Structural changes in smooth muscle cells during isotonic contraction

Summary Smooth muscle cells of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000 · mm^-2 to 18,000 · mm^-2. The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted cells are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells; on the other hand, they run obliquely and transversely around the isotonically contracted cells.This work is supported by the Medical Research Council. I thank Miss E.M. Franke and Mr S.J. Sarsfield for excellent technical assistance     

Arrangement of smooth muscle cells and intramuscular septa in the taenia coli

Summary Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10 nm) filaments; about 50% of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5° with the septa. In muscles fixed during isotonic contraction the angle increases to about 20–22°, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4–6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission.I thank Mr. S.J. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council     

The fine structure of smooth muscle cells and their relationship to connective tissue in the rabbit portal vein

Summary The smooth muscle of rabbit portal vein was studied by electron microscopy with particular emphasis on the mechanical linkage between the muscle cells and on the distribution of connective tissue.The media of this vein is composed of inner circular and outer longitudinal muscle layers which are orientated almost perpendicularly to each other. The muscle of the inner circular layer shows very irregular contours with much branching and anastomosing of the cytoplasmic processes, which often make membrane contacts with neighbouring cells to form an extensive network of cytoplasmic processes. The muscle cells of the outer longitudinal layer are arranged in densely packed bundles and are spindle-shaped, with no branching processes. Opposing dense areas from neighbouring cells, with variable gap distances (30–100 nm) and close membrane contacts (intermediate junctions) with a gap of 11 nm were observed in both circular and longitudinal muscle layers.In the terminal regions of muscle cells in both circular and longitudinal layers a specialized anchoring structure was present which was closely related to extracellular elastic tissue. Muscle cells in the longitudinal layer showed the most elaborate structure, the tapering end of the muscle cell showing a honeycomb-like structure penetrated by columns of connective tissue compounds. The functional implications of these structures are discussed.     

Effect of stretch and contraction on caveolae of smooth muscle cells

Summary The number of caveolae present at the surface of smooth muscle cells of guinea-pig taenia coli and visualized by freeze-fracture is about 35 per m². (By comparison, endothelial cells of intramuscular capillaries have about 73 caveolae per m².) The packing density of smooth muscle caveolae is not significantly different in muscle strips isotonically contracted with carbachol or stretched and relaxed in a calcium-free solution, under a range of loads varying from 1 to 15 g. Also the diameter of the fractured necks of the caveolae appears unchanged in all the experimental conditions tested. The plasma membrane of smooth muscle cells often shows a ring of intramembranous particles rimming the opening of a caveola; on the other hand, particles are rare in the membrane of the caveolae themselves. The close relation between caveolae and sarcoplasmic reticulum is readily visualized in freeze-fracture preparations. Characteristic changes of the cell surface shape accompany the contraction and relaxation of the muscle. On rare occasions small aggregates of intramembranous particles are found and it is possible that they represent punctate gap junctions. However, the characteristic clusters of particles found in the circular musculature of the caecum and ileum are not seen in taenia coli. Acknowledgements. We thank Simon Sarsfield and Eva Franke for excellent technical assistance. The work is supported by grants from the Medical Research Council     

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     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

中药甘草对大鼠肠平滑肌运动的影响

目的观察甘草对大鼠肠道平滑肌运动的影响,了解甘草与胃肠运动之间的关系。方法实验分成正常组、甘草制成煎剂4．8、16、32、64g／kg剂量组,每日1次灌胃给药。末次给药1h后观察大鼠胃半排时间;采用灌胃给予炭末,测定胃推进率。结果甘草煎剂剂量大小对大鼠小肠蠕动有直接的影响,较小剂量对大鼠小肠的推进功能有抑制作用,较大剂量对大鼠的肠推进有促进作用。     

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.     

The development and maintenance of smooth muscle in control and aneurogenic amphibians (Ambystoma)

Summary Ultrastructural investigations showed that development and maintenance of smooth muscle was similar in control and aneurogenic amphibian larvae. This applies to both multi-unit and unitary smooth muscles. The gut musculature displayed a regional variation in smooth muscle morphology and a variety of intermuscular appositions even under conditions of nervelessness.Supported by NIH grant # AM 15732-04 and funds from the Muscular Dystrophy Association of America.The author thanks Ms. Shirley June for her excellent technical assistance and Mr. H. Popiela for preparation of some of the specimens.     

