Cell and Tissue Research
Cell Tissue Res. 202, 337-341 (1979)
9 by Springer-Verlag 1979
Short Communication
Structural Analysis of Functionally Different Smooth Muscles Megumi Moriya and Eisaku Miyazaki* Department of Physiology,Sapporo Medical College, Sapporo, Japan
Summary. The ultrastructure of the longitudinal and circular muscle cells of the guinea pig stomach which show different contractile responses was compared. The extracellular space within the muscle bundles is about 12.1 ~ in the longitudinal layer and about 4.4% in the circular layer. Nexuses were consistently found in the circular muscle layer but not in the longitudinal muscle layer. Numbers of both mitochondria and microtubules per unit area of smooth muscle cell are larger in the longitudinal than in the circular muscle. Cellular area occupied by sarcoplasmic reticulum is about 4.7% in the longitudinal muscle, 2.3 % in the circular muscle. The numbers of caveolae are almost the same in both tissues. The most distinct difference between the two types of smooth muscle is the appearance of the thick filaments. The circular muscle cell contains approximately 50thick filaments per 0.5~tm 2 of cytoplasmic area, while the longitudinal muscle cell has only about 25 filaments which were usually much thinner than those of the circular muscle. These results indicate that the contractile apparatus itself is different in longitudinal and circular smooth muscles of the guinea pig stomach.
Key words: Longitudinal m u s c l e - Circular muscle - Structural analysis - Thick filaments - Guinea pig stomach.
In response to K-depolarizing solution, the longitudinal muscle strip of the guinea pig stomach contracts with a small phasic phase followed by a large tonic phase, while the circular muscle strip shows only a transient contraction (Kuriyama et a1.,1975). They also respond differently from each other to prostaglandin Send offprint requests to: Megurni Moriya, Department of Physiology,Sapporo Medical College, S.1,
W.17 Sapporo 060, Japan * We are grateful to Dr. Y, Furukawa, Physical Section, Institute of Low Temperature Science, Hokkaido University,for help in the use of their film analyzingsystem. We are also indebted to the Department of Pathology of our college, for the use of a Photo Pattern Analyzer throughout this experiment.
0302-766X/79/0202/0337/$01.00
338
M. Moriya and E. Miyazaki
( W a t a n a b e , 1972; I s h i z a w a et al., 1973; M i s h i m a a n d K u r i y a m a , 1976), a n d to N a - f r e e s o l u t i o n a n d electrical s t i m u l a t i o n ( K u r i y a m a et al., 1975). T h e p u r p o s e o f the present study is to o b t a i n i n f o r m a t i o n on the fine structure o f the s m o o t h muscle cells in the l o n g i t u d i n a l a n d circular layers o f the guinea pig stomach.
Materials and Methods Guinea pigs weighing between 300 and 450 g were stunned and bled. The longitudinal and circular muscle strips (1.5-2 mm wide, 15-20 mm long) were dissected out at the corpus region and separated from the mucosal layer. Isotonic contraction was recorded by a kymograph. After 30 min equilibration in a modified Krebs solution, the muscle was relaxed by the addition of adrenaline (final concentration; 10- 5 M), and then prefixed by replacing the medium by warm fixative (see below). The change in length following fixation was negligible. The tissues were prefixed with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at room temperature for 2 h, washed with 0.1 M cacodylate buffer containing 0.2 M sucrose more than 1 h and then postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer containing 0.2 M sucrose at low temperature (0-4~C) for I h. For the purpose of better preservation of thick muscle filaments, the following buffer solutions were used. (1) 0.1 M cacodylate buffer, pH 6.6, (2) 0.1 M cacodylate buffer, pH 6.6 plus 10 mM MgC12, and (3) 0.1 M piperazine N-N'-bis[2 ethanol sulfonic acid (PIPES)], pH 7.6. The specimens were stained en bloc with 2 % uranyl acetate for 1 h, and embedded in Epon 8t 2. Thin sections cut transversely to the longitudinal axis of muscle cells were stained with 0.2 % lead citrate. The area and the perimeter of each cell examined (magnified to • 30,000) were estimated by means of a film analyzing system (Oosawa Sho-Kai, Model 514). Total area of sarcoplasmic reticulum (SR) and extracellular space were measured by a Photo Pattern Analyzer (Applied Electric Lab. Co Ltd, Model PPA-250).
