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Junctional structure of smooth muscle cells
J. ULTRASTRUCTURE RESEARCH 10, 567-577 (1964)
567
J u n c t i o n a l S t r u c t u r e of S m o o t h M u s c l e Cells The Ultrostructure of the Regions of Junction between Smooth Muscle Cells in the Rot Small Intestine TAKEO OOSAKI AND SABURO ISHII
Department of Anatomy, Fukushima Medical College, Fukushima, Japan Received October 11, 1963 The electron micrographs obtained in this study revealed that there are two kinds of contact zones between smooth muscle cells in the rat small intestine-those with fusion of adjoining plasma membranes and those with intercellular attachments. The pattern of the former is characterized by fusion of the adjoining plasma membranes resulting in obliteration of the intercellular space. Within the obliterated areas the outer layers of the adjoining plasma membranes fuse to form a single intermediate line. The pattern of the latter is characterized by the presence of an intercellular space (30-100 A wide) occupied by a homogeneous and amorphous material of moderate density, by strict parallelism of the adjoining plasma membranes and by an increased density of the cytoplasmically opaque material attached to the plasma membranes. In electron microscopic studies, evidence in favor of protoplasmic continuity between smooth muscle cells was reported by Mark (11) and Thaemert (19). In opposition, Caesar et al. (2) and Bcrgman (1) described the architecture of smooth muscles as being cellular. In the latter studies, however, definite information regarding the fine structure of the contact regions between cells seems to have been lacking. The present paper indicates essentially that the connection between muscle cells is ephaptic in character. At high magnifications, Dewey and Barr (4) found areas of fusion of the adjacent plasma membranes at the regions of contact between the smooth muscle cells from dog intestine and proposed calling them a "nexus." The present paper supports the findings of Dewey and Barr and offers more convincing evidence concerning the structure of the contact zones of the adjoining plasma membranes.
MATERIAL AND METHODS For this investigation, smooth muscles of the jejunum and ileum of rats were used. Immediately after removal from the sacrificed animals, segments of jejunum and ileum were fixed for 2-2½ hours in ice-cold 0.6 or 1.0% potassium permanganate solution buffered
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at pH 7.4 with Verona1 acetate (9). Specimens were also fixed in 1.0% osmium tetroxide buffered at pH 7.4-7.6 with Veronal acetate (3) for 2 hours. Specimens were rapidly dehydrated in graded acetone or ethanol and finally embedded in Epon 812 (10). Thin sections prepared from blocks fixed in potassium permanganate were doubly stained, first in uranyl acetate (20), then in lead hydroxide according to Millonig (12). The other sections cut from blocks fixed in osmium tetroxide were stained with lead hydroxide. Micrographs were taken at original magnifications of 5000 to 50,000 with a Hitachi HU11 electron microscope, operating at 50 or 75 kV. OBSERVATIONS At lower magnifications the regions of contact between s m o o t h muscle cells from rat small intestine are demonstrated in Figs. 1-5. The regions of contact of adjacent cells vary in size and shape and basically represent mutual extensions of adjacent cells or extensions of single cells abutting neighboring cells (see Figs. 1-5). In some cases, short, stout processes are seen at the contact regions, extending f r o m one cell into a corresponding depression in the surface of the adjoining cell. The interlocking relationship which results is suggestive of a dovetail joint (Fig. 5). These contact regions are characterized by a close contact of adjoining plasma membranes, the gap between adjoining plasma membranes being indiscernible or appearing as an extreme narrowing of the intercellular space. N o structure of desmosomes is recognizable. At higher magnifications the patterns of fine structure in the contact regions are illustrated by Figs. 6-14. I n high resolution micrographs of KMnO4-fixed specimens the plasma m e m b r a n e of s m o o t h muscle cells is asymmetric and triple layered and has a total thickness of a b o u t 70-75 A (Fig. 7). The m e m b r a n e is composed of an inner dense layer a b o u t 25 N thick facing the cytoplasm, an outer dense layer about 20 ~ thick, and an intermediate light layer about 25-30 ~ thick. I n m a n y cases, the outer layer is slightly less dense than the inner layer (Figs. 6 and 7). Analysis of the contact regions reveals that the plasma membranes of the adjoining cells come together and fuse, with resultant obliteration of the intercellular space. The intermediate line represents the merged outer layers of the adjoining plasma membranes (Figs. 6-8). The pattern of the contact zone therefore appears five layered, with a thickness of a b o u t 125-145/k. The thickness of the intermediate line is about 25-30 A (Fig. 7). Fins. 1-5. Various shapes of the contact regions between smooth muscle cells in the rat small intestine illustrated by low magnifications. In these figures, the adjoining plasma membranes at the contact regions show a close contact. The regions between adjacent membranes are indiscernible (Figs. 1, 2, and 5) or appear as extreme narrowing of the intercellular space (Figs. 3 and 4). The interlocking appearance of the junction between cells illustrated by Fig. 5 is reminiscent of a dovetail joint. Fins. 1-3. Specimens fixed in 1% OsO4. Lead hydroxide-stained sections. Fig. 1, x 17,500; Fig. 2, x 25,500; Fig. 3, x 40,000. F1~s. 4 and 5. Specimens fixed in 1% KMnO4. Sections stained in uranyl acetate and lead hydroxide. Fig. 4, x 35,000; Fig. 5, x 25,000.
