SURFACE FEATURES OF SMOOTH MUSCLE CELLS FROM THE MESENTERIC ARTERY AND VAS DEFERENS

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1 J. Cell Set. 8, (i97i) 427 Printed in Great Britain SURFACE FEATURES OF SMOOTH MUSCLE CELLS FROM THE MESENTERIC ARTERY AND VAS DEFERENS C. E. DEVINE, F.O.SIMPSON Wellcome Medical Research Institute, Department of Medicine, University of Otago Medical School, Dunedin, New Zealand AND W. S. BERTAUD Department of Scientific and Industrial Research, Lower Hutt, New Zealand SUMMARY Smooth muscle cells of small mesenteric arteries and vas deferens of guinea-pig were examined by freeze-etching. The most striking finding was that the surface vesicles lie in roughly longitudinal rows, with areas of membrane free of vesicles in between. The areas free of vesicles are believed to correspond to areas occupied by dense bodies in conventionally fixed and sectioned material. Other cell constituents which could be identified included sarcoplasmic reticulum and, probably, thick myofilaments. INTRODUCTION Numerous vesicles are present at the cell surface of smooth muscle of blood vessels (Pease & Molinari, i960; Rhodin, 1962; Simpson & Devine, 1966) and vas deferens (Richardson, 1962; Merrillees, Burnstock & Holman, 1963). The vesicles lie in groups, usually separated from each other by densely staining areas, termed dense bodies (Pease & Molinari, i960), but the actual distribution and arrangement of vesicles on the cell surface have not been ascertained. The technique of freeze-etching developed by Moor & Miihlethaler (1963) and Moor (1964) has permitted the study of surface features of myocardium (Moore, Ruska & Ruska, 1964; Rayns, Simpson & Bertaud, 1967, 1968a) and skeletal muscle (Rayns, Simpson & Bertaud, 19686). In the present study, the surfaces of smooth muscle cells of guinea-pig mesenteric arteries and vas deferens were examined by freeze-etching techniques and the findings correlated with observations from conventional electron microscopy, including the use of lanthanum. A preliminary communication has already been published (Devine, Simpson & Bertaud, 1969). METHODS The blood vessels of the mesentery of guinea-pigs, anaesthetized with sodium pentobarbitone, 55 mg/kg intraperitoneally, were exposed and flooded with 1 % procaine hydrochloride in either Krebs-Ringer phosphate buffer or Tyrode solution. Blood vessels of approximately 300 /im diameter were removed, stripped of fat and placed in 25 % glycerol in Krebs-Ringer phosphate buffer or Tyrode solution for min. Portions of the vas deferens from the same