Results and Discussion T h e muscle layer o f the guinea pig s t o m a c h at the c o r p u s region is a b o u t 0.2 m m thick. T h e outer, l o n g i t u d i n a l muscle l a y e r occupies one third o f the thickness, a n d consists o f a r o u n d 20 a r r a y s o f muscle cells. In the inner, circular layer, muscle cells are densely p a c k e d to f o r m large b u n d l e s a r r a n g e d side b y side. The extracellular space within each muscle b u n d l e was 12.1 + 1.0% in the l o n g i t u d i n a l layer, a n d 4.4 + 0.6 % in the circular layer. Nexuses were seen between some circular muscle cells (Fig. 1B), b u t n o t in the l o n g i t u d i n a l layer. T h e largest p a r t o f each s m o o t h muscle cell is o c c u p i e d by m y o f i l a m e n t s in b o t h tissues. T h e o t h e r organelles, i.e., m i t o c h o n d r i a , s a r c o p l a s m i c reticulum (SR) a n d m i c r o t u b u l e s are d i s p e r s e d a m o n g the m y o f i l a m e n t s . T h e m i t o c h o n d r i a are always a s s o c i a t e d with the SR or caveolae o f b o t h , a n d m i c r o t u b u l e s are l o c a t e d j u s t next to the SR. T h e greatest a m o u n t o f SR is seen at the p e r i p h e r y o f the cell where clusters o f caveolae are f o r m e d (Fig. 1). The percentage a r e a o f SR in the l o n g i t u d i n a l muscle cell is 4.67 _+ 1.25 (S.D.) (n = 112; 6 animals) which is twice t h a t in the circular muscle cell (2.24 +_ 0.61%, n = 120). The c o u n t o f m i c r o t u b u l e s per 1 g m z is also greater in the l o n g i t u d i n a l t h a n in the circular muscle cell ( 4 . 9 + 1.6 c o u n t s / g m 2 vs. 1.9+ 0.5, n - = 55 a n d 57, respectively; 3 animals). The l o n g i t u d i n a l muscle c o n t a i n s a b o u t 7 . 6 m i t o c h o n d r i a l profiles per 10 g m 2 cross sectional area, a n d the circular muscle a b o u t 4.5 (counts o f m o r e t h a n t 0 0 cells f r o m
Longitudinal and Circular Muscle Cells of Guinea Pig Stomach
339
Fig, 1AandB. Transverse sections of bundles of smooth muscle cells in guinea pig stomach. A Cells of longitudinal muscle layer; B cells of circular muscle layer. Note sarcoplasmic reticulum (SR) adjacent to caveolae (arrowheads), mitochondria, and microtubules (arrows). Thick filaments more distinct in B than A. Nexus (NX) in B. Fixation PIPES-buffered (pH 7.6) glutaraldehyde and OSO4, block-stained with uranyl acetate, x 20,000. Insets: Thick filaments within electron lucent areas, x 80,000
340
M. Moriya and E. Miyazaki
6 animals for each). The distribution of caveolae was almost the same in the two types of smooth muscle cells (about 1.9 caveolae/ixm of perimeter). It is evident from these results that except for the myofilaments, the longitudinal smooth muscle contains a higher proportion of every organelle than the circular muscle. The most striking difference between the longitudinal and the circular muscle cells is the appearance of the thick filaments. In spite of the application of exactly the same procedure, thick filaments could not be demonstrated as readily in the longitudinal as in the circular muscle cells. At higher magnification, the thick filaments are clearly distinguishable even in the longitudinal muscle cells with their unique location near the center of a round or oval, electron lucent area surrounded by thin filaments (Fig. 1A, insert). However, the thick filaments in the longitudinal muscle are much thinner than those in the circular muscle. The density of distribution of thick filaments is 25.3 + 8.4/0.5 ixm2 (n = 12) in the longitudinal, and 45.8 + 3.2 (n = 12) in the circular muscle cells. The use of lower-pH fixative (pH 6.6) which was reported to prevent myosin extraction from smooth muscle (Kelly and Rice, 1968; Sobieszek and Small, 1976; N o n o m u r a , 1977) gave a similar result in that the frequency of thick filaments in the longitudinal muscle cell was half of that in the circular muscle cell (28.9 + 10.8 vs. 5 7 . 2 + 5.0/0.5~tm2). An organic buffer, PIPES, presumed to retain much cytoplasmic detail during fixation (Baur and Stacey, 1977) yielded a more regular arrangement of thin filaments but did not make any difference as to the density of thick filaments compared with that following the use of cacodylate buffer, p H 7.4 (24.5 + 7.6/0.5 ~tm2 for longitudinal muscle; 46.4 + 7.0 for circular muscle). The circular muscle cell shows clear thick filaments abundantly distributed through the cytoplasm, although the density of the thick filaments, approximately 100/Bin 2, is still small compared to that of the portal anterior mesenteric vein of the rabbit and the portal vein of the guinea pig (160/gm2; Somlyo et al., 1973). The significance of the several structural differences observed on the contractile responses of the two muscle types compared is not yet understood. However, the fact that the longitudinal muscle cell contains a smaller amount of thick filaments than the circular muscle cell illustrates that in the guinea pig stomach the structural and functional difference of the two cell types concerns the contractile apparatus.
