Nexuses between the smooth muscle cells of the guinea-pig ileum

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.     

Freeze-fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum

The freeze-fracture appearance of the nexus was compared in the smooth muscle of guinea pig sphincter pupillac, portal vein, pulmonary artery, taenia coli, uretzr, and vas diferens, mouse vas deferens, chicken gizzard and anterior mesenteric artery, and toad stomach. Nexuses are particularly numerous in the guinea pig sphincter pupillae; they are usually oval and their average area is 0.15 mum2, although some as large as 0.6 mum2 were seen. Small aggregations of particles were observed which would not be recognizable as nexuses in thin section. What constitutes the minimum size of a nexus is discussed. It is estimated that the number of nexuses per cell in this preparation is of the order of tens rather than hundreds. All nexuses examined had 6-9-nm particles in the PF face, with corresponding 3-4-nm pits on the EF face forming a polygonal tending towards a hexagonal lattice. The nexuses are arranged in rows parallel to the main axis of the cell, usually alternating with longitudinal rows of plasmalemmal vesicles. Many nexuses in the guinea pig sphincter pupillae, chicken gizzard, and toad stomach show a close relationship with sarcoplasmic reticulum. The possibility that this may have some role in current flow across this specialized junction is discussed.     

FURTHER OBSERVATIONS ON THE OCCURRENCE OF NEXUSES IN BENIGN AND MALIGNANT HUMAN CERVICAL EPITHELIUM

An estimate is made of the frequency of occurrence of nexuses ("gap junctions") in a spectrum of human cervical epithelia, ranging from normal to malignant, since a deficiency of nexuses may be important in abnormal cell-to-cell communication in malignant tissues. The normal cervical epithelium has approximately ten nexuses per cell in the basal layer of proliferating cells and 200 nexuses per cell in the more differentiated intermediate zone. Nexuses are rare between invasive malignant epithelial cells (carcinoma cells). In many areas of cell proliferation near the edge of the tumor mass, fewer than one nexus per cell is present. However, up to four nexuses per cell can be found in some well differentiated regions of invasive carcinoma. Preinvasive malignant epithelia (severe dysplasia and carcinoma-in situ) have as few nexuses as invasive carcinoma. In abnormal but benign epithelia (squamous metaplasia and mild dysplasia), nexuses are abundant. The data indicate that a decrease in number of nexuses correlates with the severity of the morphological alteration in the dysplastic epithelium. Also the deficiency of nexuses in groups of carcinoma cells can occur many cell generations before the development of invasion of the malignant epithelium into the connective tissue. The diminution of nexuses before invasion suggests that a deficiency of nexuses may be one of the important factors in eventually permitting the development of the diffusely infiltrating type of invasion which is characteristic of highly malignant tumors such as squamous carcinomas.     

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     

A STUDY OF NEXUSES IN VISCERAL SMOOTH MUSCLE

Nexuses are described between the smooth muscle cells of the gizzard of the chick and the pigeon, the vas deferens of the mouse and the guinea pig, and the taenia coli of the guinea pig. The nexuses in the gizzard were examined after osmium tetroxide and potassium permanganate had been used as fixatives. Although differences in the dimensions of the unit membranes and the nexuses were noted, the results with the two fixation techniques were complementary. The distribution of nexuses within the smooth muscle tissues examined was uneven. Nexuses were still present in both small and large pieces of tissue incubated in hypertonic solutions at varying temperatures. Other experiments showed that the degree of contraction at the time of fixation did not affect the presence of nexuses in the tissue. These results indicate that nexuses between smooth muscle cells are stable under a variety of conditions.     