2 428 C. E. Devine, F. O. Simpson and W. S. Bertaud animals were also prepared in the same way. The tissues were then frozen, fractured, etched, shadowed and replicated in a Balzers BA500R apparatus, as described by Moor & Miihlethaler (1963) and Moor (1964, 1965). For conventional electron microscopy, small mesenteric arteries of the guinea-pig were perfused via the aorta with Tyrode solution containing 2 % glutaraldehyde and 2 % formaldehyde (aldehyde fixative) and post-fixed in 1 % osmium tetroxide in the same buffer. Portions of the vas deferens were fixed in cacodylate-buffered aldehyde fixative and post-fixed in osmium tetroxide in the same buffer. Both tissues were stained en bloc with aqueous 2 % uranyl acetate solution before dehydration. In addition, blood vessels from the mesentery of guinea-pigs were fixed in cacodylatebuffered aldehyde and post-fixed in osmium tetroxide and 2 % lanthanum nitrate in collidine buffer (ph 7-7) and dehydrated rapidly in ethanol after the method of Revel & Karnovsky (1967). All tissues were embedded in epoxy resins and thin sections were stained with alkaline lead citrate. The replicas and sections were examined in a Philips EM200, a Hitachi HU 11A or a Hitachi HU 11E electron microscope. OBSERVATIONS Freeze-etch studies Orientation and identification of surfaces. The fracture planes in vascular smooth muscle were not consistent for each preparation, and often only relatively small areas of smooth muscle membrane were exposed. The orientation of the vascular smooth muscle cells was determined from the location of the vessel lumen, the presence of elastic tissue and the ratio of length to width in obliquely sectioned muscle cells. The adventitial region was recognized by the large amounts of collagen and elastic tissue and the presence of nerve bundles. The vas deferens had less connective tissue between the cells, and the preparations were easier to orientate. The cutting of the frozen tissue results in fracturing of the specimen along planes which follow natural lines of separation in the tissues. Contours of the cell surfaces, either inner or outer, or of surfaces of cell organelles are accordingly seen. When the freshly fractured surface is shadowed, deposits of metal pile up on the side of a projection nearest the shadowing source, leaving a negative shadow cast beyond the projection. When depressions are present, the metal piles up on the side of the depression furthest from the shadowing source. From the direction of the shadow on the small particles which are usually present on the cell membrane, the angle of shadowing and hence the nature of a depression or raised structure can be determined. Inner (cytoplasmic) surfaces of cell membranes can be determined by their relationship to cytoplasmic structures such as myofilaments, and outer cell surfaces can be determined by their relationship to extracellular material such as collagen. Surface features of smooth muscle cells. The general surface structures of vascular smooth muscle and vas deferens smooth muscle were similar and will be discussed together. Views of the outer aspect of the cell membrane revealed rows of depressions (30-60 nm in diameter) which were interpreted as apertures of surface vesicles (Figs. 1, 2). The rows of vesicles were one to ten vesicles wide. The length of the rows of vesicles could not be determined, as the exposed membrane surfaces were not large enough. At some points adjacent rows of vesicles appeared to fuse (Figs. 1-3). The clear spaces between the rows of vesicles were approximately /tm wide, although occasional

3 Surface features of smooth muscle 429 isolated vesicles were present in the areas between the main rows (Figs. 2, 3). The depressions appeared in many cases to be shallow (Fig. 2). Particles up to 16 nmin diameter were randomly distributed on the outer aspect of the cell membrane (Fig. 2). Views of the inner aspect of the cell membrane (Fig. 5) showed excrescences nm wide which were interpreted as complete vesicles, and 'craters' about 30 nm wide which were interpreted as the necks of vesicles which had broken off when the contents of the cell had been stripped away from the membrane. Particles up to 16 nm in diameter were occasionally found also on the inner aspect of the cell membrane. In cross-fractures of cells, as in conventional sectioned material, it could be seen that in many cases the connexion of the vesicles to the inner surface of the cell membrane was by a narrow neck (approximately nm wide), the lumen of the vesicles communicating with the extracellular space (Figs. 5, 10). In some cases multilobed vesicles could be seen (Fig. 11). There was little difference between blood vessels and the vas deferens in the arrangement of the surface vesicles (Figs. 1-4). In the vas deferens, the rows appeared to be rather straighter (Fig. 4) and wider. Cytoplasmic components. In cross-fractured smooth muscle cells, many of the cytoplasmic components recognized in sectioned material could also be found. Mitochondria were recognized as oval projections (Figs. 6, 10) or depressions (Fig. 6) in which particle-covered membranes were seen. Membranous structures, interpreted as sarcoplasmic reticulum (SR) were present close to the nucleus (Fig. 6) and also close to the cell membrane and surface vesicles (Fig. 10). Both the inner luminal and the cytoplasmic surfaces of the SR carried some particles 8-10 nm in diameter (Figs. 10, 12), but the amount of SR which could be definitely identified was too small to permit any accurate comparison of the numbers of particles on the 2 surfaces. Small projecting stumps of myofilaments, estimated to be approximately 20 nm wide and interpreted as thick filaments (Figs. 10, 12), were visible in the crossfractured cytoplasm. They had a density of about 65 per //m 2 of cross-fractured cytoplasm corresponding to a possible thick myofilament density of 130 per//.m 2 (see Discussion), within the range observed by conventional techniques in rabbit mesenteric vein (Devine & Somlyo, 1971). In none of the freeze-etch preparations were any structures seen comparable to the dense bodies seen in conventional electron microscopy, but nucleus, mitochondria, and Golgi apparatus were observed (Fig. 6). Elastic material and endothelial cells. Elastic material, seen as a granular illdefined band, was present between vascular smooth muscle cells and endothelial cells (internal elastic lamina), and in the adventitial region (Figs ). Surfaces of smooth muscle cells adjacent to the elastic tissue of the internal elastic lamina still had longitudinally arranged rows of vesicles, but regions were present where vesicles were absent and patches of elastic tissue were closely applied to muscle cells. Endothelial cells adjacent to the elastic tissue could be distinguished from smooth muscle cells by their proximity to the lumen and by the random arrangement of their surface vesicles (Fig- 7)-

