortening is accompanied by an increase of the transverse sectional area of the muscle of approximately the same amount. b) The packing density of smooth muscle cells passed from 94,000 to 18,000. mm-2 (shortening to 25 percent), and from 105,000 to 37,000. mm -2 (shortening to 40 percent). During isotonic contraction the packing density of muscle cells decreases more than the muscle increases in transverse sectional area. This may indicate that the muscle cells shorten to a greater extent than the muscle as a whole. c) The percentage of nucleated cell profiles increases when the muscle contracts isotonically. Since the shortening of the nucleus is approximately proportional to the overall shortening of the muscle, the increase in the percentage of nucleated cell profiles suggests, as seen in (b), that the individual muscle cells shorten slightly more than the muscle as a whole. There is probably a pulling-in of the tapering ends of the muscle cells. d) In the cell profiles containing a nucleus, the nucleus represents a smaller proportion of the total transverse sectional area in contracted muscle cells than in stretched cells. The nucleated cell profiles increase in transverse sectional area more than the taenia as a whole. The evidence discussed in (b), (c) and (d) indicates that the changes in shape of muscle cells do not closely mirror the changes in shape of the whole muscle. The middle parts of the muscle cells seem to fatten more than the taenia as a whole; the ends of the muscle cells are probably pulled in towards the middle portions of the cells, so that the individual muscle cells shorten more than the muscle as a whole. A more accurate study, with histograms of the cell profile sizes at various stages of contraction, may clarify this point. e) In isotonically contracted taeniae the muscle cells are no longer parallel (as they are in stretched taeniae), but run at a small angle to each other. They appear twisted and many seem to entwine around each other in a complicated pattern. A tortuous arrangement of the shortened muscle cells as opposed to a parallel one in the elongated cells has been described by Schlote (1960) in the retractor penis of Helix pomatia. However, the details of the re-arrangement of the muscle cells of taenia during isotonic contraction are not yet clear, and need further investigation. It may be assumed that this complex re-arrangement of muscle cells is a more orderly process than it appears from individual sections. f) Fay and Delise (1973) have shown that isolated smooth muscle cells, prepared from the toad stomach and suspended in vitro, present a remarkable invagination of the cell surface when they contract. The surface of fully contracted cells is packed with bulbous and mound-like evaginations, whereas that of relaxed muscle cells is generally smooth. These results by Fay and Delise (1973) are confirmed by the present observations on intact muscles. However, size, shape, and pattern of the evaginations appear different in the intact taenia coli and in the isolated muscle cells of the toad stomach. In the latter case the evaginations are usually round in profile (as seen in scanning electron micrographs). In the taenia coli many e v a g i n a t i o n s - p r o b a b l y because of the constraints
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imposed by the collagen network and the dense packing of cells-are long and slender in sectioned muscles and are part of laminar folds arranged at an angle between 90 ~ and 45 ~ to the longitudinal axis of the cells, often interdigitating with similar processes from neighbouring cells. Some folds extend for not less than half the circumference of a muscle cell, but whether they form a helicoidal system with many turns around a muscle cell could not be ascertained. This seems, however, unlikely, since plain areas, usually occupied by desmosome-like attachments, and longitudinal, wider folds are also present. However, the exact shape and arrangement of the extrusions of the cell surface could not be established, since only individual (transverse or longitudinal) sections were studied. The formation of folds at the cell surface is to some extent due to a geometrical reason. In an elongated structure such as a muscle cell, if the volume remains constant, shortening produces a decrease of the geometrical surface; the "excess' membrane is then thrown into folds. It has been reported that during isotonic