Large Gap Junctions Research Articles

Structure of intercellular junctions in different segments of the intrapulmonary vasculature.

Freeze-fracture studies have shown that there is a segmental differentiation of the structure of intercellular junctions of intraacinar pulmonary vessels. Tight junctions vary in complexity, being composed in the arterial segment of the most highly interconnected and most numerous (two to seven) rows of tight junction particles, which are present in E face grooves. In capillaries, they consist of one to three rows of particles, which, in the venular segment, show some discontinuities. Tight junctions in intraacinar veins consist of one to five rows of particles in the E face. Large gap junctions are numerous and are intimately associated with arterial tight junctions. They are absent from capillaries, and are fewer in number and smaller in size in veins than in arteries. The above observations suggest that, as in the systemic circulation, the venular segment is likely to be the most permeable to small, water-soluble solutes. The presence of numerous gap junctions, particularly in the arteries, suggests that in this segment there is a high degree of electrotonic and/or metabolic coupling between endothelial cells.

Intercellular junctions in the developing arachnoid membrane in the chick.

The arachnoid membrane of chick embryos was prepared for electron microscopic study by means of thin sections and freeze-fracture replicas. Particular attention was given to the relationships among junctional complexes during arachnoid maturation. By 14 days of incubation, the arachnoid had differentiated into morphologically distinct inner and outer zones. Both desmosomes and gap junctions were present among the cells of both layers at this time. Desmosomes were most numerous in the inner arachnoid layer and their structure remained constant. Gap junctions showed a great variation in structure. The large gap junctions contained a particle packing pattern in which rows of intramembranous particles were separated by particle-free zones. Arched gap junctions were also present. Smaller arrays of gap junctions exhibited a variety of configurations on the membrane P-face. The first tight-junctional stands clearly identifiable in freeze-fractured preparations appeared at 15-17 days. These were closely associated with the particles of gap junctions and consisted of single stands on the P-face. By hatching age (21 days) a "mature" pattern of tight-junctional strands was interwoven in several layers. In the interim, more complex arrangements of tight-junctional strands were in intimate relation with gap junctions.

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, Ca 2+-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.

Formation of gap and tight junctions between reaggregated blastomeres of the killifish, Fundulus.

Blastomeres from eggs of the killifish, Fundulus, were mechanically dissociated and reaggregated by pelleting in a simple saline solution. Formation of gap and tight junctions was followed by electron microscopy of freeze-fracture replicas. Five to eight min after pelleting, neither new nor old junctions were observed. After 10-15 min, small gap junctions were found, but these were not associated with distinct formation plaques. Larger gap junctions were observed after 45 min, and the images were consistent with growth by accretion of intramembrane particles. In aggregates, after 20 min or more, tight junctions were much more commonly found than in intact blastulae, and it seemed likely that they were being formed by cells that were not doing so in the intact embryo. Initial stages consisted of short strands that appeared to grow in length. Also, more elaborate junctions were seen than occur in situ. Particle-free membrane often occurred near incomplete junctions, and large junctions like those in situ separated particle-rich from particle-free membrane. In this system, the formation of both gap and tight junctions occurs with shorter latency, and is more precisely timed, than heretofore described.

Intercellular junctions in the full term human placenta. II. Cytotrophoblast cells, intravillous stroma cells and blood vessels.

Intercellular junctions within the villous stroma and the cytotrophoblastic layer of the human full term placenta were investigated using thin sectioning and freeze-fracturing. Numerous maculae adherentes (desmosomes) were found between the cytotrophoblast cells and the syncytiotrophoblast. This junction type was also seen connecting adjacent cytotrophoblast cells. Large gap junctions were frequently observed in contact areas of perikarya or at processes of adjacent fibroblasts. They often exhibited a peculiar pattern of their particles on the P-face of the membrane. Small rows of junctional particles were found on the P-faces of interconnected smooth muscle cells and gap junctions frequently bridged myoendothelial and interendothelial contact zones. The significance of the junctional complexes is discussed in relation to functional systems within the villous stroma of the human full term placenta.

A correlated thin-section and freeze-fracture analysis of guinea pig adrenocortical cells.