A STUDY OF THE STRUCTURE AND DISTRIBUTION OF THE NEXUS

Nexuses, that is, fusions of plasma membranes of adjacent cells, are described in mammalian smooth and cardiac muscle, median giant axon of earthworm, frog skin, and rat submandibular gland. In smooth muscle they usually occur where a process from one cell either meets a process of, or projects into a neighboring cell. On the other hand, in mammalian heart muscle and in earthworm giant axon the nexuses occur along the intercalated disc and intercellular segmental septa, respectively. Their occurrence between these excitable cells is correlated with propagation of action potentials by an electrical rather than chemical mechanism. Since the nexuses may offer pathways for electric current between cell interiors, it seems possible that they constitute a link in the structural basis for electrical transmission in these systems. In epithelia, nexuses usually appear as part of a terminal bar complex. This is true in the rat salivary gland studied here. In the epidermis of frog skin, nexuses are less numerous between the basilar columnar cells than between the subjacent squamous cells. The nexuses which occur in epithelia in frog skin and rat salivary gland are distributed as though to provide seals against electrochemical backleaks and sites of chemical exchange between cell interiors.     

Hypertrophy of intestinal smooth muscle

Proximal to an experimental stenosis of the small intestine of rats and guinea-pigs a remarkable hypertrophy of the muscle coat develops 3-5 weeks after the operation. There is no increase in the length of the intestine but an overall increase in volume of the muscularis externa up to 10 times. This increase is accounted for by an increase in size and in number (by mitosis) of smooth muscle cells of both the longitudinal and circular layers. Bundles of newly-formed smooth cells appear in the serosa and are circularly arranged. In the hypertrophic smooth muscle cells of the circular layer the ratio of surface to volume is 0.80 (0.80 mum2 of cell surface for every mum3 of cell volume) as against 1.4 in the control muscle. The hypertrophic muscle cells have a highly developed sarcoplasmic reticulum and show a large number of nexuses. The density of innervation (number of axons per given number of smooth muscle cells) is smaller than in controls. Few collagen fibrils are visible in the extracellular space.     

Quantitation of nexus junctions in the granulosa cell layer of rabbit ovarian follicles during ovulation

Electron microscopy was used in a semi-quantitative study to determine changes in the abundance and size of surface nexuses and changes in the abundance of interiorized nexuses in growing and mature ovarian follicles during the ovulatory process. Mature follicles contain larger granulosa cells than follicles in the early stage of antral formation. Also, the granulosa cells of mature follicles have a slightly greater number of surface nexuses (without a change in nexus length), and more interiorized nexuses, compared to immature follicles. As a mature follicle approaches rupture, there is an appreciable decrease in the number of surface nexuses per granulosa cell. There is also a slight reduction in the number of interiorized nexuses at this time. It is concluded that this decrease in both surface nexuses and interiorized nexuses may be a consequence of ovulatory changes during which the rate of granulosa cell division is greater than the rate of formation of new nexuses. Additionally, the disruption to cell-to-cell cohesion during the ovulatory process appears to be independent of the interiorization of surface nexuses.     

The ultrastructure of the sinu-atrial node of the bat

The sinu-atrial node (SAN) of the bat, Pipistrellus subflavus, is capable of generating a wide range of spontaneous activity varying from 20 bpm when hibernating to bursts of 800 bpm during active flight. Electrophysiological studies have shown an absence of arrhythmias even below 4 degrees C body temperature. In order to determine whether these physiological capabilities are based upon unique ultrastructural features of the bat SAN, the present study was conducted. We found that the structure of the SAN of the bat is typically mammalian. Diameters of all three cell types in the SAN (nodal, transitional, and atrial) are smaller than those observed in any other mammalian species. A morphometric analysis of cell junctions reveals that nodal-nodal and transitional-transitional cell contacts are primarily undifferentiated with few nexuses. Atrial-atrial cell contacts have a dominance of fasciae adherentes-type junctions with a small area left undifferentiated. Nexuses are much more prevalent in atrial-atrial cell contacts.     

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     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Intercellular junctions between circular and longitudinal intestinal muscle layers

Summary Different intercellular relationships between smooth muscle cells are observed in the tunica muscularis externa of the ileum of guinea-pig. a) simple adjoining of the two muscle cells; b) nexus; c) desmosome-like attachment with dense aggregations on both membranes; d) projection from one muscle cell protruding into another muscle cell. All four types are found in the circular muscle layer. On the contrary, in the longitudinal layer clear-cut nexuses are not observed. Simple adjoining of muscle cells, nexuses, and desmosome-like attachments are observed between muscle cells of the circular layer and the longitudinallayer.Dedicated to Professor Angelo Bairati, on the occasion of his sixtieth birthday.I thank Professor E. G. Gray for discussion, Professor J. Z. Young for use of facilities, and the Wellcome Trust for a fellowship.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