4 430 C. E. Devine, F. 0. Simpson and W. S. Bertaud Conventional electron microscopy With conventional electron microscopy of sections of both vascular and vas deferens smooth muscle cells, the surface vesicles were seen in groups separated by dense bodies where there were no vesicles (Fig. 8). The vesicles were often seen to be open to the extracellular space. In transverse sections the vesicle numbers and dimensions corresponded to those seen in freeze-etch preparations. Most vesicles were 30 nm wide at the openings, 20 nm at the necks and 60 nm at the widest portions, but there was considerable variation in shape, and the neck was not always so narrow. The regions occupied by dense bodies in transverse sections (Fig. 8) corresponded in dimension (approximately nm) to the clear areas of cell membrane seen in freeze-etch preparations (Figs. 1-4). SR was very sparse: it often lay close to surface vesicles and mitochondria (Fig. 8). In lanthanum-treated vascular smooth muscle, deposits of lanthanum were found in the extracellular space around the smooth muscle cells and in the surface vesicles (Fig. 9). Some lanthanum-filled vesicles were not obviously connected to the surface and their connexions were presumably out of the plane of section (Fig. 9). DISCUSSION It is evident from this freeze-etch study that the distribution of the surface vesicles in smooth muscle cells of blood vessels and vas deferens is not a random one. The vesicles may be interpreted as lying predominantly in longitudinal rows or, alternatively, as lying all over the smooth muscle surface except for longitudinal regions in which no vesicles are present. Whatever the interpretation, it is clear that there is a definite longitudinal arrangement of the areas with and without vesicles, although in some places large accumulations of vesicles do occur with the fusion of adjacent rows. In freeze-etch preparations there are no recognizable cytoplasmic structures beneath the cell membrane at areas devoid of surface vesicles, but in conventional preparations the dense bodies, which are possibly attachment areas for the myofilaments (Pease & Molinari, i960), are present between the groups of surface vesicles. It seems likely therefore that the clear areas of the cell membrane correspond to attachment areas of the myofilaments. Thin sections, and freeze-etch preparations where the fracture is more or less normal to the cell membrane, show that the vesicles frequently have a narrow neck and wider body. Such structures might not remain attached to inner cell membrane surfaces where these are exposed by the fracture. Thus it may be that 2 distinct stages of vesicle development are illustrated in fractures such as those shown in Figs Where the complete vesicle remains on the membrane, this might be an indication that it was approximately hemispherical, and hence presumably at an early stage of development. Where only a crater remains, presumably this is an indication that the vesicle had attained the more mature amphora shape, and hence broke off at the neck as the fracture advanced along the membrane. The presence of compound branched vesicles may indicate a third and later stage of development.