References Baur, P.S., Stacey, T.R.: The use of PIPES buffer in the fixation of mammalian and marine tissues for electron microscopy.J. Microsc. 109, 315-327 (1977) Ishizawa, M., Sakabe, K., Miyazaki,E.: Action of prostaglandin on gastrointestinalmotility in vivo and in vitro. Proc. Syrup. Chemical Physiologyand Pathology, 13, 53-56 (1973) Kelly, R.E., Rice, R.V.: Localization of myosin filaments in smooth muscle. J. Cell Biol. 37, 105-116 (1968) Kuriyama, H., Mishima, K., Suzuki, H.: Some differences in contractile responses of isolated longitudinal and circular muscle from the guinea pig stomach. J. Physiol. 251, 317-331 (1975) Mishima, K., Kuriyama, H.: Effects of prostaglandins on electrical and mechanical activities of the guinea pig stomach. Jpn. J. Physiol. 26, 537-548 (1976) Nonomura, Y.: Structure of smooth muscle. In: The function and structure of muscle (T. Sakai, M. Endo and H. Sugita eds.), pp. 149-163. Tokyo: Igaku Shoin 1977
Longitudinal and Circular Muscle Cells of Guinea Pig Stomach
341
Sobieszek, A., Small, J.V.: Myosin-linked calcium regulation in vertebrate smooth muscle. J. Mol. Biol. 102, 75-92 (1976) Somlyo, A.P., Devine, C.E., Somlyo, A.V., Rice, R.V.: Filament organization in vertebrate smooth muscle. Philos. Trans. R. Soc. Lond [Biol.] 265, 223-229 (1973) Watanabe, S.: Action of prostaglandin E and F types on the circular muscle preparations of the gastrointestinal tract from the guinea pig. The Sapporo Med. J. 41, 57-70 (1972) Accepted August 31, 1979
Cell Tissue Res. 202, 337-341 (1979)
9 by Springer-Verlag 1979
Short Communication
Structural Analysis of Functionally Different Smooth Muscles Megumi Moriya and Eisaku Miyazaki* Department of Physiology,Sapporo Medical College, Sapporo, Japan
Summary. The ultrastructure of the longitudinal and circular muscle cells of the guinea pig stomach which show different contractile responses was compared. The extracellular space within the muscle bundles is about 12.1 ~ in the longitudinal layer and about 4.4% in the circular layer. Nexuses were consistently found in the circular muscle layer but not in the longitudinal muscle layer. Numbers of both mitochondria and microtubules per unit area of smooth muscle cell are larger in the longitudinal than in the circular muscle. Cellular area occupied by sarcoplasmic reticulum is about 4.7% in the longitudinal muscle, 2.3 % in the circular muscle. The numbers of caveolae are almost the same in both tissues. The most distinct difference between the two types of smooth muscle is the appearance of the thick filaments. The circular muscle cell contains approximately 50thick filaments per 0.5~tm 2 of cytoplasmic area, while the longitudinal muscle cell has only about 25 filaments which were usually much thinner than those of the circular muscle. These results indicate that the contractile apparatus itself is different in longitudinal and circular smooth muscles of the guinea pig stomach.
Key words: Longitudinal m u s c l e - Circular muscle - Structural analysis - Thick filaments - Guinea pig stomach.
In response to K-depolarizing solution, the longitudinal muscle strip of the guinea pig stomach contracts with a small phasic phase followed by a large tonic phase, while the circular muscle strip shows only a transient contraction (Kuriyama et a1.,1975). They also respond differently from each other to prostaglandin Send offprint requests to: Megurni Moriya, Department of Physiology,Sapporo Medical College, S.1,
W.17 Sapporo 060, Japan * We are grateful to Dr. Y, Furukawa, Physical Section, Institute of Low Temperature Science, Hokkaido University,for help in the use of their film analyzingsystem. We are also indebted to the Department of Pathology of our college, for the use of a Photo Pattern Analyzer throughout this experiment.