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In sections cut normal to the plane of the junction, the individual plasma membranes can be traced to their point of mergence, where the two outer layers of the plasma membranes join to become the intermediate line of the junction (Figs. 6 and 7). Sometimes a cytoplasmically dense material attached to the plasma membranes along the junction is observed (Fig. 6). A rather frequent observation of the contact regions is that the intermediate fusion line is discontinuous. This variation consists of the splitting of the fusion line within the junction followed by refusion (Figs. 9-11). In some cases, the space separating the inner layers of the fusion membranes from one another is about 125 A wide, and no intermediate fusion line is visible (Fig. 10). Contact zones of the other type have also been observed. This type of contact is characterized by the presence of a true intercellular space, which measures 30-100 A across, occupied by a homogeneous and amorphous material of moderate density. There is also an increased density of the cytoplasmically opaque components of the plasma membranes. Throughout the contact regions between adjacent cells, the apposed outer layers of the adjoining plasma membranes run strictly parallel to one another and are separated by a narrow light zone of intercellular space that is almost constant in width (Figs. 12-14). The plasma membranes of the contact regions measure about 70-80 A in thickness; the inner, middle, and the outer layers accounting for 25-30 A, 25-30 A, and about 20/~, respectively. These dimensions remain essentially the same along the cell surface. At these regions of contact there is no structure of desmosomes, which is characterized by high local concentration of dense amorphous and fibrillar materials in the subjacent cytoplasmic matrix. DISCUSSION The electron micrographs obtained in this study demonstrate the occurrence of a characteristic junction of the plasma membranes at the regions of contact between the smooth muscle cells in rat small intestine. Whether or not protoplasmic continuity occurs between smooth muscle cells has been a subject of controversy. In published electron micrographs of smooth muscle cells from the gastrointestinal tracts of rats, Thaemert (19) observed that their cells are connected by small anastomotic intercellular bridges. M a r k (11) interpreted gaps in the sarcolemma as suggestive of continuity in the smooth muscle cells of rat uteri.
FIGS. 6 and 7. Detailed structure of the junction with fusion of the plasma membranes. A five-layered pattern is clearly shown all along the junction. At the point shown by arrows the outer layers of the converging membranes fuse to form the intermediate line of the junction. In Fig. 6, it should be noticed that a dense cytoplasmic material is visible attached to the plasma membranes along the junction. Materials fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining. Fig. 6, × 185,000; Fig. 7, × 500,000.