Comparison of the fine structural features of guinea pig adrenocortical cells as seen in thin sections with those revealed by freeze-fracture confirms the structural appearance of steroid-secreting cells as interpreted from thin sections and reveals significant new features of the membranous organelles. Smooth-surfaced endoplasmic reticulum appears as a network of tubules, interwoven or in parallel, and as cisternae, fenestrated and non-fenestrated. These elements are tightly packed in the deeper cortical cells, excluding other organelles from their domain. Tubules and fenestrated cisternae possess randomly distributed intramembranous particles on their PF faces, while closely packed non-fenestrated cisternae possess aggregates of particles interspersed with aparticulate regions on their PF faces. These differences in particle distribution suggest functional specialization among the various forms of reticulum. Mitochondria appear as elongated structures of varying shape. Freeze-fracture reveals that all their cristae have circular origins from the inner membrane. Sinuous tubules, which appear as tubules in section, and straight tubules, which appear as lamellae in section, arise from single sites. Flattened sac-like cristae may have multiple circular origins. Definite contact points seen between inner and outer membranes may facilitate passage of molecules, including steroids, into the mitochondrial compartments. Lysosomes and peroxisomes, which are easily identified in thin sections with the aid of cytochemistry, are difficult to identify with certainty by freeze-fracture. Single membrane-bound granules of slightly smaller diameter than mitochondria may represent lysosomes. Smaller granules interconnected with the tubular reticulum, as well as dilated regions of this organelle, may represent peroxisomes. Plasma membranes show no indication of tight junctions but do have abundant gap junctions which show a zonal differentiation: small gap junctions throughout the cortex, medium-sized regularly shaped gap junctions in zona fasciculata externa, and large irregular gap junctions in zona fasciculata interna and zona reticularis. The large junctions cover planar areas as well as surfaces of projections of one cell into another. Such junctions may allow passage of ions as well as of low-molecular-weight substances between the cells, facilitating or even amplifying the response to trophic hormone stimulation.

Hormonal Control of GAP Junction Formation in Sheep Myometrium during Parturition

The ultrastructure of myometrial tissues from nonpregnant, pregnant and postpartum sheep was quantitatively examined for the presence of gap junctions between smooth musde cells. Serum concentrations of estrone, estradiol and progesterone were determined in some of the sheep prior to and at the time of sacrifice. Gap junctions were not present in any tissue from nonpregnant animals or in tissues of pregnant animals at 74-100 days gestation. Small gap junctions were present in some of the tissues from pregnant animals at 127-132 days gestation. Larger gap junctions were present in increased frequency in animals at term and immediately postpartum. The formation of gap junctions correlated with a rise in fetal estrogens, a fall in maternal progesterone and increases in fetal and maternal estrogen/progesterone ratios. These results are consistent with the hypothesis that the hormonal changes which occur near the end of pregnancy bring about the formation of gap junctions and the activation of the myometrium.

Cell junctions in the endothelia and connective tissue of the rat choroid plexus.

AbstractCell junctions between endothelial cells and between connective tissue cells (leptomeninges) of the rat choroid plexus were studied by freeze‐fracture. Special attention was paid to the problems of identifying fractured cell membranes and fracture faces. Tight junctions in arterioles are comprised of continuous small‐meshed networks of strands appearing as shallow grooves with aligned particles on E‐faces, and low ridges with some particles on P‐faces. In the fenestrated capillaries the tight junctions are represented by discontinuous, isolated slightly elevated ridges with some particles on P‐faces, and corresponding shallow grooves devoid of particles on E‐faces. Junctions in venules appeared as a less developed version of the junctions in the fenestrated capillaries. Extended areas of the meningeal cell membranes exhibit cell junctions. Tight junctions in this tissue are comprised of a loose discontinuous arrangement of isolated, often branched junctional elements. On P‐faces the elements appear as elongated crests with a distinct furrow running along the edge of the crest, occupied by a few particles. On E‐faces corresponding elongated impressions are present and occupied by aligned particles and a few short bars. The arrangement of these tight junction elements are not supportive of any significant barrier function. Small gap junctions occur “freely” in the membrane, or they are sometimes surrounded by tight junction elements. Very large gap junctions (up to about 10,000 particles) are mostly surrounded and interconnected by tight junctional elements. The possible significance of the variability of gap junction size, and the relation to tight junction elements is discussed.

The formation of gap and tight junctions between retinal pigment cells in cell cultures.

The reformation of the junctional complex of retinal pigment cells was studied after trypsin disaggregation and in vitro reaggregation. Control specimens show a zonula occludens (tight junction) with integrated gap junctions and very large macular gap junctions. Isolation after trypsination results in disaggregation of the large gap junctions and fragmentation of the tight junctions with disaggregation of their integrated gap junctions. After two to four days of incubation the restoration of the zonula occludens is complete. After approximately five days of incubation, large gap junctions are found with a patchy arrangement of particles similar to that seen in vivo.

