An ultrastructural study of mitotic division in differentiated gastric smooth muscle cells

Summary An ultrastructural examination of tissue from the gizzards of chicks just before and just after hatching showed numerous mitotic divisions in the well differentiated and functional smooth muscle. The nuclei in the very elongate, dividing cells were located centrally. The cytoplasm immediately adjacent to the nuclei contained the normal fully differentiated complement of myofilaments. During the active stages of division, after the breakdown of the nuclear membrane, myofilaments were shown to lie between the individual chromosomes. The process of division only occupied a small portion of the long muscle cells; the ultrastructural changes seen appeared similar to those described in other cell types.This work was supported by grants from the National Heart Foundation of Australia and the Australian Research Grants Committee. Part of this study was completed while J.L.S.C. was in receipt of a Queen Elizabeth II Research Fellowship. T. B. was supported by a Commonwealth Postgraduate Award.     

An ultrastructural study of regeneration of minced smooth muscle in the vas deferens of the guinea-pig

Summary Electron microscopic studies were made of the regeneration of minced smooth muscle of the vas deferens of the guinea-pig 3 days to 15 weeks after operation. At 3–5 days the mince contained degenerating smooth muscle cells and dedifferentiating cells showing characteristics of embryonic smooth muscle cells: numerous free ribosomes, well developed rough endoplasmic reticulum and Golgi apparatus with few peripherally placed myofilaments associated with dense bodies. During the first two weeks of regeneration, scattered cells surrounded by debris and collagen were separated by a large extra-cellular space. After three weeks, extracellular space was reduced to near normal values. Regenerating cells had a shorter length than normal cells, but during later stages of regeneration they showed an increase in diameter. Muscle effector bundles began to form after 2 to 3 weeks. Initially there were large gaps between the muscle cells, but at later stages of bundle formation, the extracellular space between the muscle cells was much reduced. From 3 weeks, arterioles appeared between the smooth muscle bundles in the regenerating areas. Regeneration of individual smooth muscle cells was complete by 15 weeks after the operation.This work was supported by grants from the Wellcome Trust and the Medical Research Council     

Fine structure of smooth muscle and neuromuscular junctions in the foot of Helix aspersa

Summary The smooth muscle cells in the foot of Helix aspersa are arranged in bundles which interweave to form a complex mesh. In the peripheral cytoplasm of the muscle cells there is a system of interconnected obliquely and longitudinally orientated tubules. The full extent of this system has not been determined; its possible function in relation to Ca⁺⁺ storage and excitation-contraction coupling is discussed. Longitudinal tubules are present among the myofilaments and in association with mitochondria. Distributed throughout the myofilaments are elliptically shaped dense bodies, the fine structure of which resembles an accumulation of thin filaments. Located on the plasma membrane of the muscle cells are dense areas; the fine structure and relationships of these cellular elements resemble desmosomes. They may serve as attachment points for thin, cytoplasmic filaments (not necessarily myofilaments). The muscle cells are innervated by axons which diverge from a coarse, neural plexus (the sole plexus). The axons initially come into close contact with the muscle cells and then pass over their surfaces for up to 35 before being gradually enveloped by flange-like protrusions of the muscle cells. These axons contain either, (i) agranular vesicles (600 Å in diameter), (ii) agranular and very dense granular vesicles (1000 Å in diameter) or (iii) agranular and less dense, granular vesicles (1000 Å in diameter). The possible role of these inclusions as sites of excitatory and inhibitory transmitters is discussed.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

Regulation of mitochondrial hexokinase in cultured HT 29 human cancer cells. An ultrastructural and biochemical study

The involvement of the mitochondrial bound hexokinase in aerobic glycolysis was investigated in two subpopulations of the HT 29 human colon cancer cell line: a poorly differentiated one with high aerobic lactate production (referred as undifferentiated or standard cells), and an enterocyte-like differentiated one with lower lactate production (referred as differentiated or Glc- cells). After mild digitonin treatment, 85% of the total cellular hexokinase activity remained in the particulate fraction in both cell types. In both cases mitochondria appeared to be tightly coupled but the Glc- cells exhibited a significantly higher oxidation rate in the presence of glucose. Electron microscopy of freeze-fractured cells revealed the absence of contacts between the two limiting mitochondrial membranes in the highly glycolytic standard cells, whereas the contacts were present in the Glc- cells. Furthermore, we investigated the functional relationship between bound hexokinase (as hexokinase-porin complex) and the inner compartment of mitochondria isolated from standard and Glc- HT 29 cells. In contrast to the differentiated cells the hexokinase in undifferentiated standard cells was not functionally coupled to the oxidative phosphorylation. This suggests that the high rate of lactate formation in neoplastic cells is not caused by an increase of particulate hexokinase activity but rather by a disregulation of the hexokinase-porin complex caused by the absence of contact sites between the two mitochondrial membranes. In agreement with this interpretation, the hexokinase-porin complex could be completely removed by digitonin treatment in standard HT 29 cells, while this was not possible in mitochondria from Glc- cells.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

An ultrastructural study of tettigoniid muscle in relation to function

The modian dorsal longitudinal indirect flight muscles from the mesothorax and metathorax of Homorocoryphus nitidulus vicinus have been studied to determine whether structural differences might offer an explanation for reports that the mesothoracic musculature effects a wing-beat rate of 140 beats/sec during stridulation, whereas during flight, it, like that of the metathorax, effects wing-beat frequencies of 14 to 20 beats/sec. No differences were observed and it is concluded that the high wing-beat rate, reported during stridulation, is not reflected in any specific modification of mesothoracic muscle fine structure.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.     