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These findings s u p p o r t the syncytial c o n c e p t of s m o o t h muscles. Caesar et al. (2) described their m i c r o g r a p h s of s m o o t h muscle cells f r o m m o u s e u r i n a r y b l a d d e r as d i s p l a y i n g o n l y cells t h a t are c o m p l e t e l y s e p a r a t e d f r o m each o t h e r by a cytolemma c o m p a r a b l e to the s a r c o l e m m a of s t r i a t e d muscles. I n m i c r o g r a p h s of rat ureter, B e r g m a n (1) r e c o g n i z e d the presence of i n t e r c e l l u l a r bridges between s m o o t h muscle cells, which are transversed b y i n t r a b r i d g e m e m b r a n e s . Their m i c r o g r a p h s reveal that the architecture of s m o o t h muscles is cellular. O u r findings also s u p p o r t essentially the e p h a p t i c concept of s m o o t h muscle cells m o r p h o l o g i c a l l y d e m o n s t r a t e d b y Caesar et al. (2), B e r g m a n (1), a n d others (7). I n their m i c r o g r a p h s , however, definite inf o r m a t i o n r e g a r d i n g the fine structure of the c o n t a c t regions between cells seems to have been lacking. I n l o w resolution m i c r o g r a p h s the p a t t e r n of the c o n t a c t regions between adjacent ceils is c h a r a c t e r i z e d by a close c o n t a c t of a d j o i n i n g p l a s m a m e m b r a n e s . The region between a d j o i n i n g p l a s m a m e m b r a n e s is discernible as a n a r e a of extreme narrowing of the intercellular space. N o structure of d e s m o s o m e s is recognized. In K M n Q fixed m a t e r i a l s the high r e s o l u t i o n m i c r o g r a p h s reveal the five-layered structures of the c o n t a c t zones where the a d j o i n i n g p l a s m a m e m b r a n e s c o m e t o g e t h e r and fuse with resultant o b l i t e r a t i o n of the intercellular space. The i n t e r m e d i a t e line thus f o r m e d represents the m e r g e d outer layers of the a d j o i n i n g p l a s m a m e m b r a n e s . The thickness of the i n t e r m e d i a t e line is a b o u t 25-30 •. If this were m e r e l y j u x t a p o s i t i o n of the a d j a c e n t p l a s m a m e m b r a n e s , the i n t e r m e d i a t e dense line s h o u l d be nearly d o u b l e the thickness of the o u t e r layers of a d j o i n i n g p l a s m a m e m b r a n e s , which is a b o u t 20 A wide. A p p a r e n t l y there is a true fusion of the o u t e r layers of adjoining
FrGs. 8 and 9. Detailed structure of the junction between adjacent plasma membranes. In Fig. 8, a fusion of the plasma membranes at the junctional zones is complete. A wavy appearance of the junctional zone seems to have been caused by preparatory factors. In Fig. 9, the several splittings of the fusion line within the junction followed by their refusion are recognized. The intermediate fusion line is visible within the areas between arrows 1 and 2, 3 and 4, 5 and 6, and 7 and 8. Specimens fixed in 1% KMnO~. Sections stained in uranyl acetate and lead hydroxide. Fig. 8, x 225,000; Fig. 9, x 170,000. FIGS. 10 and 11. Detailed structure of the junction, which is reminiscent of a dovetail joint. In Fig. 10, arrows I and 2 show an area at which the space separating the inner layers of the fused membranes is narrowed to 125 ~ and no intermediate line is visible. The single dense lines within the areas shown by arrows 3, 4, and 5 are suggestive of the fusion line. In Fig. 11, a fairly wide area of the splitting of the fusion line from arrow I to arrow 2 is visible. F~G. 10. Specimens fixed in 0.6% KMnO4. Sections stained in lead hydroxide, x 137,500. FIG. 11. Specimens fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining, x 175,000. FIGS. 12-14. Detailed structure of the junction with an intercellular attachment device. A moderately dense material appears in the intercellular space between the adjoining plasma membranes. Furthermore, the opaque material closely associated with the plasma membranes along the junction is visible. No structure of desmosomes is discernible. Materials fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining. Fig. 12, x 175,000; Yig. 13, ×240,000; Fig. 14, x 175,000.
