A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross- sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.     

An ultrastructural study of isolated rat skeletal muscle mitochondria in various metabolic states

Summary An electron microscopic examination was made of isolated rat skeletal muscle mitochondria in various functional states. Shifts in the inner membrane ultrastructure of populations of mitochondria were observed under certain conditions. However, ultrastructural transformations reported by others during rapid changes in biochemical states were not observed in skeletal muscle mitochondria. There does not appear to be a strict correlation between metabolic states and ultrastructural states in isolated rat skeletal muscle mitochondria as has been observed in isolated mitochondria from several other tissue types.     

Morphology, ultrastructure and contractile properties of muscles responsible for superior tentacle movements of the snail

Bending, twitching and quivering are different types of tentacle movements observed during olfactory orientation of the snail. Three recently discovered special muscles, spanning along the length of superior tentacles from the tip to the base, seem to be responsible for the execution of these movements. In this study we have investigated the ultrastructure, contractile properties and protein composition of these muscles. Our ultrastructural studies show that smooth muscle fibers are loosely embedded in a collagen matrix and they are coupled with long sarcolemma protrusions. The muscle fibers apparently lack organized SR and transverse tubular system. Instead subsarcolemmal vesicles and mitochondria have been shown to be possible Ca2+ pools for contraction. It was shown that external Ca2+ is required for contraction elicited by high (40 mM) K+ or 10-4 M ACh. Caffeine (5 mM) induced contraction in Ca2+-free solution suggesting the presence of a substantial intracellular Ca2+ pool. High-resolution electrophoretic analysis of columellar and tentacular muscles did not reveal differences in major contractile proteins, such as actin, myosin and paramyosin. Differences were observed however in several bands representing presumably regulatory enzymes. It is concluded that, the ultrastructural, biochemical and contractile properties of the string muscles support their special physiological function.     

Effects of rotator cuff ruptures on the cellular and intracellular composition of the human supraspinatus muscle

Ruptures of the rotator cuff tendons of the human shoulder are a common incidence and lead to functional impairment of the four muscles connected to the cuff, entailing profound changes of their cellular tissue composition. Most importantly, such tendon tears lead to atrophy, fatty degeneration and fibrosis of the corresponding muscles. The muscle most commonly affected with such changes is the M. supraspinatus. The present study uses biopsy samples from the supraspinatus muscle of 12 elderly patients and 6 controls to examine the rupture-induced muscle change at both the cellular and the intracellular (ultrastructural) levels. Amounts of fatty tissue, connective tissue and muscle were assessed by light microscopy-based morphometry and stereology. Stereology of electron micrographs was employed to determine volume densities of muscle fibre mitochondria, myofibrils and intracellular lipid. Results demonstrate that the supraspinatus muscles of patients with a massive rupture contain significantly higher amounts not only of fatty tissue but also of intracellular lipid than those of control subjects. These patients further exhibit a major decrease in relative amounts of myofibrils, thus confirming that change of intracellular composition is a major component of the observed muscle degeneration. The results contribute to establish the true spectrum of supraspinatus muscle damage in humans induced by tendon rupture.     

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.     

Terminal axons ensheathed in smooth muscle cells of the vas deferens

Summary The ultrastructure of axon profiles which were completely ensheathed in smooth muscle cells has been described in the guinea pig, mouse and rat vas deferens. The axon profiles contained both small (500 Å) and large (1,000 Å) vesicles, neurotubules and mitochondria. Adrenergic axons were clearly identified within smooth muscle cells after treatment of the tissue with 5-or 6-hydroxydopamine, drugs which cause specific ultrastructural changes in adrenergic axons. The ensheathed axons were separated from the surrounding muscle cells by narrow, regular gaps, usually about 100–300 Å wide. Schwann cells seldom accompanied the ensheathed axons. Axons often penetrated the muscle cells in the nuclear region and profiles were sometimes observed among the perinuclear organelles.     

Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

Summary The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.     