Changes in biophysical and biochemical properties of single bronchial smooth muscle cells from asthmatic subjects

Whether contractility of bronchial smooth muscle cells (BSMC) from asthmatic subjects is significantly altered has never been validated. We tested the hypothesis that such BSMC show increased contractility. Cells were isolated from endobronchial biopsies. BSMC shortening was measured under an inverted microscope. Statistically significant increases in maximum shortening capacity (Delta L max) and velocity (Vo) were found in asthmatic BSMC compared with normal cells. Mean Delta L max in asthmatic BSMC was 39.05 +/- 1.99% (SE) of resting cell length compared with 28.6 +/- 1.1% in normal cells; mean Vo was 7.2 +/- 0.8% of resting cell length/s in asthmatic cells and 5.23 +/- 0.46% in normal cells. To investigate the mechanism of the increased contractility, we measured mRNA abundance of smooth muscle types of myosin light chain kinase (smMLCK) and myosin heavy chain. RT-PCR data revealed that smMLCK mRNA was higher in asthmatic BSMC (0.106 +/- 0.021 arbitrary densitometric units, n = 7) than in control cells (0.04 +/- 0.008, n = 11; P < 0.05). Messages for myosin heavy chain isoforms showed no difference. Increased kinase message content is an index of the mechanism for the increased velocity and capacity of shortening we report.     

The ultrastructure of smooth cardiac muscle in the clam,Venus mercenaria

A fine structural study of the ventricular muscle of Venus mercenaria has revealed that it is an invertebrate smooth muscle. In the relaxed state induced by acetylcholine, both thick (350 Å) and thin (80 Å) myofilaments are observed. These are loosely distributed in bundles in the periphery of the mononucleated myocytes. The central core of the cell contains an ovoid nucleus, α-glycogen rosettes, round mitochondria and numerous smooth surfaced vesicles of the endoplasmic reticulum. After exposure to serotonin, all myofilaments are compacted in the peripheral cytoplasm and become oriented parallel to the longitudinal cellular axis. This produces contraction bands visible in phase contrast microscopy. Because these myofilaments attach to the cell membrane at sites of attachment plaques, contraction of the cell results in the serial evagination or blebbing of the cell surface. The above features are clearly demonstrable in this invertebrate smooth muscle and strongly suggest a sliding filament model as the contractile mechanism in this tissue. Moreover, the cell surface is thought to play an active and major role in that process.     

An ultrastructural study of the stretch-induced hypertrophy of skeletal muscle

The teres minor muscle of the adult chicken was studied ultrastructurally following tonic stretch-induced hypertrophy. The contralateral control muscle fibres showed compact myofibrils and proliferation of normal Z-bands. Myofibrils of the hypertrophied muscle however, showed Z-band alterations as Z-band expansions and Z-band streaming. Thus Z-band is a highly responsive structure to tonic stretch. Since a number of neuromuscular conditions display Z-band anomalies, the latter occurring in response to a variety of metabolic and physiologic stimuli, including tonic stretch as shown here, represents a non-specific phenomenon.     

A morphometric study of vascular smooth muscle cells in culture

Summary Cultured arterial smooth muscle cells derived from different times in culture, different passages, and different species were evaluated by a combination of transmission electron microscopy and morphometry. The morphometric studies focused on point counting and monitored the following cellular components: lysosomes, myofilaments, mitochondria, ribosomes, and rough endoplasmic reticulum (RER). Percent volume composition values for the organelles involved in protein synthesis, namely ribosomes and RER, show significant fluctuations with time. Consistent with these observations, the cells showed increasing myofilaments during the early weeks in culture, which subsequently decreased significantly. The data also indicate that rabbit cells in culture may become synthetically quiescent with time and the distribution of cellular components is altered with each succeeding passage. Cultured calf (bovine) cells exhibit similar activity periods compared to rabbit but show a significantly higher lysosomal and lower myofilament content than rabbit. Calf cells could not be maintained for longer than 21 days in the absence of ascorbate, whereas ascorbate affects the ultrastructure of rabbit cells less dramatically. Age, passage, and donor, among others, are important considerations for studying in vitro smooth muscle cells. With proper morphologic and morphometric monitoring, these smooth muscle cell culture systems can be important tools in the study of aging or pathologic processes, or both. This work was presented as partial fulfillment for the degree of Ph.D. This work was supported by National Institutes of Health Grants HL-13262, HL-19717, and AG-00001.     

Calcium sensitivy of foot muscle myosin from clam (Meretrix lusoria).

1. It was found that Mg-ATPase of clam foot myosin is strongly activated by calcium or strontium ions and is as sensitive to those divalent cations as the Mg-ATPase and superprecipitation of rabbit skeletal acto-clam foot myosin are. 2. It was also found that desensitization and resensitization of clam foot myosin result in the loss of superprecipitation activity with acto-desensitized myosin and in its recovery with acto-resensitized myosin, respectively. However, the ATP-ASE activity in the absence of calcium ions rises with acto-desensitized myosin and falls again with acto-resensitized myosin. 3. It is thus proposed that the primary role of the EDTA-light chain component in calcium regulation is to inhibit myosin-ATPase rather than to inhibit the actin-myosin interaction.