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plasma membranes at the contact zones. This five-layered pattern of the plasma membrane corresponds to that of the structure of junction described by Dewey and Barr (4) as "nexuses" between the smooth muscle cells in dog intestine. Also, this junctional pattern essentially corresponds to that between the intestinal epithelial cells originally described by Zetterqvist (21) and Sj/Sstrand and Zetterqvist (18). In addition, this is basically the same as the structure described by Robertson (15) as "external compound membranes" between intestinal epithelial cells; by Karrer (8) as "quintuple-layered cell interconnections" between cells of the human cervical epithelium; by Muir and Peters (13) as "quintuple-layered membrane junctions" between endothelial cells of blood capiUaries; by Peters (14) as "quintuple-layered units" between glial cells and between myelin sheaths and glia; by Sj6strand and Elfvin (17) as "five- or seven-layered membrane attachments" between exocrine pancreas cells; by Farquhar and Palade (6) as "zonule occludens" between cells of various epithelia; and by Elfvin (5) as "five- or seven-layered cell boundaries" between preand postganglionic neurons and between pre-ganglionic nerve fibers and satellite cells. Furthermore, most recently, junctions of similar structures have been illustrated by Sj/Sstrand (I6) in epithelium of the mouse intestine. It is occasionally observed that the fusion of the plasma membranes at the contact zone is not complete but partial. This variation consists of the splitting of the fusion line within the junction followed by its refusion. The presence of these variations of junction are suggestive of ultrastructural changes of the contact zones in connection with cellular conditions. Variations of the same kind at the contact regions between epithelial cells have been reported by Farquhar and Palade (6). In addition to the connection of the plasma membranes described above, the other type of junction has been observed. The pattern of this type is characterized by the presence of a narrow intercellular space occupied by a homogeneous and amorphous material of moderate density, by an increased density of the cytoplasmic opaque components of the plasma membranes, and by strict parallelism of the adjoining plasma membranes. As pointed out by Elfvin (5), the dense materials attached to the plasma membranes show a variation in density and thickness. The difference of density may be due to difference in staining ability caused by preparatory factors. This type of junction should be distinguished from the usual desmosome, and no detailed description of this type regarding the connection between smooth muscle cells is found in the literature. However, a junction of similar structure has been clearly described by Sj6strand and Elfvin (17) at the contact zones between exocrine pancreas cells; by Farquhar and Palade (6) at the contact regions between various epithelial cells; and by Elfvin (5) at the junctional areas between pre- and postganglionic neurons. The possibility of correlation between variation of the junctional structure of the
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plasma membranes at the contact regions between cells and s m o o t h muscle stimulus conduction will have to be explored further. The authors gratefully acknowledge the technical assistance of Mr. M. Honda, Mr. Y. Ono, Mr. T. Kurosu, and Mrs. T. Naito.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BERGMAN, R. A., Bull. Johns Hopkins Hosp. 102, 195 (1958).
CAESAR,R., EDWARDS,G. A., and RUSKA, H., J. Biophys. Biochem. Cytol. 3, 867 (1957). CAULFIELD,J. B., J. Biophys. Biochem. Cytol. 3, 827 (1957). DEWEY, M. M. and BARR, L., Science 137, 670 (1962). ELFVlN, L.-G., J. Ultrastruct. Res. 8, 441 (1963). FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). GANSLER,H., Z. Zellforsch. 55, 724 (1961). KARRER, H. E., J. Biophys. Biochem. Cytol. 7, 181 (1960). LUFT, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). - - - - ibid. 9, 409 (1961). MARK, J. S. T., Anat. Record125, 473 (1956). MILLONIG, G., 3-. Biophys. Biochem. Cytol. 11, 739 (1961). MUIR, A. R. and PETERS, A., J. CellBiol. 12, 443 (1962). PETERS, A., J. Anat. 96 (Pt. 2), 237 (1962). ROBERTSON,J. B., Progr. Biophys. Biophys. Chem. 10, 343 (1960). SJ(3STRAND,F. S., J. Ultrastruct. Res. 8, 517 (1963). SJ6STRAND, F. S. and ELFVlN, L.-G., J. Ultrastruct. Res. 7, 504 (1963). SJ()STRAND,F. S. and ZETTERQVIST, I-I., Electron Microscopy, Proc. Stockholm Conf. 1956, p. 150 (1957). Academic Press, New York. 19. THAEMERT,J. C., or. Biophys. Biochem. Cytol. 6, 67 (1959). 20. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). 21. ZETTERQVlST, H., The Ultrastructural Organization of the Columnar Absorbing Cells of the Mouse Intestine. Aktiebolaget Godvil, Stockholm, 1956.