A comparative study of the regeneration of the striated and smooth-muscle tissues of the esophagus]

By means of electron microscopy and observational histological techniques, using a similar experimental model, regeneration of the striated and smooth muscle tissues of the esophagus has been studied in rats. During early periods after lesion in both muscle tissues destructive-necrotic changes develop. Beginning from the 2nd-3d days regeneration processes are observed. The course and periodicity of the regenerative processes are specific for the types of the muscle tissues studied. Each of the muscle tissues of the esophagus has its own source of regeneration. For the smooth muscles those are myoblasts, that convert into smooth myocytes, for the striated ones--myosatellites, which after activation get out of the muscle fiber. During the restorative process of the muscular membrane no tissue interconnections are observed. This also proves certain specificity of the striated and smooth muscle tissues of the esophagus.     

The ultrastructure of normal and denervated human facial muscle

The ultrastructure of normal human facial muscles from 25 nonparalytic and 17 paralytic patients revealed normal features in nondenervated human facial muscles, identical to the fine structure of other normal human and mammalian cross-striated muscle fibers. However, in denervated facial muscle, a broad spectrum of ultrastructural lesions had affected sarcomeres, abnormal inclusions, and organelles. A large variety of inclusion bodies, some of which have not been described, were also found. The spectrum of ultrastructural changes showed no dependence on the length of the denervation period. There were no inclusion bodies in all the normal facial muscle biopsies. To our knowledge, this study represents the first systematic electron microscopic investigation of normal and denervated human facial muscles.     

Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum.

Calcium uptake by the microsomal and mitochondrial fractions of pig coronary artery and guinea pig ileum was studied in the presence of ATP, ATP plus oxalate and without ATP and oxalate. Microsomes and mitochondria of both smooth muscles were found to be unable to accumulate appreciable amounts of calcium in the absence of ATP. Oxalate noticeably stimulated the calcium uptake of the mitochondrial fraction from pig coronary artery but had little effect on calcium uptake by the microsomal fraction of this smooth muscle. The calcium uptake of microsomes and mitochondria from guinea pig ileum was not or only slightly enhanced by oxalate. There are typical kinetics regarding the time course and the extent of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. In comparison, considerable qualitative and quantitative differences between both smooth muscles are observed. The high ATP-dependent calcium uptake capacity of the mitochondria from pig coronary artery and guinea pig ileum are a further argument for the hypothesis that these organelles may play an important role in the contraction-relaxation mechanism of smooth muscle.     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

Morphometric ultrastructural evaluation of satellite cells in musculus soleus and musculus extensor digitorum longus of rats subjected to hypokinesia

Soleus and extensor digitorum longus muscles were studied in rats confined to cages which restricted their movement. Ultrastructural study by electron microscopy of satellite cells did not reveal major differences between the cytoplasmic organelles of confined animals and those of controls. Some changes in mitochondria were noted. Results of the ultrastructural analysis are discussed.     

Comparative aspects of cardiac and skeletal muscle sarcoplasmic reticulum.

While differing in numerous physiological and biochemical parameters, mammalian cardiac and skeletal muscles exhibit many common ultrastructural characteristics. General subcellular organization is similar with longitudinal disposition and organization of the myofibrils as well as subcellular organelles such as mitochondria, sarcoplasmic reticulum and transverse tubules. Significant differences are more readily discerned in terms of degree, not only with respect to relative amounts of various organelles, but also in regard to membrane composition. It is these macromolecular variations in membrane components which may, at least in part, provide the basis for differences in overall functional characteristics in the muscles.In cardiac, as well as skeletal muscle, the concentration of Ca²⁺ ions at specific intracellular sites regulates the contractile state of the muscle. The differences in mechanism and sources of Ca²⁺ for contraction in cardiac and skeletal muscle are but a few of the unsolved areas which are now being addressed. We shall focus primarily on research advances involving cardiac and skeletal SR emphasizing the contrasting features related to their functional roles in control of contraction and metabolic events.     