Further observations on upper urinary tract smooth muscle

Summary Light and electron microscopic techniques have been employed to study the arrangement and distribution of two types of muscle in the upper urinary tract of the rat. An outer layer of cells has been identified in the wall of the renal calix and pelvis. These cells are separated by connective tissue but possess numerous processes which make close contacts with adjacent cells. A layer of similar cells has not been observed in the wall of the upper ureter. The inner layer of muscle in the calix and pelvis is composed of larger cells similar to and apparently continuous with ureteric muscle. These cells are closely related to one another without intervening connective tissue and possess numerous bundles of myofilaments which extend along the length of the cell. The two types of muscle are closely related and, in the junctional region, cells of the outer layer are arranged along the length and make close contacts with one or more of the inner smooth muscle cells. A quantitative estimation has been made of nerve bundles associated with smooth muscle forming the outer layer of the calix and pelvis and with the muscle of the ureter. The results have shown a five fold increase in nerves associated with the caliceal muscle when compared with the ureter. The results are discussed in relation to the concept of a ureteric pacemaker.The authors wish to thank Professor G. A. G. Mitchell for his useful advice and encouragement.     

Human smooth muscle cell cultures of the stomach morphologic and biochemical studies

Summary Smooth muscle cell cultures were prepared from stomach explants obtained surgically from 10 patients with duodenal ulcer. The cultured cells grew in either overlapping layers in “hills and valleys” or in parallel arrays. The ultrastructure studies showed plasmalemmal vesicles, bundles of myofilaments associated with dense bodies, and gap junctions. The synthesis of contractile proteins illustrated the preponderance of actin on myosin and tropomyosin. The synthesis of contractile proteins in stomach smooth muscle cell cultures is significantly higher than in skin fibroblast cultures, i.e. 20 x higher for myosin, 10 x higher for actin, and 30 x higher for tropomyosin.     

Solubility-determining domain of smooth muscle myosin rod

Chymotryptic digestion of chicken gizzard light meromyosin (LMM) produced a 72 kDa core fragment, which was fully soluble at 150 mM KCl, pH 6.5–7.5. The fragment showed weak self-association at 50 mM KCl. The homology of the N-terminus amino acid sequence of this fragment with the sequence of the rabbit skeletal myosin rod suggested that the N-terminus of the core fragment originated 5 kDa from the hinge common to both smooth and skeletal myosin rod. Sedimentation experiments indicated that the domain specifying the insolubility of the intact LMM was 13 kDa long. Progressive proteolytic shortening of this region produced LMM fragments of progressively increasing solubility. Electron microscopy of segments formed from full-length LMM and from LMM core suggested that this 13 kDa domain specified the 43 nm parallel and antiparallel molecular overlaps characteristic of self-assembled intact myosin.     

Ultrastructural comparison of smooth muscle from ovarian follicle and oviduct of the golden hamster

Summary Ultrastructural characteristics of smooth muscle taken from ovarian follicles and oviducts of hamsters are compared. Differences between the two muscle types are more quantitative than qualitative, thus confirming that follicular muscle is a true smooth muscle with no unique characteristics. While both muscle types contain 50–80 Å filaments, -glycogen deposits, and organelles characteristically found in smooth muscle, the oviductal cells have substantially more sacs, tubular structures, sarcoplasmic reticulum, and mitochondria. Another difference concerns the cellular junctions; the oviductal cells exhibit nexuses, whereas the follicular cells show desmosomelike junctions. Based on ultrastructural differences, follicular smooth muscle seems to be a relatively toneless muscle suited for short, infrequent contractions, whereas oviductal smooth muscle is probably involved in more active tonic contractions.Supported by an Institutional Research Grant from Texas Women's University, by NIH Grant HD 12988, and by the Department of Anatomy at Wright State University     

Proteomic dataset of mouse aortic smooth muscle cells

In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.     