contraction single isolated smooth muscle cells decrease in volume (Fay and Delise, 1973; Kominz and Gr6schelSteward, 1973). Whether the same holds true for the muscle cells of an intact muscle could not be investigated in the present study, since cell volume of shortened muscle cells could not be estimated to the same degree of accuracy as that of stretched muscle cells, by means of stereological methods (Gabella, 1976). g) A further indication of the complex structural changes occurring during shortening of the taenia coli, is given by the arrangement of collagen fibrils. These run mainly longitudinally along stretched muscle cells, but obliquely or transversely around contracted muscle cells. Apparently the collagen runs parallel to the folds of the cell surface. These observations indicate that during shortening the muscle cell undergoes some torsion around its longitudinal axis and it twists with neighbouring cells. The cell surface is thrown into folds which are parallel to the lines of force. The collagen fibrils are caught between folds, form bundles running as helices of small pitch around the muscle cells, and remain (or become more) taut in spite of the considerable shortening of the cells. During isometric contraction dramatic changes occur also at the level of the filaments, which are indeed the primum movens of the changes in shape and arrangement of the muscle cells. They will be examined in a following paper. It may be worth stressing that the results discussed in this paper are confined to the taenia coli of the guinea-pig and should not be generalized to other smooth muscles. h) Finally, isotonically contracted muscle cells show characteristic striations. Both the longitudinal and the transverse striations were related to the folds, processes, dense patches and invaginations present at the cell surface. A longitudinal striation of similar appearance has been seen in isolated muscle cells grown in vitro (Small, 1974). Small (1974) interpreted such striation as evidence that smooth muscle cells contain contractile units, which he calls myofibrils. It is possible that the striation observed by Small (1974) is the same as the longitudinal striation described here. If this is the case, it is here suggested that the striation should be interpreted as due to characteristics of the cell surface and not to the presence of special contractile units.
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References Bagby, R.M~, Young, A.M., Dotson, R.S., Fisher, B.A., McKinnon, K.: Contraction of single smooth muscle cells from Bufo marinus stomach. Nature (Lond.) 234, 351-352 (1971) Fay, F.S., Delise, C.M.: Contraction of isolated smooth-muscle cells. Structural changes. Proc. nat. Acad. Sci. (Wash.) 70, 641-645 (1973) Gabella, G.: Quantitative morphological study of smooth muscle cells in the guinea-pig taenia coli. Ceil Tiss. Res. 170, 161 - 1 8 6 (1976) Kelly, R.E., Rice, R.V.: Ultrastructural studies on the contraction mechanism of smooth muscle. J. Cell Biol. 42, 683 694 (1969) Kominz, D.R., Gr6schel-Stewart, U.: Antibody-dependent size changes of myofibrils and isolated smooth muscle cells. J. Mechanochem. Cell Motility 2, 181 191 (1973) 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) Schlote, F.-W.: Die Kontraktion glatter Muskulatur auf Grund yon Torsionsspannungen in den Myofilamenten. Z. Zellforsch. 52, 362-395 (1960) Small, J.V.: Contractile units in vertebrate smooth muscle cells. Nature (Lond.) 249, 324-327 (1974) Wilder, H~C.: An improved technique for silver impregnation of reticulin fibers. Amer. J. Path. ll, 817-820 (1935)
Received March 22, 1976
Cell and Tissue Research ',~}by Springer-Verlag 1976
Structural Changes in Smooth Muscle Cells during Isotonic Contraction Giorgio Gabella* Department of Anatomy, University College London, England
Summary. Smooth muscle ceils of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000. m m - 2 to 18,000" m m - 2 . The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted ceils are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells: on the other hand, they run obliquely and transversely around the isotonically contracted cells. Key words: Smooth muscle - Contraction - Taenia coli - Ultrastructure Collagen.
Introduction In spite of the growing interest in smooth muscle morphology in the past few years, few studies on the structural changes in smooth muscle ceils during conSend off~vrint requests to." Dr.