Neuro-glial relationships at the external limiting membrane of the newt retina

The ultrastructure of the external limiting membrane of the adult newt retina and of the seleral, outer portions of the Müller cells in this region, has been examined in detail. The radial fibers of Müller expand in a conical fashion at their scleral ends to compensate for the narrowing of the photoreceptor myoids in this region. The Müller cell conical expansions are divided into two regions, a scleral mitochondria-free zone and a more vitreal mitochondrial zone. Apical Müllerian microvilli, contain fine filaments which are continuous with the filamentous mitochondria-free zone, where coated vesicles and multivesicular bodies are found. Lateral extensions of the mitochondria-free zone appear in longitudinal sections to overlap, and in tangential sections to interlock with adjacent Müller cells and so fit together like the pieces of a jig-saw puzzle. The external limiting membrane is composed of both interglial and neuroglial junctions. The most scleral component of the interglial junction consists of large gap junctions which form an extensive network between the interlocking apical processes of the Müller cells and are consistently observed in a plane perpendicular to the long axis of the Müller fibers. A second interglial junctional component, resembling a zonula adherens, is found vitreal to the interglial gap junction network. The neuroglial junctions resemble zonulae adherentes but display a frequent narrowing of the intercellular cleft and gap junctions were never observed between these cells. A second population of large interglial gap junctions are observed but these are distinguished from gap junctions of the external limiting membrane, by their proximity to the mitochondria-filled portions of the conical Müller cell expansions. Glio-glial and neuro-glial relationships are discussed with regard to the nutritional role of the Müller cell in this avascular retina.

Photoreceptor coupling in retina of the toad, Bufo marinus. I. Anatomy.

1. Red rod photoreceptors in the toad retina, which are known to be physiologically coupled, were examined for interreceptor contacts. 2. A dense network of large gap junctions was found between the inner segments of red rods, this being the only specialized site of contact that was observed between rods. Each red rod contacts an average of about four neighboring red rods with a junctional area of approximately 0.75 micrometer2. From freeze-fracture micrographs, the density of junctional particles was found to be 5 X 10(3)/micrometer2. 3. The large gap junctions were found only to connect red rods to each other in agreement with physiological data. Only small focal gap junctions were seen between red rods and cones and no junctions were observed between red and green rods. 4. It is concluded that these gap junctions are the site of coupling between the red rods and that the coupling is electrical.

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.

Loss and reappearance of gap junctions in regenerating liver.

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.

Freeze-fracture images of the zonula occludens in the mouse oviduct epithelium.

Freeze-fracture images of zonulae occludentes of the oviduct epithelium of adult mice were observed in the electron microscope. The structure of zonulae occludentes varies depending on the types of oviduct epithelial cells. Those between two secretory cells consist of 5--14 (9.2 +/- 2.0) strands interposed between the luminal and the lateral plasma membrane. The strands run irregularly making well developed anastomoses with one another. The tight junction between two ciliated cells consists of 4--14 (8.5 +/- 2.3) strands. Among them the upper 3--9 strands are unique in shape. The strands run regularly, compactly and parallel to the luminal surface and to each other with a few anastomoses. The distance between the adjacent strands is about 40 nm. Under this characteristic part several strands are very loosely distributed and run irregularly. The pattern of the strands in the zonula occludens between a secretory cell and a ciliated one resembles that between adjacent ciliated cells. The junction consists of 4--13 (8.3 +/- 2.0) strands. The characteristically dense and parallel arrangement of the strands demonstrated in this study is presumed to be related to the ciliary movement. All the zonulae occludentes in the mouse oviduct epithelium are "very tight" in type. No large gap junctions are seen in the mouse oviduct epithelium.

Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis

ABSTRACTThe fine structure of the apical ectodermal ridge of five phylogenetically divergent orders of mammals and two orders of birds was examined using transmission and freeze fracture electron microscopy. Numerous large gap junctions were found in all apical ectodermal ridges studied. This was in contrast to the dorsal and ventral limb ectoderms where gap junctions were always very small and sparsely distributed. Thus, gap junctions distinguish the inductively active apical epithelium from the adjacent dorsal and ventral ectoderms.The distribution of gap junctions in the ridge was different between birds and mammals but characteristic within the two classes. Birds, with a pseudostratified columnar apical ridge, had the heaviest concentration of gap junctions at the base of each ridge cell close to the point where contact was made with the basal lamina. Whereas mammals, with a stratified cuboidal to squamous apical ridge, had a more uniform distribution of gap junctions throughout the apical epithelium. The difference in distribution for each class may reflect structural requirements for coupling of cells in the entire ridge. We propose that all cells of the apical ridges of birds and mammals are electrotonically and/or metabolically coupled and that this may be a requirement for the integrated function of the ridge during limb morphogenesis.