0302-766X/79/0202/0337/$01.00
338
M. Moriya and E. Miyazaki
( W a t a n a b e , 1972; I s h i z a w a et al., 1973; M i s h i m a a n d K u r i y a m a , 1976), a n d to N a - f r e e s o l u t i o n a n d electrical s t i m u l a t i o n ( K u r i y a m a et al., 1975). T h e p u r p o s e o f the present study is to o b t a i n i n f o r m a t i o n on the fine structure o f the s m o o t h muscle cells in the l o n g i t u d i n a l a n d circular layers o f the guinea pig stomach.
Materials and Methods Guinea pigs weighing between 300 and 450 g were stunned and bled. The longitudinal and circular muscle strips (1.5-2 mm wide, 15-20 mm long) were dissected out at the corpus region and separated from the mucosal layer. Isotonic contraction was recorded by a kymograph. After 30 min equilibration in a modified Krebs solution, the muscle was relaxed by the addition of adrenaline (final concentration; 10- 5 M), and then prefixed by replacing the medium by warm fixative (see below). The change in length following fixation was negligible. The tissues were prefixed with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at room temperature for 2 h, washed with 0.1 M cacodylate buffer containing 0.2 M sucrose more than 1 h and then postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer containing 0.2 M sucrose at low temperature (0-4~C) for I h. For the purpose of better preservation of thick muscle filaments, the following buffer solutions were used. (1) 0.1 M cacodylate buffer, pH 6.6, (2) 0.1 M cacodylate buffer, pH 6.6 plus 10 mM MgC12, and (3) 0.1 M piperazine N-N'-bis[2 ethanol sulfonic acid (PIPES)], pH 7.6. The specimens were stained en bloc with 2 % uranyl acetate for 1 h, and embedded in Epon 8t 2. Thin sections cut transversely to the longitudinal axis of muscle cells were stained with 0.2 % lead citrate. The area and the perimeter of each cell examined (magnified to • 30,000) were estimated by means of a film analyzing system (Oosawa Sho-Kai, Model 514). Total area of sarcoplasmic reticulum (SR) and extracellular space were measured by a Photo Pattern Analyzer (Applied Electric Lab. Co Ltd, Model PPA-250).
Results and Discussion T h e muscle layer o f the guinea pig s t o m a c h at the c o r p u s region is a b o u t 0.2 m m thick. T h e outer, l o n g i t u d i n a l muscle l a y e r occupies one third o f the thickness, a n d consists o f a r o u n d 20 a r r a y s o f muscle cells. In the inner, circular layer, muscle cells are densely p a c k e d to f o r m large b u n d l e s a r r a n g e d side b y side. The extracellular space within each muscle b u n d l e was 12.1 + 1.0% in the l o n g i t u d i n a l layer, a n d 4.4 + 0.6 % in the circular layer. Nexuses were seen between some circular muscle cells (Fig. 1B), b u t n o t in the l o n g i t u d i n a l layer. T h e largest p a r t o f each s m o o t h muscle cell is o c c u p i e d by m y o f i l a m e n t s in b o t h tissues. T h e o t h e r organelles, i.e., m i t o c h o n d r i a , s a r c o p l a s m i c reticulum (SR) a n d m i c r o t u b u l e s are d i s p e r s e d a m o n g the m y o f i l a m e n t s . T h e m i t o c h o n d r i a are always a s s o c i a t e d with the SR or caveolae o f b o t h , a n d m i c r o t u b u l e s are l o c a t e d j u s t next to the SR. T h e greatest a m o u n t o f SR is seen at the p e r i p h e r y o f the cell where clusters o f caveolae are f o r m e d (Fig. 1). The percentage a r e a o f SR in the l o n g i t u d i n a l muscle cell is 4.67 _+ 1.25 (S.D.) (n = 112; 6 animals) which is twice t h a t in the circular muscle cell (2.24 +_ 0.61%, n = 120). The c o u n t o f m i c r o t u b u l e s per 1 g m z is also greater in the l o n g i t u d i n a l t h a n in the circular muscle cell ( 4 . 9 + 1.6 c o u n t s / g m 2 vs. 1.9+ 0.5, n - = 55 a n d 57, respectively; 3 animals). The l o n g i t u d i n a l muscle c o n t a i n s a b o u t 7 . 6 m i t o c h o n d r i a l profiles per 10 g m 2 cross sectional area, a n d the circular muscle a b o u t 4.5 (counts o f m o r e t h a n t 0 0 cells f r o m
Longitudinal and Circular Muscle Cells of Guinea Pig Stomach
339