Giorgio Gabella, Department of Anatomy, University College London, Gower Street, London, WC1E 6BT, England * This work is supported by the Medical Research Council. I thank Miss E.M. Franke and Mr S.J. Sarsfield for excellent technical assistance
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traction have been published. Lane (1965) described structural changes in the muscle cells of the m o u s e j e j u n u m in various stages of c o n t r a c t i o n . D u r i n g s h o r t e n i n g the muscle cell becomes ellipsoid a n d its borders show i n v a g i n a t i o n s of the area of a t t a c h m e n t of the myofilaments. Kelly a n d Rice (1969) c o n f i r m e d that some of the isotonically c o n t r a c t e d muscle cells of the guinea-pig t a e n i a coli have n u m e r o u s cytoplasmic projections interdigitating with projections f r o m n e i g h b o u r i n g cells. Recently, studies have been p u b l i s h e d o n single muscle cells isolated from the toad s t o m a c h according to the technique of Bagby et al. (1971). F a y a n d Delise (1973) observed that the isolated c o n t r a c t e d muscle cells have the surface entirely occupied by b u l b o u s a n d m o u n d - l i k e evaginations, whereas the relaxed muscle cells have a fairly s m o o t h surface. In view of the scarsity of i n f o r m a t i o n on the structural changes in muscle cells actively shortened (isotonic contraction), the present investigation was carried out. This paper reports the changes observed at the cell surface a n d in the geometry of muscle cells in the whole muscle d u r i n g isotonic c o n t r a c t i o n . Changes in the a r r a n g e m e n t of filaments, which have received m o r e a t t e n t i o n in previous investigations, will be reported later.
Material and Methods Guinea-pigs of either sex, weighing 480-600 grams, were used. Lengths of the free taenia coli of the caecum, of about 5-20 mm in vivo length, were dissected with part of the underlying circular muscle, loaded with 1 gram weight and incubated in vitro at 24~ in an oxygenated Krebs solution. After 5-20 minutes some taeniae were incubated for 3-6 minutes in a Krebs solution with no calcium, or in a Krebs solution with 1mM atropine; they were then fixed in glutaraldehyde. The taeniae fixed after incubation in the presence of atropine or in the absence of calcium were considered to be mildly stretched. The load of 1 gram was small compared with a maximal tension of between 8 and 11 grams developed by these strips during an isometric contraction. Other taeniae were made to contract isotonically against 1 gram load with a Krebs solution containing 10-5 M carbachol, or with a solution containing 80 mM K+; after 0.5-1 minute they were fixed in glutaraldehyde. In some experiments a long strip of taenia was prepared and mounted in such a way that half of it was made to contract isotonically and the other half made to shorten, as described above. All tissues were fixed with 5 percent glutaraldehyde in 100 mM Na cacodylate at pH 7.4 at room temperature for 2 16 h. After a brief wash in buffer the specimenswere osmicated for 1 2 h in 2 percent osmium tetroxide in cacodylate buffer, then block-stained for 30 minutes in an aqueous saturated solution of uranyl acetate, dehydrated in ethanol and epoxypropane and embedded in Araldite. Sections 1-3 gm thick, cut with glass knives were examined unstained in a phase contrast light microscope. Thin sections cut with glass knives were stained with lead citrate and examined in Philips 300 and 301 electron microscopes. Other technical details were as reported in a previous paper (Gabella, 1976). 'Reticular' fibres were stained with the Wilder's (1935) method, in stretched or in contracted taeniae fixed in formalin and embedded in paraffin.
Results
a) Cell Size and Packing Density I n phase contrast microscopy the muscle cells of taeniae fixed d u r i n g isotonic c o n t r a c t i o n were m u c h larger (in transverse section) t h a n those of taeniae fixed
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when mildly stretched (Fig. 1 a, b). Most cell profiles of the shortened muscle had a rough surface with numerous spines and extrusions (Fig. 1 b). The extrusions from adjacent cells often interdigitated (indeed, on the sole light microscopic evidence it would be hard to exclude that they were intercellular bridges). In view of the evidence reported in (d.) a transverse section of an isotonically contracted taenia does not cut the muscle cells exactly transversely. The cells do, however, appear transverselysectioned in light micrographs, since the angles formed by the longitudinal axes of the cells are small. It has previously been shown that muscle cells cut at 30~ from the transverse plane still appear as if they were transversely cut (Gabella, 1976). In one experiment two contiguous portions of the same taenia were fixed, respectively during a moderate stretch and during isotonic contraction against 1 gram load: the latter muscle shortened from 80 to 19 mm, i.e. to about a quarter its resting length. The transverse sectional area of the two muscles was about 0.19mm 2 and 0.85mm 2, respectively. The number of muscle cell profiles on a full transverse section, counted on phase contrast micrographs, was 17,700 and 15,400 in the stretched and the isotonically contracted taenia, respectively. The packing density of the muscle cells was 94,000.ram 2 and 18,000 2, respectively. In another similar experiment, the contracting taenia shortened to about 40 percent its resting length. The packing density of muscle cells was in this case 105,000.mm -2 in the stretched taenia and 37,000-mm -2 in the isotonically contracted taenia.