5 Surface features of smooth muscle 431 Views of the outer aspect of the cell membrane also appear to distinguish between these stages. For instance, it is sometimes possible to determine that a depression indicating the presence of a vesicle is shallow and smooth-contoured, probably corresponding to the 'early stage'. In many cases, however, the shadowing process has not reached the bottom of the vesicle, which therefore may have been more amphorashaped. Communication between the vesicles and the extracellular space is of course confirmed by the use of lanthanum as a tracer, by which the colloidal lanthanum deposits are also found in the vesicles, even when the actual line of communication is out of the plane of the section. No function has as yet been assigned to the surface vesicles. They have been termed pinocytotic, but very few definitive vesicles are seen away from the cell membrane in either freeze-etch or conventional preparations and a pinocytotic function seems therefore unlikely. The vesicles are also more elongate than those seen in endothelial cells. Adenosinetriphosphatase has been demonstrated histochemically in a few surface vesicles in cerebral vessels (Santos-Buch, 1966; Hoff, 1968) at regions close to the internal elastic lamina, but the concentration of this enzyme was low compared with that in vesicles of endothelial cells or endothelial cell junctions. The vesicles may in some ways be analogous to the T-system of striated muscle, and the many vesicle shapes, especially the multilobed vesicles, appear similar to early stages of inpocketings of the sarcolemma in developing chicken breast skeletal muscle as shown by Ishikawa (1968). The vesicles certainly must increase the surface area of the smooth muscle cell considerably, as Rhodin (1962) pointed out. The sparse SR, containing 8-10 nm particles on the inner (non-cytoplasmic) surface, was in many cases found close to vesicles or to the cell membrane. It may have the same function in smooth muscle as in striated muscle, that is, a calcium store (Somlyo & Somlyo, 1968; Devine & Somlyo, 1970). Bertaud et al. (1970), in a freeze-etch study of fish skeletal muscle, reported a very high concentration of particles, about 9 nm in diameter, on the inner (non-cytoplasmic) surfaces of the SR and no particles on the outer surfaces, while a relatively sparse distribution of particles was seen on both surfaces of the T-tubule system. These observations are consistent with those of Baskin & Deamer (1969) who used both freeze-etching and histochemical methods to demonstrate that the sarcoplasmic fraction from skeletal muscle with a high Ca 2+ content carried a greater concentration of particles than the corresponding fraction from cardiac muscle with a lower Ca 2+ content. The particle population found on inner surfaces of SR of smooth muscle in the present study appears to be smaller than that reported by the above authors, but the total area of SR seen in the present work was too small to allow any definite conclusion at this stage. The small raised projections on fracture faces traversing the cytoplasm of the smooth muscle cells possibly correspond to the broken-off stumps of thick myofilaments seen in striated muscle (Bertaud, Rayns & Simpson, 1968). They had a density of about 65 per /tm 2 of cross-fractured cytoplasm, which corresponded to slightly less than half the density noted by Pease (1968) in cross-sectioned vascular smooth muscle processed by inert dehydration methods. This difference could simply represent a

6 432 C. E. Devine, F. O. Simpson and W. S. Bertaud difference in the type of vascular smooth muscle studied, or it could be due to the fact that in freeze-etch preparations half of the myofilaments would not be seen because they would be oriented in such a way as to be plucked out of the cytoplasm during the fracturing procedure, leaving depressions merging with the background granularity of the preparations. This latter view is based on the interpretations of Bertaud et al. (1968), who found in frozen-etched skeletal muscle that the appearance of the brokenoff thick filaments varied according to the polarity of the myosin molecules: in the process of fracturing, molecules with their 'heads' sticking up would be removed, while those with their ' heads' down would be retained and their ' tails' would remain as projections in the replicas. The authors wish to thank Miss J. M. Ledingham, Mrs S. O'Kane for skilful technical assistance and the Medical Research Council of New Zealand for financial assistance. The use of the Hitachi HU 11E electron microscope provided by General Research Support Grant NIH FR05610 to the Presbyterian-University of Pennsylvania Medical Center, and partial support from NIH Grant HE are gratefully acknowledged. REFERENCES BASKIN, R. J. & DEAMER, D. W. (1969). Comparative ultrastructure and calcium transport in heart and skeletal muscle microsomes. J. Cell Biol. 43, BERTAUD, W. S., RAYNS, D. G. & SIMPSON, F. O. (1968). Myofilaments in frozen-etched muscle. Nature, Lond. 220, 381. BERTAUD, W. S., RAYNS, D. G. & SIMPSON, F. O. (1970). Freeze-etch studies on fish skeletal muscle. J. Cell Sci. 6, DEVINE, C. E., SIMPSON, F. O. & BERTAUD, W. S. (1969). Surface vesicles in vascular smooth muscle cells: A freeze-etch study. Proc. Univ. Otago med. Sell. 47, DEVINE, C. E. & SOMLYO, A. P. (1970). Ultrastructure of vascular smooth muscle studied with lanthanum. Fedn Proc. Fedn Am. Socs exp. Biol. 29, 455. DEVINE, C. E. & SOMLYO, A. P. (1971). Thick filaments in vascular smooth muscle. J. Cell Biol. (in the Press). HOFF, H. F. (1968). A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits. Histocliemie 13, ISHIKAWA, H. (1968). Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. J. Cell Biol. 38, MERRILLEES, N. C. R., BURNSTOCK, G. & HOLMAN, M. E. (1963). Correlation of fine structure and physiology of smooth muscle in the guinea pig vas deferens. J. Cell Biol. 19, MOOR, H. (1964). Die Gefrierfixation lebender Zellenund ihre Anwendung in der Elektronenmikroskopie. Z. Zellforsch. mikrosk. Anat. 62, MOOR, H. (1965). Freeze-etching. Balzers High Vacuum Report 2, MOOR, H. & MOHLETHALER, K. (1963). Fine structure of frozen-etched yeast cells. J. Cell Biol. 17, MOOR, H., RUSKA, C. & RUSKA, H. (1964). Elektronenmikroskopische Darstellung tierisher Zellen mit de Gefrieratztechnik. Z. Zellforsch. mikrosk. Anat. 62, PEASE, D. C. (1968). Structural features of unfixed mammalian smooth and striated muscle prepared by glycol dehydration. J. Ultrastruct. Res. 23, PEASE, D. C. & MOLINARI, S. (i960). Electron microscopy of muscular arteries; pial vessels of the cat and monkey. J. Ultrastruct. Res. 3, RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (1967). Transverse tubule apertures in mammalian myocardial cells: surface array. Science, N.Y. 156, RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (1968a). Surface features of striated muscle cells. I. Guinea-pig cardiac muscle. J. Cell Sci. 3,