Fig, 1AandB. Transverse sections of bundles of smooth muscle cells in guinea pig stomach. A Cells of longitudinal muscle layer; B cells of circular muscle layer. Note sarcoplasmic reticulum (SR) adjacent to caveolae (arrowheads), mitochondria, and microtubules (arrows). Thick filaments more distinct in B than A. Nexus (NX) in B. Fixation PIPES-buffered (pH 7.6) glutaraldehyde and OSO4, block-stained with uranyl acetate, x 20,000. Insets: Thick filaments within electron lucent areas, x 80,000
340
M. Moriya and E. Miyazaki
6 animals for each). The distribution of caveolae was almost the same in the two types of smooth muscle cells (about 1.9 caveolae/ixm of perimeter). It is evident from these results that except for the myofilaments, the longitudinal smooth muscle contains a higher proportion of every organelle than the circular muscle. The most striking difference between the longitudinal and the circular muscle cells is the appearance of the thick filaments. In spite of the application of exactly the same procedure, thick filaments could not be demonstrated as readily in the longitudinal as in the circular muscle cells. At higher magnification, the thick filaments are clearly distinguishable even in the longitudinal muscle cells with their unique location near the center of a round or oval, electron lucent area surrounded by thin filaments (Fig. 1A, insert). However, the thick filaments in the longitudinal muscle are much thinner than those in the circular muscle. The density of distribution of thick filaments is 25.3 + 8.4/0.5 ixm2 (n = 12) in the longitudinal, and 45.8 + 3.2 (n = 12) in the circular muscle cells. The use of lower-pH fixative (pH 6.6) which was reported to prevent myosin extraction from smooth muscle (Kelly and Rice, 1968; Sobieszek and Small, 1976; N o n o m u r a , 1977) gave a similar result in that the frequency of thick filaments in the longitudinal muscle cell was half of that in the circular muscle cell (28.9 + 10.8 vs. 5 7 . 2 + 5.0/0.5~tm2). An organic buffer, PIPES, presumed to retain much cytoplasmic detail during fixation (Baur and Stacey, 1977) yielded a more regular arrangement of thin filaments but did not make any difference as to the density of thick filaments compared with that following the use of cacodylate buffer, p H 7.4 (24.5 + 7.6/0.5 ~tm2 for longitudinal muscle; 46.4 + 7.0 for circular muscle). The circular muscle cell shows clear thick filaments abundantly distributed through the cytoplasm, although the density of the thick filaments, approximately 100/Bin 2, is still small compared to that of the portal anterior mesenteric vein of the rabbit and the portal vein of the guinea pig (160/gm2; Somlyo et al., 1973). The significance of the several structural differences observed on the contractile responses of the two muscle types compared is not yet understood. However, the fact that the longitudinal muscle cell contains a smaller amount of thick filaments than the circular muscle cell illustrates that in the guinea pig stomach the structural and functional difference of the two cell types concerns the contractile apparatus.
References Baur, P.S., Stacey, T.R.: The use of PIPES buffer in the fixation of mammalian and marine tissues for electron microscopy.J. Microsc. 109, 315-327 (1977) Ishizawa, M., Sakabe, K., Miyazaki,E.: Action of prostaglandin on gastrointestinalmotility in vivo and in vitro. Proc. Syrup. Chemical Physiologyand Pathology, 13, 53-56 (1973) Kelly, R.E., Rice, R.V.: Localization of myosin filaments in smooth muscle. J. Cell Biol. 37, 105-116 (1968) Kuriyama, H., Mishima, K., Suzuki, H.: Some differences in contractile responses of isolated longitudinal and circular muscle from the guinea pig stomach. J. Physiol. 251, 317-331 (1975) Mishima, K., Kuriyama, H.: Effects of prostaglandins on electrical and mechanical activities of the guinea pig stomach. Jpn. J. Physiol. 26, 537-548 (1976) Nonomura, Y.: Structure of smooth muscle. In: The function and structure of muscle (T. Sakai, M. Endo and H. Sugita eds.), pp. 149-163. Tokyo: Igaku Shoin 1977
Longitudinal and Circular Muscle Cells of Guinea Pig Stomach
341
Sobieszek, A., Small, J.V.: Myosin-linked calcium regulation in vertebrate smooth muscle. J. Mol. Biol. 102, 75-92 (1976) Somlyo, A.P., Devine, C.E., Somlyo, A.V., Rice, R.V.: Filament organization in vertebrate smooth muscle. Philos. Trans. R. Soc. Lond [Biol.] 265, 223-229 (1973) Watanabe, S.: Action of prostaglandin E and F types on the circular muscle preparations of the gastrointestinal tract from the guinea pig. The Sapporo Med. J. 41, 57-70 (1972) Accepted August 31, 1979