b) Nuclei and Nucleated Cell Profiles The nuclei of the shortened muscle cells usually occupied the centre of the cell profile, and showed deep indentations and a characteristic convoluted surface which has been described by other authors in contracted muscle cells (Lane, 1965; Kelly and Rice, 1969). The shortening of the nuclei during isotonic contraction was roughly proportional to the overall shortening of the muscle strip. Nucleated muscle cell profiles, counted on low-power electron micrographs, were 5.9 percent of all muscle cell profiles in a mildly stretched taenia and 8.5 percent in an adjacent portion of the same taenia, isotonically contracted (2,000 cell profiles were counted). The transverse sectional area of the nucleated profiles averaged 65.6 ~tm2 in isotonically contracted muscle cells as opposed to 10.8 ~tm2 in stretched muscle cells, a difference of about six times. Since in the contracted taeniae the muscle cells are not exactly transversely sectioned, the nucleated cell profiles must be larger than expected from the total area of the taenia. However, the difference observed was too big to be accounted for entirely by the direction of the plane of section (see Discussion, d). Whereas in the mildly stretched taenia the nucleus constituted about 33.4 percent of the corresponding cell profile (in terms of transverse sectional area), in the isotonically contracted taenia the nucleus represented about 25.1 percent of the muscle cell profile (50 muscle cell profiles were measured in both cases; the standerd error was 1.05 and 1.27, respectively).
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c) Longitudinal Arrangement of Muscle Cells Longitudinal sections o f mildly stretched taeniae showed in phase contrast microscopy a very regular arrangement. The muscle cells were almost perfectly parallel to each other over long distances and t h r o u g h the full thickness o f the taenia (Fig. 1 c). By contrast, in longitudinal sections o f isotonically contracted taeniae, the muscle cells were no longer parallel to each other (Fig. 1 d). Even when the plane o f section coincided with the longitudinal axis of the taenia, all muscle cells were obliquely sectioned; the axis o f each cell made a small angle with both the plane of section and the neighbouring cells. They appeared twisted and m a n y seemed to entwine a r o u n d each other. The outline of the cells was very rough, studded with n u m e r o u s processes and folds covering most o f the cell surface. On rare occasions, in isotonically contracted taeniae single muscle cells were found whose surface showed a regular ondulation (Fig. 1e). Other authors have based their description of contracted muscle cells on cells of this appearance (e.g. Figs. 11 and 12 in Kelly and Rice, 1969). These cells, however, were different from the majority of the shortened muscle cells (and in the few preparations in which they were found they were less than 1 percent of all the muscle cells), and it was not clear whether they were genuinely contracted muscle cells. They might have been cells which had been shortened and deformed by the contraction of the neighbouring cells.
d) Striation of Muscle Cells In stretched taeniae the muscle cell surface appeared s m o o t h in longitudinal sections examined in phase contrast, but a very faint and ill-defined longitudinal striation was visible in a n u m b e r o f cells (Fig. 2 a). On the other hand, the cells f r o m taeniae fixed after shortening showed prominent striations of two types.