7 Surface features of smooth muscle 433 RAYNS, D. G., SIMPSON, F. O. & BERTAUD, W. S. (19686). Surface features of striated muscle cells. II. Guinea-pig skeletal muscle, jf. Cell Set. 3, REVEL, J. P. & KARNOVSKY, M. J. (1967). Hexagonal array of subunits in intercellular junctions of mouse heart and liver, J. Cell Biol. 33, RHODIN, J. A. G. (1962). Fine structure of vascular walls in mammals. Physiol. Rev. 42, Suppl. 5, RICHARDSON, K. C. (1962). The fine structure of autonomic nerve endings in smooth muscle of the rat vas deferens. J. Anat. 96, SANTOS-BUCH, C. A. (1966). Extrusion of ATPase activity from pinocytotic vesicles of abutting endothelium and smooth muscle to the internal elastic membrane of the major arterial circle of the iris of rabbits. Nature, Lond. 211, SIMPSON, F. O. & DEVINE, C. E. (1966). The fine structure of autonomic neuromuscular contacts in arterioles of the sheep renal cortex. J. Anat. 100, SOMLYO, A. P. & SOMLYO, A. V. (1968). Vascular smooth muscle I. Normal structure, pathology, biochemistry, and biophysics. Pharmac. Rev. 20, ABBREVIATIONS ON PLATES (Received 17 August 1970) col collagen myo myofilaments db dense body n nucleus el elastic tissue pi inner aspect of plasmalemma end endothelial cell po outer aspect of plasmalemma g Golgi apparatus sm smooth muscle / lumen sr sarcoplasmic reticulum m mitochondria sri inner aspect of sarcoplasmic reticulum mi inner aspect of mitochondria sro outer aspect of sarcoplasmic reticulum mo outer aspect of mitochondria D vesicle All the freeze-etch replicas are from small mesenteric arteries and vas deferens of guinea-pigs. The direction of shadowing is indicated by ($). 28 CEL 8

