Cytoskeletal features in longitudinal and circular smooth muscles during development of the rat portal vein

Immunohistochemistry of -smooth muscle actin and desmin, two markers of smooth muscle cell differentiation, and electron-microscopic observation of thick filaments of myosin were performed on the media of the developing rat hepatic portal vein to gain insights into the chronology of differentiation of its longitudinal and circular smooth muscles. In accordance with the ultrastructural distribution of thin filaments, staining of -smooth muscle actin is lightly positive in the myoblasts at postnatal day 1 and then extends in probably all muscle cells of the developing vessel. Desmin, which appears later than -smooth muscle actin in the two muscles, is distributed throughout the longitudinal layer at day 8, whereas the first arrangements of thick filaments are detectable in most longitudinal muscle cells; at this stage, desmin and thick filaments are absent from the poorly differentiated circular muscle cells. The longitudinal muscle cells differentiate in a strikingly synchronized way from day 8 onwards, conferring a homogeneous structure to the developing and mature longitudinal layer. Several desmin-positive cells and a heterogeneous distribution of thick filaments occur in the circular muscle at day 14; the subsequent extension of these filaments in this layer results in a persisting heterogeneous distribution in the young 7-week-old adult. Many features of the mature smooth muscle cells are established within the third week in the longitudinal muscle, approximately one week before those of the circular layer. These results are consistent with the function of the longitudinal muscle as a spontaneously contractile smooth muscle unit, and emphasize the need for its fast maturation to fulfil its major role in the control of portal blood flow.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Force-velocity characteristics of stomach muscle: a comparison between longitudinal and circular muscle strips

The longitudinal and circular muscle preparations from guinea pig stomach were compared in their property of shortening. The highest shortening speed was attained only for about 1 sec for longitudinal muscle and about 2 sec for circular muscle from the onset of stimulation in the course of tetanic contraction. Dynamic constants calculated from a force-velocity curve were almost independent of the muscle length, being set near its optimum length. Mean dynamic constants of the longitudinal muscle were: Vmax: 0.205 L/sec, b: 0.056 L/sec, a/Po: 0.292 and that of the circular muscle were: Vmax: 0.576 L/sec, b: 0.056 L/sec, a/Po: 0.107. The difference in Vmax of these muscles are discussed along with the difference in ultrastructure of the contractile filaments.     

缩胆囊素和促胰液素对豚鼠离体胃平滑肌运动的影响

用８个肌槽同时记录豚鼠胃不同部位肌条的收缩活动,以观察八肽缩胆囊素（ＣＣＫ－８）和促胰液素的影响。结果表明：ＣＣＫ－８能（１）增高各部位纵行肌和环行肌的张力;（２）加快胃体纵行肌,胃窦纵行肌、环行肌和幽门环行肌的收缩频率;（３）增大胃窦环行肌收缩波平均振幅和（４）增加幽门环行肌收缩波运动指数,但减少胃体和胃窦纵行肌收缩波平均振幅。上述作用均不能被阿托品和消炎病所阻断。而促胰液素对各部位肌条的收缩活动没有明显的影响。     

Gap junctions of the muscles of the small and large intestine

Summary The distribution of gap junctions (nexuses) in various parts of the small and large intestines of the guinea-pig was studied using the freeze-fracture technique and in thin sections. The percentage area of smooth muscle cell surface occupied by gap junctions varies from 0.50% in the circular muscle of the duodenum to zero in the longitudinal muscle of the ileum. In the circular muscle of the jejunum and ileum the area occupied by nexuses is 0.22% (or about 11 m² per cell). The sizes of junctions range from less than 0.01 m² to 0.20 m², with two-thirds of them being smaller than 0.05 m². In the colon, gap junctions are rare, very small and confined to the circular muscle layer. Even the smallest aggregates of intramembrane particles correspond to areas of close apposition between the membranes of adjacent cells; it is therefore justified to interpret them as being gap junctions. Some gap junctions are formed between a smooth muscle cell and an interstitial cell. Gap junctions are not found in the longitudinal muscle of the small intestine; this is in sharp contrast to the abundance of gap junctions in the adjacent circular layer.In the small intestine of cats and rabbits, gap junctions are abundant in the circular muscle layer, whereas they are very small in size and very few in number in the longitudinal muscle layer.The authors wish to thank Mr Peter Trigg and Miss Eva Franke for help and support. This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