37-- 641835 J . Ultrastvucture Research
567
J u n c t i o n a l S t r u c t u r e of S m o o t h M u s c l e Cells The Ultrostructure of the Regions of Junction between Smooth Muscle Cells in the Rot Small Intestine TAKEO OOSAKI AND SABURO ISHII
Department of Anatomy, Fukushima Medical College, Fukushima, Japan Received October 11, 1963 The electron micrographs obtained in this study revealed that there are two kinds of contact zones between smooth muscle cells in the rat small intestine-those with fusion of adjoining plasma membranes and those with intercellular attachments. The pattern of the former is characterized by fusion of the adjoining plasma membranes resulting in obliteration of the intercellular space. Within the obliterated areas the outer layers of the adjoining plasma membranes fuse to form a single intermediate line. The pattern of the latter is characterized by the presence of an intercellular space (30-100 A wide) occupied by a homogeneous and amorphous material of moderate density, by strict parallelism of the adjoining plasma membranes and by an increased density of the cytoplasmically opaque material attached to the plasma membranes. In electron microscopic studies, evidence in favor of protoplasmic continuity between smooth muscle cells was reported by Mark (11) and Thaemert (19). In opposition, Caesar et al. (2) and Bcrgman (1) described the architecture of smooth muscles as being cellular. In the latter studies, however, definite information regarding the fine structure of the contact regions between cells seems to have been lacking. The present paper indicates essentially that the connection between muscle cells is ephaptic in character. At high magnifications, Dewey and Barr (4) found areas of fusion of the adjacent plasma membranes at the regions of contact between the smooth muscle cells from dog intestine and proposed calling them a "nexus." The present paper supports the findings of Dewey and Barr and offers more convincing evidence concerning the structure of the contact zones of the adjoining plasma membranes.
MATERIAL AND METHODS For this investigation, smooth muscles of the jejunum and ileum of rats were used. Immediately after removal from the sacrificed animals, segments of jejunum and ileum were fixed for 2-2½ hours in ice-cold 0.6 or 1.0% potassium permanganate solution buffered
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at pH 7.4 with Verona1 acetate (9). Specimens were also fixed in 1.0% osmium tetroxide buffered at pH 7.4-7.6 with Veronal acetate (3) for 2 hours. Specimens were rapidly dehydrated in graded acetone or ethanol and finally embedded in Epon 812 (10). Thin sections prepared from blocks fixed in potassium permanganate were doubly stained, first in uranyl acetate (20), then in lead hydroxide according to Millonig (12). The other sections cut from blocks fixed in osmium tetroxide were stained with lead hydroxide. Micrographs were taken at original magnifications of 5000 to 50,000 with a Hitachi HU11 electron microscope, operating at 50 or 75 kV. OBSERVATIONS At lower magnifications the regions of contact between s m o o t h muscle cells from rat small intestine are demonstrated in Figs. 1-5. The regions of contact of adjacent cells vary in size and shape and basically represent mutual extensions of adjacent cells or extensions of single cells abutting neighboring cells (see Figs. 1-5). In some cases, short, stout processes are seen at the contact regions, extending f r o m one cell into a corresponding depression in the surface of the adjoining cell. The interlocking relationship which results is suggestive of a dovetail joint (Fig. 5). These contact regions are characterized by a close contact of adjoining plasma membranes, the gap between adjoining plasma membranes being indiscernible or appearing as an extreme narrowing of the intercellular space. N o structure of desmosomes is recognizable. At higher magnifications the patterns of fine structure in the contact regions are illustrated by Figs. 6-14. I n high resolution micrographs of KMnO4-fixed specimens the plasma m e m b r a n e of s m o o t h muscle cells is asymmetric and triple layered and has a total thickness of a b o u t 70-75 A (Fig. 7). The m e m b r a n e is composed of an inner dense layer a b o u t 25 N thick facing the cytoplasm, an outer dense layer about 20 ~ thick, and an intermediate light layer about 25-30 ~ thick. I n m a n y cases, the outer layer is slightly less dense than the inner layer (Figs. 6 and 7). Analysis of the contact regions reveals that the plasma membranes of the adjoining cells come together and fuse, with resultant obliteration of the intercellular space. The intermediate line represents the merged outer layers of the adjoining plasma membranes (Figs. 6-8). The pattern of the contact zone therefore appears five layered, with a thickness of a b o u t 125-145/k. The thickness of the intermediate line is about 25-30 A (Fig. 7). Fins. 1-5. Various shapes of the contact regions between smooth muscle cells in the rat small intestine illustrated by low magnifications. In these figures, the adjoining plasma membranes at the contact regions show a close contact. The regions between adjacent membranes are indiscernible (Figs. 1, 2, and 5) or appear as extreme narrowing of the intercellular space (Figs. 3 and 4). The interlocking appearance of the junction between cells illustrated by Fig. 5 is reminiscent of a dovetail joint. Fins. 1-3. Specimens fixed in 1% OsO4. Lead hydroxide-stained sections. Fig. 1, x 17,500; Fig. 2, x 25,500; Fig. 3, x 40,000. F1~s. 4 and 5. Specimens fixed in 1% KMnO4. Sections stained in uranyl acetate and lead hydroxide. Fig. 4, x 35,000; Fig. 5, x 25,000.