8 434 C- E- Devine, F. O. Simpson and W. S. Bertaud Fig. i. Low-power view of the outer surface of a vascular smooth muscle cell showing rows of depressions (30-50 nm in diameter) which correspond to apertures of surface vesicles. At one point (arrow) the rows fuse. In the extracellular space, collagen can be seen projecting as stumps and elastic tissue is also present. To the left the fracture traverses the cytoplasm of the cell, x Fig. 2. View of the outer surface of a smooth muscle cell of the vas deferens showing vesicle apertures (30-60 nm in diameter) lying predominantly in longitudinal rows. The apertures do not penetrate very deeply. Small particles (up to 16 nm in diameter) are also present on the membrane surface, x

9 Surface features of smooth muscle 435

10 436 C. E. Devine, F. O. Simpson and W. S. Bertaud Fig. 3. View of the cytoplasmic surface of a vascular smooth muscle cell membrane (cell axis horizontal). Predominantly longitudinal rows of vesicles are seen as excrescences (50-80 nm in diameter) or craters (about 30 run in diameter). Clear areas (up to 1 /im wide) are present between the rows of vesicles. Some small areas of cell membrane have been removed in the fracturing process, so that extracellular space can be seen at these points. Particles are present on the cytoplasmic aspect of the cell membrane, x Fig. 4. View of the cytoplasmic surface of a smooth muscle cell membrane of the vas deferens showing longitudinal rows of vesicles, 2-10 vesicles wide, with areas (up to 600 nm wide) comparatively free of vesicles between them. Most of the vesicles have been fractured at the 'necks', leaving craters, x

11 Surface features of smooth muscle 437

12 438 C. E. Devine, F. O. Simpson and W. S. Bertaud Fig. 5. Portion of a vascular smooth muscle cell showing the cytoplasmic surface of the cell membrane (above) and the cross-fractured membrane (below). Excrescences are seen (50-80 nm in diameter) corresponding to complete vesicles (double arrow), craters (30 nm wide) corresponding to broken-off necks of vesicles (single arrow), and a vesicle ( ) connected to the inner cytoplasmic surface by a narrow neck (20 nm in width at the narrowest point). The cross-fractured region (below) shows surface vesicles (v) in communication with the extracellular space, x Fig. 6. Portion of a cross-fractured smooth muscle cell of vas deferens near the nucleus showing inner and outer aspects of mitochondrial membranes. Parts of the Golgi apparatus and SR are also seen, x Fig. 7. Outer surface of an endothelial cell showing random depressions in contrast to the longitudinal arrangement in smooth muscle. Particles (up to 16 nm in diameter) are also present, x

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14 44 C. E. Devine, F. 0. Simpson and W. S. Bertaud Fig. 8. Conventionally fixed and sectioned vascular smooth muscle cell showing groups of vesicles (arrows) separated from each other by dense bodies ( ). Only thin myofilaments are seen. The arrangement of groups of vesicles and dense bodies is essentially similar in the vas deferens. Stained with alkaline lead citrate, x Fig. 9. Tangential section through a vascular smooth muscle cell showing lanthanumfilled vesicles connected to the extracellular space (arrow) where deposits of lanthanum are also present. Large numbers of apparently 'free floating' vesicles are also present; they contain lanthanum, and presumably must be in communication with the extracellular space but their communication is not in the plane of this section. Lanthanum treatment, alkaline lead citrate stain, x

15 Surface features of smooth muscle 44* SI I sm 1

16 442 C. E. Devine, F. O. Simpson and W. S. Bertaud Fig. 10. Cross-fractured vascular smooth muscle cell. In the extracellular space broken-off stumps of collagenfibrilscan be seen, and also amorphous granular patches indicating the position of elastic tissue. A portion of the outer surface of a mitochondrion is visible. In the cross-fractured cytoplasm, raised stumps of thick myofilaments are seen. Several structures are present which possibly represent the surfaces of the sparse sarcoplasmic reticulum; they have a few particles 8-10 run wide on both the inner (luminal) and the outer (cytoplasmic) surfaces, x Fig. 11. Cross-fractured vascular smooth muscle cell showing a multilobed vesicle, x Fig. 12. Cross-fractured vascular smooth muscle cell showing stumps of myofilaments in the cytoplasm and a portion of the inner surface of the SR with 8-10 nm particles. Elastic material is present in the extracellular space, x

17 Surface features of smooth muscle 443 mo \ sr;

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