Longitudinal striation. Most muscle cells showed along part o f their length longitudinal, parallel bands, alternately light and dark (Fig. 2 b, c, d). In some cells this striation formed a small angle (up to 20 ~ with the cell longitudinal axis (Fig. 2 b, c). The dense bands were ill-defined, o f variable density and often showing discontinuities along their length. The centre-to-centre distance between adjacent dark bands or stripes was about 1.1-1.4 lam, their length up to 25 tam. In m a n y instances the stripes clearly appeared associated with the superficial parts of the muscle cells; this occurred, for example, where muscle cells were seen 'entering' or 'leaving' the section or where the knife had 'grazed' the surface o f a cell (Fig. 2 d). In obliquely sectioned cells, light and dark bands (similar to those o f the striation, but o f shorter length) clearly appeared related to the cell surface (Fig. 2e). By cutting a taenia at various degrees o f tilt in the microtome, all the transition aspects between a regular longitudinal striation (in longitudinally sectioned cells) and a regular arrangement of extrusions or processes at the cell Fig. I a-e. Phase contrast micrographs of plastic embedded taeniae coli. a Transverse section of a
mildly stretched taenia, x 1,500. b Transverse section of an isotonically contracted taenia. • 1,500. c Longitudinal section of a mildly stretched taenia. • 600. d Longitudinal section of an isotonically contracted taenia. • 600. e Longitudinal section of an isotonically contracted taenia; the arrowpoints to a cell of unusual appearance (see text for description). • 1,500
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surface (in transversely sectioned cells) were obtained. The longitudinal striation was therefore related to structures present at the cell surface (see, below, the evidence in electron microscopy). The 'striated' parts of a cell were those where the cell surface was included in the section, the smooth parts were those were the section passed well within the cell. The dark bands would seem to correspond to evaginated parts of the cell surface, the light bands to invaginated parts. Occasionally, a criss-cross appearance was observed (Fig. 2 b), each set of bands on a slightly different plane of focus, which was interpreted as due to extrusions on the surface of two adjacent muscle cells (or on opposed surfaces of one, very slender cell) both included for a short length within one section. Transverse Striation. Other, more irregular stripes run across longitudinally sectioned muscle cells (from taeniae fixed after shortening) or at an angle between 90 ~ and 45 ~ to their longitudinal axis (Fig. 2b, f). The centre-to-centre distance between successive dark bands was about 1.0-1.2~tm, but in many cases the pattern was too irregular for the bands to be measured. This transverse or oblique striation was related to laminar cell processes running in that direction and clearly seen in electron microscopy (Fig. 5a). Here again the striation was better visible where the microtome knife had 'grazed' the surface of a muscle cell. The transverse or oblique striation was often superimposed on the longitudinal striation described above. The dark bands of the latter were interrupted by thinner light bands running across or obliquely (Fig. 2 f).
e) Fine Structure
In the electron microscope muscle cells of stretched taeniae cut in longitudinal section lay parallel to each other and had a fairly smooth surface (Fig. 3a). In transverse section the cells partly moulded their shapes upon one another, but at the level of the nucleus they tended to have a circular profile. Various types of intercellular contacts were observed, as described in a previous paper (Gabella, 1976). In isotonically contracted taeniae, the muscle cells were much larger in transverse section (Fig. 4). Their surfaces were very rough and irregular with
Fig. 2 a - L Phase contrast micrographs of plastic embedded taeniae coli. All x 1,500. a Longitudinal section of a mildly stretched taenia. Some muscle cells show an ill-defined longitudinal striation (arrow). h - d Longitudinal sections of isotonically contracted taeniae. Many muscle cells have a crenated profile and show a striation. Longitudinal stripes are visible in d (arrow), transverse stripes in b (arrow), oblique stripes in c (arrow), and a criss-cross pattern in b (arrowhead). e Obliquely sectioned muscle cells, isotonically contracted, to show an intermediate appearance between the longitudinal striation (e.g. in d) of longitudinally sectioned muscle cells and the crenations (Fig. 1 b) of transversely sectioned muscle cells, f Longitudinal section of an isotonically contracted taenia. The cell in the centre has longitudinal stripes superimposed on an oblique striation. To the left a capillary Fig. 3. a Longitudinal section of a mildly stretched taenia, showing muscle cells with a fairly smooth surface, x6,300, h Longitudinal section of an isotonically contracted taenia, showing numerous processes, of various sizes and shapes, covering most of the cell surface. Nuclei have deep indentations. x 2,300