Freeze-fracture replication of junctional complexes in unincubated and incubated chick embryos

Junctional complexes have been investigated in the epiblast of young chick embryos by examination of freeze-fracture replicas and of sections of comparable specimens stained with lanthanum nitrate. By means of freeze-fracture, tight junctions were shown to be present in the unincubated embryo (stage 1 of Hamburger and Hamilton). The number of ridges or grooves was found to vary between 2 and 10 near the dorsal border, whereas isolated ridges were found more ventrally. Lanthanum was unable to penetrate between the cells in the region of the dorsally situated tight junctions. Similar tight junctions were found in incubated embryos (stage 3) examined by both techniques. Tight junctions were also seen in cleavage (pre-laying) embryos examined in section. Gap junctions were extremely uncommon in unincubated embryos, though occasional aggregates of gap junction particles were seen on the lateral cell membranes close to the dorsal surface. In only one instance were associated pits visible. By contrast, gap junctions were more frequently encountered by stage 3, and these junctions possessed both pits and particles. Desmosomes were never seen in the freeze-fracture replicas at either stages 1 or 3, though structures which might be developing desmosomes were visible in sections. The functions of both the tight and gap junctions in the young chick embryo are discussed. The results are also considered in relation to recent theories about the way in which gap junctions are formed.     

Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle and neurons

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate and Purkinie cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.     

Is the nexus necessary for cell-to-cell coupling of smooth muscle?

Summary Electronmicroscopic study of electrically coupled smooth muscles was undertaken to determine the distribution of nexuses in various types of smooth muscle. The study revealed that while nexal structures were commonplace in some types of smooth muscle, they were very rare or absent in others, even though in some cases these cells were only a few nanometers distant from one another. The persistence in thin section of these structures in the main circular muscle of dog intestine after poor fixation, fixation under strain, cell shrinkage, and metabolic damage of various sorts seems to rule out the thesis that they are labile. The absence of nexuses in longitudinal muscle of dog intestine examined both by thin section and by freeze fracture suggests that in this tissue they are absent or very rarein vivo and cannot account for electrical coupling.Nexuses were discernible in thin sections of main circular muscle after a variety of experimental conditions of fixation. Metabolic inhibition orin vitro permanganate fixation partially destroyed nexal contacts. These procedures induced tissue, membrane apposition and an accompanying increase in the number of structures which resemble nexuses at low magnification (nexus-like structures). Nexus-like structures occurred in all smooth muscle fixed byin vitro permanganate associated with apposition of membranes and poor preservation of basement membrane. A technique ofin vitro permanganate fixation was developed which prevented tissue swelling; consequently nexus-like structures were absent in tissues so treated. The suggestion is made that some structures described in the literature as nexuses, following permanganate fixation, may represent nexus-like structures.The balance of evidence suggests that nexuses need not be present for electrical coupling of some smooth muscle cells, in which other types of cell-to-cell contacts must be invoked.     

Nexus of frog ventricle

Here were demonstrate in Rana pipiens ventricle a nexus with very unusual morphology. This tissue has been reported previously to lack nexuses. The nexus appears in thin sections of ventricle, fixed in aldehyde and OsO4 or permanganate as a series of punctate membrane appositions regularly alternating with regions of membrane separation. The junctional width at membrane appositions, as determined by microdensitometry and optical measurements, is 15-17 nm, and the width of the electron-translucent region between the junctional membranes is 1.8 nm. These values correspond closely to similar measurements of the more typical nexues in frog liver. Along the nexus the mean distance between punctate appositions is 74.5 nm. Freeze-cleave replicas of the nexuses between myocardial cells show particles 10.4 nm in diameter arranged in arrays of up to nine linked circles or partial circles on the PF-face and similar arrays of pits of shallow grooves on the EF-face. The mean diameter of the circles on both membrane fracture faces is 76.7 nm comparsion of the thin-sectioned and freeze-cleaved nexuses demonstrates an excellent correspondence between the spacing of membrane appositions along the junction and the diameters of the freeze-cleaved circles of particles and pits or grooves.     