Fine structure of the nephridial muscle layer cells of Phascolosoma granulatum (Sipuncula)

The nephridial muscle layer of Phascolosoma granulatum consists of a network of longitudinal and circular cells separated by connective tissue matrix. The muscle fibers are densely packed with thick and thin myofilaments, among which are scattered cytoplasmic dense bodies. The nucleus and noncontractile cytoplasmic organelles occupy a lateral projection from the contractile portion of the fiber. Cytoplasmic dense bodies are the result of a clustering of an indeterminate number of the thin actin filaments that fill the cytoplasm between thick filaments. Attached to the cytoplasmic face of the cell membrane are membrane-associated electron-dense plaques. These sites are linked to the contractile myofilaments by narrow filamentous bridges. Extracellular narrow filaments extend from these plaques to collagen fibers of the connective tissue matrix. Differences in length of the dense plaques may be related to differences in thick myofilament diameter in three types of muscle fiber, types A, B and C, statistically distinguished by mean fiber size differences. The plaques may serve as connecting links for the transmission of tension from contractile units to the connective tissue of the muscle layer. © 1993 Wiley-Liss, Inc.     

Ultrastructurally different muscle cell types in Eisenia foetida (Annelida,Oligochaeta)

Muscles in the body wall, intestinal wall, and contractile hemolymphatic vessels (pseudohearts) of an oligochaete anelid (Eisenia foetida) were studied by electron microscopy. The muscle cells in all locations, except for the outer layer of the pseudohearts, are variants of obliquely striated muscle cells. Cells comprising the circular layer of the body wall possess single, peripherally located myofibrils that occupy most of the cytoplasm and surround other cytoplasmic organelles. The nuclei of the cells lie peripherally to the myofibrils. The sarcomeres consist of thin and thick myofilaments that are arranged in parallel arrays. In one plane of view, the filaments appear to be oriented obliquely to Z bands. Thin myofilaments measure 5–6 nm in diameter. Thick myofilaments are fusiform in shape and their width decreases from their centers (40–45 nm) to their tips (23–25 nm). The thin/thick filament ratio in the A bands is 10. The Z bands consist of Z bars alternating with tubules of the sarcoplasmic reticulum. Subsarcolemmal electron-dense plaques are found frequently. The cells forming the longitudinal layer of the body wall musculature are smaller than the cells in the circular layer and their thick filaments are smaller (31–33 nm centrally and 21–23 nm at the tips). Subsarcolemmal plaques are less numerous. The cells forming the heart wall inner layer, the large hemolymphatic vessels, and the intestinal wall are characterized by their large thick myofilaments (50–52 nm centrally and 27–28 nm at the tips) and abundance of mitochondria. The cells forming the outer muscular layer of the pseudohearts are smooth muscle cells. These cells are richer in thick filaments than vertebrate smooth muscle cells. They differ from obliquely striated muscle cells by possessing irregularly distributed electron-dense bodies for filament anchorage rather than sarcomeres and Z bands and by displaying tubules of smooth endoplasmic reticulum among the bundles of myofilaments. © 1995 Wiley-Liss, Inc.     

Primary Structure and Tissue Distribution of Guinea Pig Gastrin-Releasing Peptide

The primary structure of gastrin-releasing peptide from the guinea pig stomach has been determined by automated Edman degradation and shown to be identical to porcine gastrin-releasing peptide. Extracts of guinea pig brain and small intestine contained both gastrin-releasing peptide and its COOH-terminal decapeptide (neuromedin C) but the stomach extracts contained only gastrin-releasing peptide. Within the small intestine, highest concentrations of gastrin-releasing peptide-like immunoreactivity were found in extracts of the circular and longitudinal smooth muscle layers.     