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Fig. 4. Transverse section of an isotonically contracted taenia. Muscle cells have a rough surface with numerous extrusions and interdigitations. There are also many desmosome-like attachments between muscle cells (e.g. at arrows), n Nerve bundles: c capillary, x 2,300
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some finger-like and bulbous projections and many, short extrusions. Some processes from adjacent cells interdigitated, some penetrated deep into a neighbouring cell. Intermediate or desmosome-like contacts between shortened muscle cells were often found. In longitudinal section the contracted muscle cells showed myriad processes, over most of their surface (Fig. 3 b). These varied greatly in shape and size (Fig. 5 b, c, d, f); slender processes only 0.15 gm thick and 1 ~tm long; stout, triangular or square processes, 0.5-1 gm at their base and 1-1.5 gm long; Y-shaped processes studded with caveolae; small extrusions of the cell surface only a few tenth of a micron in size; and all intermediate appearances. In the areas where the microtome knife had 'grazed' the surface of a muscle cell, it appeared that many cell processes were not cylindrical or conical projections of the cell surface but laminar projections arranged at an angle of 90 ~ to 45 ~ to the longitudinal axis of the cell (Fig. 5 a). As in stretched muscle cells, the two major structural specializations associated to the cell membrane were caveolae (plasmalemmal vesicles) and dense patches. Both were obvious in isotonically contracted muscle cells. Quantitative studies on these structures, such as those carried out on stretched muscle cells (Gabella, 1976), were not feasible because of the irregular surface of the cells. From qualitative observations there was no indication of large changes in the number of caveolae or dense patches during cell shortening. Whether more subtle changes occurred, such as small changes in the caveolar volume or in the size of the neck or in the relation to the sarcoplasmic reticulum, remains to be seen. Caveolae were mainly found on the extrusions of the cell surface; dense patches were mainly found on the invaginated parts of the cell surface. However, it was not uncommon to observe dense patches on cell evaginations (Fig. 5 e). The slimmest membrane folds could lodge only one set of caveolae, originating from both surfaces (Fig. 5d). The large cell extrusions and all those which had dense patches contained myofilaments. Cisternae of sarcoplasmic reticulum were often present within the folds, closely associated to caveolae (Fig. 5c, d). In the stretched muscle cells the cisternae intervened between caveolae and myofilaments; those present in the thin folds of shortened cells were situated at a greater distance from the contractile structures. Occasionally, cisternae of sarcoplasmic reticulum were stacked at the base of an extrusion of the cell surface (Fig. 5 b).
f) Arrangementof Collagen In taeniae mildly stretched, collagen fibrils run parallel or at a small angle to the longitudinal axis of the muscle cells (Fig. 5 e). On the other hand, in taeniae which Fig. 5. a Longitudinal section of an isotonically contracted taenia. Sets of parallel membrane folds from different muscle cells interdigitate, x4,200, b Membrane evaginations of various sizes and shapes in isotonically contracted cells. The a r r o w points to a stack of sarcoplasmic reticulum cisternae located at the base of a cell process, x 19,000. c A cell process, covered with caveolae and with cisternae of sarcoplasmic reticulum in the centre, x 46,000. d A thin process with caveolae arising from both its surfaces and a cisterna of sarcoplasmic reticulum, x 37,000. e Two isotonically contracted muscle cells in longitudinal section. Between them there are collagen fibrils mainly transversely cut. x 27,000. f Two mildly stretched muscle cells in longitudinal section. The collagen fibrils mainly run parallel to the cell axis. x 50,000
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Fig. 6a and b. Longitudinal sections of taeniae coli impregnated with a silver method for 'reticular'
fibres. Both • a Mildly stretched muscle, with the fine fibres mainly running longitudinally. b Isotonically contracted muscle, with fine fibres running across muscle cell, occasionally giving a ladder-like appearance
were fixed after shortening the collagen fibrils had a more complicated arrangement. In transversely sectioned muscles many fibrils were longitudinally cut; in muscles in longitudinal section m a n y fibrils appeared transversely sectioned (Fig. 5 f). Collagen fibrils were present between the folds and processes of the cell surface, a basal lamina always intervening between cell m e m b r a n e and collagen. After silver impregnation for 'reticular' fibres, the stretched taenia showed fine fibres longitudinally arranged (Fig. 6 a). On the other hand, in the shortened taenia m a n y 'reticular' fibres were arranged transversely to the longitudinal axis of the muscle cells. Around most muscle cells the 'reticular' fibres appeared arranged in a ladder-like fashion (Fig. 6 b).