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In sections cut normal to the plane of the junction, the individual plasma membranes can be traced to their point of mergence, where the two outer layers of the plasma membranes join to become the intermediate line of the junction (Figs. 6 and 7). Sometimes a cytoplasmically dense material attached to the plasma membranes along the junction is observed (Fig. 6). A rather frequent observation of the contact regions is that the intermediate fusion line is discontinuous. This variation consists of the splitting of the fusion line within the junction followed by refusion (Figs. 9-11). In some cases, the space separating the inner layers of the fusion membranes from one another is about 125 A wide, and no intermediate fusion line is visible (Fig. 10). Contact zones of the other type have also been observed. This type of contact is characterized by the presence of a true intercellular space, which measures 30-100 A across, occupied by a homogeneous and amorphous material of moderate density. There is also an increased density of the cytoplasmically opaque components of the plasma membranes. Throughout the contact regions between adjacent cells, the apposed outer layers of the adjoining plasma membranes run strictly parallel to one another and are separated by a narrow light zone of intercellular space that is almost constant in width (Figs. 12-14). The plasma membranes of the contact regions measure about 70-80 A in thickness; the inner, middle, and the outer layers accounting for 25-30 A, 25-30 A, and about 20/~, respectively. These dimensions remain essentially the same along the cell surface. At these regions of contact there is no structure of desmosomes, which is characterized by high local concentration of dense amorphous and fibrillar materials in the subjacent cytoplasmic matrix. DISCUSSION The electron micrographs obtained in this study demonstrate the occurrence of a characteristic junction of the plasma membranes at the regions of contact between the smooth muscle cells in rat small intestine. Whether or not protoplasmic continuity occurs between smooth muscle cells has been a subject of controversy. In published electron micrographs of smooth muscle cells from the gastrointestinal tracts of rats, Thaemert (19) observed that their cells are connected by small anastomotic intercellular bridges. M a r k (11) interpreted gaps in the sarcolemma as suggestive of continuity in the smooth muscle cells of rat uteri.
FIGS. 6 and 7. Detailed structure of the junction with fusion of the plasma membranes. A five-layered pattern is clearly shown all along the junction. At the point shown by arrows the outer layers of the converging membranes fuse to form the intermediate line of the junction. In Fig. 6, it should be noticed that a dense cytoplasmic material is visible attached to the plasma membranes along the junction. Materials fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining. Fig. 6, × 185,000; Fig. 7, × 500,000.