Vagal inhibition in the antral region of guinea pig stomach

The effects of vagal stimulation in the presence of a muscarinic antagonist were examined on three distinct rhythmically active cells located in guinea pig antrum. Vagal stimulation inhibited contractions of the circular muscle layer but did not change their rate of occurrence. With the use of intracellular recording techniques, these stimuli were found to initiate inhibitory junction potentials in the circular layer but produced smaller potential changes in driving and follower cells. Inhibition of the circular muscle layer involved two separate components. The dominant component was independent of changes in membrane potential and was abolished by nitro-L-arginine. After abolishing Ca(2+) entry into smooth muscle cells with a Ca(2+) antagonist, vagal stimulation continued to inhibit the residual contractions associated with each slow wave. When the cyclic changes in intracellular Ca(2+) concentration associated with each slow wave were measured, they were found to be unchanged by vagal stimulation. The observations suggest that vagal inhibition of stomach movements does not alter pacemaker activity in the stomach; rather, it results from a change in the sensitivity of smooth muscle contractile proteins to Ca(2+).     

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the chemical coding of each class of ICs.     

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Hypertrophic smooth muscle

The smooth muscle cells of the circular musculature of the guinea pig ileum are connected by gap junctions (nexuses) which occupy about 0.21% of the cell surface. When the muscle hypertrophies in the portions of the ileum oral to an experimental stenosis, the muscle cells increase in size and number. Gap junctions become markedly larger than in control muscles and occupy 0.49% of the cell surface. While the cells double their surface area, the number of nexuses per unit surface remains unchanged (47--48 per 1000 microns2). The packing density of intramembrane particles (or pits) in the nexuses of hypertrophic muscle cells is 6700 . microns-2, which is slightly less than in control muscle cells (7200 . microns-2). A characteristic grouping of the particles (or the pits) within the nexus is often observed. Nexuses between two processes originating from the same cell are common. Nexuses do not occur in the longitudinal muscle.     

Scanning electron microscopy of the muscle coat of the guinea-pig small intestine

Summary We have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) stretch preparations of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.     

Fine structure of the mammalian renal capsule: The atypical smooth muscle cell and its functional meaning

Summary The present electron microscopic study on the fine structure of the renal capsule of some mammals (mouse, rat, mole, guinea pig and rabbit) shows that, although there are some variations in the structure, the general morphology is the same.The renal capsule of these animals consists of two layers, a connective tissue layer and an atypical smooth muscle cell layer, and is bound to the renal parenchyma by a thin peritubular loose connective tissue. The atypical smooth muscle cell is characterized by the existence of fine cytoplasmic filaments usually arranged along the long axis of the cell, and the cells also show a complicated interlocking among adjacent cells. The atypical smooth muscle cells gradually undergo a transition to fibroblasts of the upper connective tissue layer, losing their similarities to smooth muscle cells.When intrarenal pressure is elevated and the renal capsule is distended, the intercellular space among interdigitating or overlapping atypical smooth muscle cells is extensively dilated.Tracers such as horseradish peroxidase and ferritin injected intravenously or intraperitoneally can transverse the renal capsule.From the present study, it is concluded that the renal capsule of mammals possesses common structures, and contains atypical smooth muscle cells. These morphological characteristics suggest that the renal capsule could play a certain role related to the renal function.The author wishes to acknowledge the helpful advices of Prof. T. Yamamoto     

Spawning period and migration of three species of cyprinids in a stream with Mediterranean regimen (SW Spain)

These studies investigated the effects of somatostatin on gastric motility in the rainbow trout. Two experimental models were used, the isolated vascularly-perfused stomach and isolated strips of gastrointestinal smooth muscle. Both models demonstrated that somatostatin can inhibit gastrointestinal motility and may therefore modulate gastric emptying in fish.
In the vascularly-perfused stomach, somatostatin (10–1000 n m ) decreased maximum and baseline intragastric pressure by 10–20% in the presence of stimulatory doses of carbachol or 5-hydroxytryptamine. In addition, somatostatin (1 μ m ) inhibited by 50% the magnitude of spontaneous contractions generated by distension. Somatostatin had little effect on the pressure gradient or contractile frequency. These results suggest that somatostatin may negatively modulate gastric emptying in the rainbow trout.
In isolated gastric smooth muscle strips, somatostatin (100 pmol) inhibited tension stimulated by carbachol (circular orientation of muscle) or 5-hydroxytryptamine (longitudinal orientation). These results correlated with those observed in the vascularly perfused stomach preparation. Somatostatin also decreased tension stimulated by carbachol and 5-hydroxytryptamine in intestinal smooth muscle strips, suggesting that under some conditions somatostatin could increase gastric emptying rate by relaxing intestinal musculature.     