Discussion
Smooth muscle contraction is a complex process effected by a number of mechanisms at sub-cellular, cellular and tissue level. The present experiments have cast some light on the following aspects:
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a) During isotonic contraction against a moderate load (1 gram) the taenia undergoes a remarkable reduction in length. Shortening to less than a quarter the initial length can be observed in vitro. The shortening is accompanied by an increase of the transverse sectional area of the muscle of approximately the same amount. b) The packing density of smooth muscle cells passed from 94,000 to 18,000. mm-2 (shortening to 25 percent), and from 105,000 to 37,000. mm -2 (shortening to 40 percent). During isotonic contraction the packing density of muscle cells decreases more than the muscle increases in transverse sectional area. This may indicate that the muscle cells shorten to a greater extent than the muscle as a whole. c) The percentage of nucleated cell profiles increases when the muscle contracts isotonically. Since the shortening of the nucleus is approximately proportional to the overall shortening of the muscle, the increase in the percentage of nucleated cell profiles suggests, as seen in (b), that the individual muscle cells shorten slightly more than the muscle as a whole. There is probably a pulling-in of the tapering ends of the muscle cells. d) In the cell profiles containing a nucleus, the nucleus represents a smaller proportion of the total transverse sectional area in contracted muscle cells than in stretched cells. The nucleated cell profiles increase in transverse sectional area more than the taenia as a whole. The evidence discussed in (b), (c) and (d) indicates that the changes in shape of muscle cells do not closely mirror the changes in shape of the whole muscle. The middle parts of the muscle cells seem to fatten more than the taenia as a whole; the ends of the muscle cells are probably pulled in towards the middle portions of the cells, so that the individual muscle cells shorten more than the muscle as a whole. A more accurate study, with histograms of the cell profile sizes at various stages of contraction, may clarify this point. e) In isotonically contracted taeniae the muscle cells are no longer parallel (as they are in stretched taeniae), but run at a small angle to each other. They appear twisted and many seem to entwine around each other in a complicated pattern. A tortuous arrangement of the shortened muscle cells as opposed to a parallel one in the elongated cells has been described by Schlote (1960) in the retractor penis of Helix pomatia. However, the details of the re-arrangement of the muscle cells of taenia during isotonic contraction are not yet clear, and need further investigation. It may be assumed that this complex re-arrangement of muscle cells is a more orderly process than it appears from individual sections. f) Fay and Delise (1973) have shown that isolated smooth muscle cells, prepared from the toad stomach and suspended in vitro, present a remarkable invagination of the cell surface when they contract. The surface of fully contracted cells is packed with bulbous and mound-like evaginations, whereas that of relaxed muscle cells is generally smooth. These results by Fay and Delise (1973) are confirmed by the present observations on intact muscles. However, size, shape, and pattern of the evaginations appear different in the intact taenia coli and in the isolated muscle cells of the toad stomach. In the latter case the evaginations are usually round in profile (as seen in scanning electron micrographs). In the taenia coli many e v a g i n a t i o n s - p r o b a b l y because of the constraints