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These findings s u p p o r t the syncytial c o n c e p t of s m o o t h muscles. Caesar et al. (2) described their m i c r o g r a p h s of s m o o t h muscle cells f r o m m o u s e u r i n a r y b l a d d e r as d i s p l a y i n g o n l y cells t h a t are c o m p l e t e l y s e p a r a t e d f r o m each o t h e r by a cytolemma c o m p a r a b l e to the s a r c o l e m m a of s t r i a t e d muscles. I n m i c r o g r a p h s of rat ureter, B e r g m a n (1) r e c o g n i z e d the presence of i n t e r c e l l u l a r bridges between s m o o t h muscle cells, which are transversed b y i n t r a b r i d g e m e m b r a n e s . Their m i c r o g r a p h s reveal that the architecture of s m o o t h muscles is cellular. O u r findings also s u p p o r t essentially the e p h a p t i c concept of s m o o t h muscle cells m o r p h o l o g i c a l l y d e m o n s t r a t e d b y Caesar et al. (2), B e r g m a n (1), a n d others (7). I n their m i c r o g r a p h s , however, definite inf o r m a t i o n r e g a r d i n g the fine structure of the c o n t a c t regions between cells seems to have been lacking. I n l o w resolution m i c r o g r a p h s the p a t t e r n of the c o n t a c t regions between adjacent ceils is c h a r a c t e r i z e d by a close c o n t a c t of a d j o i n i n g p l a s m a m e m b r a n e s . The region between a d j o i n i n g p l a s m a m e m b r a n e s is discernible as a n a r e a of extreme narrowing of the intercellular space. N o structure of d e s m o s o m e s is recognized. In K M n Q fixed m a t e r i a l s the high r e s o l u t i o n m i c r o g r a p h s reveal the five-layered structures of the c o n t a c t zones where the a d j o i n i n g p l a s m a m e m b r a n e s c o m e t o g e t h e r and fuse with resultant o b l i t e r a t i o n of the intercellular space. The i n t e r m e d i a t e line thus f o r m e d represents the m e r g e d outer layers of the a d j o i n i n g p l a s m a m e m b r a n e s . The thickness of the i n t e r m e d i a t e line is a b o u t 25-30 •. If this were m e r e l y j u x t a p o s i t i o n of the a d j a c e n t p l a s m a m e m b r a n e s , the i n t e r m e d i a t e dense line s h o u l d be nearly d o u b l e the thickness of the o u t e r layers of a d j o i n i n g p l a s m a m e m b r a n e s , which is a b o u t 20 A wide. A p p a r e n t l y there is a true fusion of the o u t e r layers of adjoining
FrGs. 8 and 9. Detailed structure of the junction between adjacent plasma membranes. In Fig. 8, a fusion of the plasma membranes at the junctional zones is complete. A wavy appearance of the junctional zone seems to have been caused by preparatory factors. In Fig. 9, the several splittings of the fusion line within the junction followed by their refusion are recognized. The intermediate fusion line is visible within the areas between arrows 1 and 2, 3 and 4, 5 and 6, and 7 and 8. Specimens fixed in 1% KMnO~. Sections stained in uranyl acetate and lead hydroxide. Fig. 8, x 225,000; Fig. 9, x 170,000. FIGS. 10 and 11. Detailed structure of the junction, which is reminiscent of a dovetail joint. In Fig. 10, arrows I and 2 show an area at which the space separating the inner layers of the fused membranes is narrowed to 125 ~ and no intermediate line is visible. The single dense lines within the areas shown by arrows 3, 4, and 5 are suggestive of the fusion line. In Fig. 11, a fairly wide area of the splitting of the fusion line from arrow I to arrow 2 is visible. F~G. 10. Specimens fixed in 0.6% KMnO4. Sections stained in lead hydroxide, x 137,500. FIG. 11. Specimens fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining, x 175,000. FIGS. 12-14. Detailed structure of the junction with an intercellular attachment device. A moderately dense material appears in the intercellular space between the adjoining plasma membranes. Furthermore, the opaque material closely associated with the plasma membranes along the junction is visible. No structure of desmosomes is discernible. Materials fixed in 1% KMnO4. Uranyl acetate and lead hydroxide staining. Fig. 12, x 175,000; Yig. 13, ×240,000; Fig. 14, x 175,000.