The ultrastructural effects of triparanol on rat atrial muscle

Summary The atrial musculature of rats given the cholesterol inhibitor triparanol (MER/29) (250 mg/kg daily) for 8 days was examined under the electron microscope and compared with that from untreated animals. The sarcoplasmic core of muscle fibers from animals given triparanol exhibited a new formation of sarcoplasmic granules which displayed a crystalline latticework with opaque lines approximately 40–60 Å separated by clear spaces 50–70 Å. They were partially or completely surrounded by a membrane. The crystalline bodies in cardiac muscle fibers were not as numerous as those observed in adrenocortical, testicular interstitial, or luteal cells as reported earlier by the investigators.This research was supported by USPHS Grants HE 12751, NS 05665, and 00690.Recipient of Career Research Development Award 1 K 3 GM 28064.     

Functional architecture of the SR calcium store in uterine smooth muscle

Sarcoplasmic reticulum (SR) is abundant in uterine smooth muscle cells. The functional role of this organelle in the regulation of uterine myocytes is not fully understood. The data available in the literature suggest that SR plays a dual role: as a source of calcium and as a calcium sink shaping calcium transients produced by membrane depolarisation and uterotonic agonists. Advances in digital imaging techniques including confocal microscopy of isolated living cells, and the development of methods for direct measurement of intraluminal calcium, has triggered a substantial increase in the number of publications elucidating the role of intracellular stores in calcium signalling. In this paper we review the literature and our own work on the SR calcium store in uterine smooth muscle cells.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

Viscoelastic properties of isolated rat colon smooth muscle cells

The measurement of the biomechanical properties of gastrointestinal smooth muscle cells is important for the basic understanding of digestive function and the interaction of muscle cells with the matrix. Externally applied forces will deform the cells depending upon their mechanical properties. Hence, the evoked response mediated through stretch-sensitive ion-channels in the smooth muscle cell membrane will depend upon membrane properties and the magnitude of the external force. The aim of this study was to test the hypothesis that gastrointestinal smooth muscle cells behave in a viscoelastic manner. Smooth muscle cells were dissociated from the muscle layers of the descending colon. The viscoelastic properties of the isolated cells were characterized by measuring the mechanical deflection response of the cell membrane to a negative pressure of 1cm H(2)O applied across the cell through a micropipette and fitting the response to a theoretical viscoelastic solid model. The viscoelastic mechanical constants of the isolated cells (N=9) were found to be as follows: k(1)=19.99+/-2.86 Pa, k(2)=7.19+/-1.21 Pa, mu=25.36+/-6.14 Pas and tau=4.84+/-0.95 s. This study represents, to the best of our knowledge, the first quantitative mechanical properties of isolated living smooth muscle cells from the gastrointestinal tract. The mechanical properties determined in this study will be of use in future analytical and numerical smooth muscle cell models to better predict the mechanism between the magnitude of mechanical stimuli, mechanosensitivity and the evoked afferent responses.     

Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli

A quantitative study of muscle cells of the guinea-pig taenia coli is reported. Stereological methods were used on electron micrographs and phase contrast micrographs. Smooth muscle cells of taeniae fixed under 1 gram load were about 515 m long. Muscle cell volume was about 3,500 m³ and cell surface 5,300 m². About 168,000 caveolae were found at the surface of each muscle cell, covering about 29 percent of its surface. They produced a 73 percent increase of the cell membrane compared to a smooth-surfaced cell. The ratio surface-to-volume is about 10.67 if the geometrical surface is considered, or 10.39 if the total surface of the cell membrane (including the caveolae) is considered. Mitochondria constituted 3.5–4 percent of the cell volume. A few nexuses were observed, both between two muscle cells and between a muscle cell and an interstitial cell. In serial sections septa of connective tissue and groups of muscle cells were found to disappear within few tens of microns or to merge with other septa, and the taenia did not appear to be divided into clear-cut muscle cell bundles. Bundles of smooth muscle cells were seen passing from the taenia to the underlying circular muscle. The transverse sectional area of the taenia ranged between 0.14 and 0.39 mm²; it showed about 526 blood vessels · mm^-2.