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imposed by the collagen network and the dense packing of cells-are long and slender in sectioned muscles and are part of laminar folds arranged at an angle between 90 ~ and 45 ~ to the longitudinal axis of the cells, often interdigitating with similar processes from neighbouring cells. Some folds extend for not less than half the circumference of a muscle cell, but whether they form a helicoidal system with many turns around a muscle cell could not be ascertained. This seems, however, unlikely, since plain areas, usually occupied by desmosome-like attachments, and longitudinal, wider folds are also present. However, the exact shape and arrangement of the extrusions of the cell surface could not be established, since only individual (transverse or longitudinal) sections were studied. The formation of folds at the cell surface is to some extent due to a geometrical reason. In an elongated structure such as a muscle cell, if the volume remains constant, shortening produces a decrease of the geometrical surface; the "excess' membrane is then thrown into folds. It has been reported that during isotonic contraction single isolated smooth muscle cells decrease in volume (Fay and Delise, 1973; Kominz and Gr6schelSteward, 1973). Whether the same holds true for the muscle cells of an intact muscle could not be investigated in the present study, since cell volume of shortened muscle cells could not be estimated to the same degree of accuracy as that of stretched muscle cells, by means of stereological methods (Gabella, 1976). g) A further indication of the complex structural changes occurring during shortening of the taenia coli, is given by the arrangement of collagen fibrils. These run mainly longitudinally along stretched muscle cells, but obliquely or transversely around contracted muscle cells. Apparently the collagen runs parallel to the folds of the cell surface. These observations indicate that during shortening the muscle cell undergoes some torsion around its longitudinal axis and it twists with neighbouring cells. The cell surface is thrown into folds which are parallel to the lines of force. The collagen fibrils are caught between folds, form bundles running as helices of small pitch around the muscle cells, and remain (or become more) taut in spite of the considerable shortening of the cells. During isometric contraction dramatic changes occur also at the level of the filaments, which are indeed the primum movens of the changes in shape and arrangement of the muscle cells. They will be examined in a following paper. It may be worth stressing that the results discussed in this paper are confined to the taenia coli of the guinea-pig and should not be generalized to other smooth muscles. h) Finally, isotonically contracted muscle cells show characteristic striations. Both the longitudinal and the transverse striations were related to the folds, processes, dense patches and invaginations present at the cell surface. A longitudinal striation of similar appearance has been seen in isolated muscle cells grown in vitro (Small, 1974). Small (1974) interpreted such striation as evidence that smooth muscle cells contain contractile units, which he calls myofibrils. It is possible that the striation observed by Small (1974) is the same as the longitudinal striation described here. If this is the case, it is here suggested that the striation should be interpreted as due to characteristics of the cell surface and not to the presence of special contractile units.
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References Bagby, R.M~, Young, A.M., Dotson, R.S., Fisher, B.A., McKinnon, K.: Contraction of single smooth muscle cells from Bufo marinus stomach. Nature (Lond.) 234, 351-352 (1971) Fay, F.S., Delise, C.M.: Contraction of isolated smooth-muscle cells. Structural changes. Proc. nat. Acad. Sci. (Wash.) 70, 641-645 (1973) Gabella, G.: Quantitative morphological study of smooth muscle cells in the guinea-pig taenia coli. Ceil Tiss. Res. 170, 161 - 1 8 6 (1976) Kelly, R.E., Rice, R.V.: Ultrastructural studies on the contraction mechanism of smooth muscle. J. Cell Biol. 42, 683 694 (1969) Kominz, D.R., Gr6schel-Stewart, U.: Antibody-dependent size changes of myofibrils and isolated smooth muscle cells. J. Mechanochem. Cell Motility 2, 181 191 (1973) 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) Schlote, F.-W.: Die Kontraktion glatter Muskulatur auf Grund yon Torsionsspannungen in den Myofilamenten. Z. Zellforsch. 52, 362-395 (1960) Small, J.V.: Contractile units in vertebrate smooth muscle cells. Nature (Lond.) 249, 324-327 (1974) Wilder, H~C.: An improved technique for silver impregnation of reticulin fibers. Amer. J. Path. ll, 817-820 (1935)
Received March 22, 1976