Detection of gap junctions between the progeny of a canine macrophage colony-forming cell in vitro

An in vitro monocyte-macrophage colony-forming cell (M-CFC) has been detected in canine bone marrow (BM). The colonies derived from these progenitor cells were similar to murine-derived M-CFC (MacVittie and Porvaznik, 1978, J. Cell Physiol. 97:305--314) colonies, since they showed a singular macrophage line of differentiation, a lag of 14--16 days before initiating colony formation, and they survived significantly longer in culture in the absence of colony-stimulating factor (CSF) than granulocyte-macrophage colony-forming cells (GM-CFC). Endotoxin (Salmonella typhosa lipopolysaccharide W)-stimulated dog serum was used as the CSF (7% vol/vol). Canine-derived M-CFC progeny were identified as macrophages on the basis of morphology, phagocytosis, and the presence of Fc receptors for IgG. Gap junctions were observed only in canine BM, M-CFC-derived colonies using freeze-fracture and lanthanum tracer techniques. They were not observed in any GM-CFC-derived colonies. The number of gap junctions observed in freeze-fracture replicas of BM, M-CFC-derived colonies (21 colonies from three different dogs) showed a significantly positive correlation (Kendall's tau = 0.70, P less than 0.001) with the size of the colony fracture plane area. Gap junctions were observed displaying hexagonal lattices of 9.3 nm +/- 0.08 (SE) particles with a center-to-center spacing of 10.4 nm +/- 1.0 (SE) on membrane P-fracture faces. On membrane E-fracture faces, highly ordered arrays of pits with 8.7 nm +/- 0.12 (SE) center-to-center spacing were observed. Arrays of both particles and pits were also observed in fracture-face breakthroughs within a gap junction. Thus, gap junctions can form in vitro between the cells of macrophage progeny of a canine M-CFC under appropriate growth conditions. The significance of this observation is that there may be a structural basis for cell-to-cell collaboration between BM macrophages and other capable cells that either pass into the tissue for modification or develop there into mature cell forms.     

Gap junctions in the cardiac muscle cells of the lamprey

Summary Electron microscopy of both thin sections and freeze-fracture replicas has demonstrated the occurrence of gap junctions (nexuses) in the cardiac muscle cells of the lamprey. These gap junctions are identical in basic structure with those found in the mammalian heart. However, they are much smaller (less than 0.5 in diameter), and more irregularly distributed than the typical gap junction in the mammalian heart. These small gap junctions seem to provide a structural basis for the electrical coupling between cardiac muscle cells in the lower vertebrates.In addition, the well developed sarcoplasmic reticulum and subsurface cisternae, which contain an electron dense spheroidal cast, are frequently observed in the cardiac muscles of the lamprey.This work is supported by a research grant from the Ministry of Education, Japan     

Freeze-fracture studies on unmyelinated axolemma of rat cervical sympathetic trunk: correlation with saxitoxin binding

The density and diameter distributions of intramembranous particles (IMPs) within unmyelinated axolemma from rat cervical sympathetic trunk were examined with freeze-fracture electron microscopy. The axolemma displays a highly asymmetrical partitioning of IMPs with ca. 1200 IMPs microns-2 on P-faces and ca. 100 IMPs microns-2 on E-faces. Particle sizes (diameters) are unimodally distributed on both fracture faces, with a range from 2.4 nm to 15.6 nm. Approximately 16% of the particles on P-faces and 28% of particles on E-faces are of a large (greater than 9.6 nm) diameter. On both fracture faces, the IMPs appear to be randomly distributed; no aggregations of particles were observed. The results indicate that there are ca. 230 large IMPs microns-2 of unmyelinated axolemma from rat cervical sympathetic trunk. The density of these IMPs is similar to the density of saxitoxin binding sites on unmyelinated axolemma from rat cervical sympathetic trunk (Pellegrino et al. 1984 (Brain Res. 305, 357-360)), which suggests that many of the large diameter particles may be the morphological correlate of voltage-sensitive Na+ channels.