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plasma membranes at the contact zones. This five-layered pattern of the plasma membrane corresponds to that of the structure of junction described by Dewey and Barr (4) as "nexuses" between the smooth muscle cells in dog intestine. Also, this junctional pattern essentially corresponds to that between the intestinal epithelial cells originally described by Zetterqvist (21) and Sj/Sstrand and Zetterqvist (18). In addition, this is basically the same as the structure described by Robertson (15) as "external compound membranes" between intestinal epithelial cells; by Karrer (8) as "quintuple-layered cell interconnections" between cells of the human cervical epithelium; by Muir and Peters (13) as "quintuple-layered membrane junctions" between endothelial cells of blood capiUaries; by Peters (14) as "quintuple-layered units" between glial cells and between myelin sheaths and glia; by Sj6strand and Elfvin (17) as "five- or seven-layered membrane attachments" between exocrine pancreas cells; by Farquhar and Palade (6) as "zonule occludens" between cells of various epithelia; and by Elfvin (5) as "five- or seven-layered cell boundaries" between preand postganglionic neurons and between pre-ganglionic nerve fibers and satellite cells. Furthermore, most recently, junctions of similar structures have been illustrated by Sj/Sstrand (I6) in epithelium of the mouse intestine. It is occasionally observed that the fusion of the plasma membranes at the contact zone is not complete but partial. This variation consists of the splitting of the fusion line within the junction followed by its refusion. The presence of these variations of junction are suggestive of ultrastructural changes of the contact zones in connection with cellular conditions. Variations of the same kind at the contact regions between epithelial cells have been reported by Farquhar and Palade (6). In addition to the connection of the plasma membranes described above, the other type of junction has been observed. The pattern of this type is characterized by the presence of a narrow intercellular space occupied by a homogeneous and amorphous material of moderate density, by an increased density of the cytoplasmic opaque components of the plasma membranes, and by strict parallelism of the adjoining plasma membranes. As pointed out by Elfvin (5), the dense materials attached to the plasma membranes show a variation in density and thickness. The difference of density may be due to difference in staining ability caused by preparatory factors. This type of junction should be distinguished from the usual desmosome, and no detailed description of this type regarding the connection between smooth muscle cells is found in the literature. However, a junction of similar structure has been clearly described by Sj6strand and Elfvin (17) at the contact zones between exocrine pancreas cells; by Farquhar and Palade (6) at the contact regions between various epithelial cells; and by Elfvin (5) at the junctional areas between pre- and postganglionic neurons. The possibility of correlation between variation of the junctional structure of the
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plasma membranes at the contact regions between cells and s m o o t h muscle stimulus conduction will have to be explored further. The authors gratefully acknowledge the technical assistance of Mr. M. Honda, Mr. Y. Ono, Mr. T. Kurosu, and Mrs. T. Naito.
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CAESAR,R., EDWARDS,G. A., and RUSKA, H., J. Biophys. Biochem. Cytol. 3, 867 (1957). CAULFIELD,J. B., J. Biophys. Biochem. Cytol. 3, 827 (1957). DEWEY, M. M. and BARR, L., Science 137, 670 (1962). ELFVlN, L.-G., J. Ultrastruct. Res. 8, 441 (1963). FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). GANSLER,H., Z. Zellforsch. 55, 724 (1961). KARRER, H. E., J. Biophys. Biochem. Cytol. 7, 181 (1960). LUFT, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). - - - - ibid. 9, 409 (1961). MARK, J. S. T., Anat. Record125, 473 (1956). MILLONIG, G., 3-. Biophys. Biochem. Cytol. 11, 739 (1961). MUIR, A. R. and PETERS, A., J. CellBiol. 12, 443 (1962). PETERS, A., J. Anat. 96 (Pt. 2), 237 (1962). ROBERTSON,J. B., Progr. Biophys. Biophys. Chem. 10, 343 (1960). SJ(3STRAND,F. S., J. Ultrastruct. Res. 8, 517 (1963). SJ6STRAND, F. S. and ELFVlN, L.-G., J. Ultrastruct. Res. 7, 504 (1963). SJ()STRAND,F. S. and ZETTERQVIST, I-I., Electron Microscopy, Proc. Stockholm Conf. 1956, p. 150 (1957). Academic Press, New York. 19. THAEMERT,J. C., or. Biophys. Biochem. Cytol. 6, 67 (1959). 20. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). 21. ZETTERQVlST, H., The Ultrastructural Organization of the Columnar Absorbing Cells of the Mouse Intestine. Aktiebolaget Godvil, Stockholm, 1956.
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