Cell calcium and its regulation in smooth muscle

Two novel methods used to study smooth muscles-electron probe X-ray microanalysis and Ca2+-sensitive indicators (which are used for resolving, respectively, the spatial distribution and temporal distribution of calcium)-are briefly reviewed and the major findings obtained are summarized. In smooth muscle the sarcoplasmic reticulum is the major intracellular source of Ca2+; mitochondria do not play a significant role in the physiological regulation of [Ca2+]i. Under pathological conditions mitochondria can reversibly accumulate large amounts of calcium. Resting [Ca2+]i generally ranges from 80 to 200 nM, and is lower in phasic than in tonic smooth muscles. Removal of extracellular Ca2+ and Ca2+ entry blockers can reduce [Ca2+]i, but the effects of beta-adrenergic agents are variable. Increases in [Ca2+]i are triggered by electrical stimulation, depolarization with high K+, and excitatory agonists. Stretch, after a delay of several seconds, can cause an increase in [Ca2+]i in some smooth muscles. There is also a delay of approximately 200-400 ms between the initiation of the rise of Ca2+ and contraction that follows spontaneous action potentials or electrical stimulation. Agonist-induced Ca2+ release, a major mechanism of pharmacomechanical coupling, has been demonstrated in smooth muscles depolarized with high K; evidence suggests that it is mediated by G proteins that couple receptors to phospholipase C. Ca2+ release can be triggered directly in permeabilized smooth muscle with inositol 1,4,5-trisphosphate. Even though Ca2+ is the major physiological regulator of contraction, Ca2+ sensitivity of the regulatory-contractile apparatus differs in different (phasic and tonic) smooth muscles, and can be modulated in a given smooth muscle. The force [Ca2+]i ratio is higher during agonist-stimulated than during high K+-induced contractions, owing to agonist-induced increases in Ca2+ sensitivity mediated by G proteins. In some phasic smooth muscles (guinea pig ileum), the time course of the initial myosin light chain phosphorylation is extremely rapid and returns to basal levels while force remains elevated. In these smooth muscles there is also a marked decrease in the Ca2+ sensitivity of the regulatory-contractile apparatus during maintained depolarization in Ca2+-free or low Ca2+ solutions. It has been suggested that regulation of myosin light chain phosphatase plays a major role in the modulation of the Ca2+ sensitivity manifested as either potentiation or desensitization to [Ca2+]i.     

Ultrastructural aspects of cytoplasmic male sterility inPetunia hybrida

Summary Anther development of isogenic male fertile and cytoplasmic male sterile types ofPetunia hybrida cv. Blue Bedder is studied by electron microscopy. First deviation in sporogenesis of the sterile type, is observed during leptotene stage of the meiocytes. Initial aberration is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells. These vacuoles reveal the first aspects of degeneration; no other ultrastructural differences are observed. Vacuolation is accompanied by the condensation of cytoplasmic organelles. The tapetal cells become distorted and ultrastructural aberrations in mitochondria do occur. The mitochondria elongate and contain several tubular cristae.Substantial evidence suggests, that cytoplasmic male sterility in petunia is encoded by the mitochondrial genome (Boeshore el al. 1983). However, before degeneration becomes manifest, no consistent ultrastructural differences in mitochondrial organization are observed.Abortion of the tapetum and the sporogenous tissue in cytoplasmic male sterile plants, generally follows a corresponding pattern. Ultimately, the cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.     

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     

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.     

Some observations on the fine structure of light and dark cells in the gall bladder epithelium of the mouse

Summary Electron microscopic observations have been made of the two epithelial cell types, light barrel-shaped and dark rod-shaped cells in the gall bladder of the mouse.The light cells have a voluminous cytoplasm of low electron opacity in which cell organelles such as mitochondria, elements of granular endoplasmic reticulum, and free ribosomes undergo more or less degenerative changes. However, there are a relatively abundant Golgi apparatus and numerous lysosomal dense bodies. The ultrastructural features of the light cells suggest that they are an aged, degenerative cell type with declining functional activity and a high degree of hydration.The dark cells are characterized by a high concentration of mitochondria and free ribosomes, more or less distinctive elements of granular endoplasmic reticulum, and well developed components of the Golgi apparatus. Such ultrastructural characteristics indicate that the dark cell type has a high synthetic activity.What has been observed in the present study can well be correlated with the results of previous studies on the same cells by methods of light microscopic histochemistry.