Ultrastructure and differentiation of the larval esophageal muscle cells of the starfish Pisaster ochraceus

The muscle cells that cause constriction of the starfish larval esophagus (esophageal muscle cells) are one of the first cell types to express their differentiated morphological characteristics during development. Ultrastructurally these muscle cells resemble vertebrate and invertebrate smooth muscles. They contain a nucleus, a Golgi apparatus, contractile myofilaments, hemidesmosome-like structures, and what appears to be a simple sarcoplasmic reticulum. In asteroid embryos, this muscle layer originates during mouth formation when mesenchyme cells migrate from the tips of the coeloms to the esophagus. Once there, they elongate, forming processes. Over the next few days, the processes become filled with arrays of longitudinally arranged thick and thin myofilaments and thin sacs of smooth endoplasmic reticulum. The latter appear between the bundles of contractile filaments and the cell membranes. Contractile activity begins at approximately this time. The cisternae may represent a sarcoplasmic reticulum that is required for contraction. The majority of the esophageal muscle cell processes extend around the circumference of the developing esophagus, but occasional cells may be oriented in other directions. The latter cells are always farther away from the basal lamina and probably have little or no contact with it. Contact with basal lamina may serve to direct the migration of the cells and the orientation of the processes. J. Morphol. 237:1–18, 1998. © 1998 Wiley-Liss, Inc.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Morphological changes during ontogeny of the canine proximal colon

The development of the canine proximal colon from the completion of organogenesis through 43 days after birth was studied using light microscopy, immunofluorescence and electron microscopy. During this period the tunica muscularis increased in thickness from 42±6 m in animals midway through the gestation period to 317±29 m in animals 25–30 days old. This increase in thickness resulted from an increase in the number and size of smooth muscle cells in the circular and longitudinal muscle layers. The cross-sectional thickness of the circular muscle layer increased from 10±2 smooth muscle cells midway through the gestation period to 92±7 cells in animals 25–30 days old. The longitudinal layer increased in thickness from 1.5±1 cells in animals midway through the gestation period to 44±2 cells in animals 25–30 days old. Smooth muscle cells from both layers also increased in diameter and length. Ultrastructural and immunohistochemical studies suggested that many of the smooth muscle cells were undergoing development throughout the fetal period. Midway through the gestation period, the circular layer was positive for desmin-like immunoreactivity (D-LI), while both the circular and longitudinal layers were positive for vimentinlike immunoreactivity (V-LI). By birth, V-LI was suppressed in the circular and longitudinal layers, and both layers expressed D-LI. The enteric nervous system was already established midway through the gestation period, and submucosal and myenteric ganglia could be identified, although the chemical coding and mature morphology of neurons were incomplete. NADPH-diaphorase-positive neurons, indicating the expression of nitric oxide synthase, developed by the time of birth. Interstitial cells of Cajal (IC) could not clearly be identified midway through gestation, however, potential precursors to ICs were observed. Several classes of ICs were identifiable at birth.     

Models of contractile units and their assembly in smooth muscle

It is believed that the contractile filaments in smooth muscle are organized into arrays of contractile units (similar to the sarcomeric structure in striated muscle), and that such an organization is crucial for transforming the mechanical activities of actomyosin interaction into cell shortening and force generation. Details of the filament organization, however, are still poorly understood. Several models of contractile filament architecture are discussed here. To account for the linear relationship observed between the force generated by a smooth muscle and the muscle length at the plateau of an isotonic contraction, a model of contractile unit is proposed. The model consists of 2 dense bodies with actin (thin) filaments attached, and a myosin (thick) filament lying between the parallel thin filaments. In addition, the thick filament is assumed to span the whole contractile unit length, from dense body to dense body, so that when the contractile unit shortens, the amount of overlap between the thick and thin filaments (i.e., the distance between the dense bodies) decreases in exact proportion to the amount of shortening. Assembly of the contractile units into functional contractile apparatus is assumed to involve a group of cells that form a mechanical syncytium. The contractile apparatus is assumed malleable in that the number of contractile units in series and in parallel can be altered to accommodate strains on the muscle and to maintain the muscle's optimal mechanical function.