Tight and gap junctions in a vertebrate inner ear.

The auditory organ of the alligator lizard has been investigated with the transmission electron microscope using methods which distinguish between tight and gap junctions. There is a continuous zone of tight junctions located near the endolymphatic surface of the organ forming a boundary between the endolymph in scala media and the interstitial spaces between the cells. No such tight junctions were observed between the perilymph of scala tympani and the interstitial fluid within the organ. Small gap junctions occur between hair cells and supporting cells and large gap junctions occur between adjacent supporting cells. The locations of the tight junctions suggest that the composition of the intercellular fluid in the receptor organ is probably more like perilymph than like endolymph. The presence of gap junctions between hair cells and supporting cells provides a possible morphological basis for the occurrence of intracellular responses to sound in supporting cells, and for elctric coupling of receptor cells.

Characteristic endothelial junctions in different segments of the vascular system

Among the structural features potentially important in the control of the integrity and permeability of the endothelial lining are its intercellular junctions. The points of interest in relation to the endothelial junctions are: their permeability to molecules of different sizes, the extent of cell to cell adhesion, and the existence of intercellular communications. Recent data have shown that alterations in endothelial junctions permeability and integrity may play an important role in inflammation, atherosclerosis as well as in the production of both hemorrhage and thrombosis. The present study was carried out to obtain more information on the fine structural organization of the endothelial junctions in different segments of the vascular system. The identification of small vessels in sectioned tissues is sometimes uncertain and since it could be of considerable importance for structural-functional correlations, we have started to study well defined sequential segments of the microvasculature. Small units consisting of an arteriole, its capillaries and the emerging venule (ACV units) were identified in the rat omentum fixed in situ . Such units were processed for thin sectioning and freeze-fracturing either as a whole or as isolated segments. On ACV units we have examined, so far, the organization of the endothelial junctions. The results showed that the intercellular junctions of the endothelium has continuous and elaborate tight junctions with enclosed large gap junctions (communicating junctions or maculae communicantes). The capillary endothelium is provided with tight junctions formed by staggered strands. The pericytic venules and the muscular venules exhibit loosely organized endothelial junctions with low profile ridges and grooves, usually devoid of particles. No gap (communicating) junctions were found in capillaries and pericytic venules, whereas the muscular venules display small, isolated gap (communicating) junctions. By extending the investigation to the large vessels, we found out that the endothelium of arteries and aorta exhibits a variable combination of well organized tight junctions and gap (communicating) junctions, whereas in the venous part of the circulation (veins, vena cava) there are less elaborate tight junctions and the gap (communicating) junctions are in low frequency. The loose venular junctions may explain low local resistance to stress and correlated increased permeability.

Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature.

Small vascular units consisting of an arteriole, its capillaries, and the emerging venule (ACV units) were identified in the rat omentum and mesentery. They were fixed in situ and processed for electron microscopy either as whole units or as dissected segments. Systematic examination of the latter (in thin sections, as well as in freeze-cleaved preparations) showed that the intercellular junctions of the vascular endothelium vary characteristically from one segment to another in the microvasculature. In arterioles, the endothelium has continuous and elaborate tight junctions with interpolated large gap junctions. The capillary endothelium is provided with tight junctions formed by either branching or staggered strands; gap junctions are absent at this level. The pericytic venules exhibit loosely organized endothelial junctions with discontinuous low-profile ridges and grooves, usually devoid of particles. No gap junctions were found in these vessels. The endothelium of muscular venules has the same type of junctions (discontinuous ridges and grooves of low profile); in addition, it displays isolated gap junctions of smaller size and lower frequency than in arterioles. The term communicating junction (macula communicans) is proposed as a substitute for gap junctions, since the latter is inappropriate, in general, and confusing in the special case of the vascular endothelium.

Ultrastructural analysis of the granulosa--luteal cell transition in the ovary of the dog.

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.

Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse.

By use of lanthanum tracer and freeze-fracture procedures it was found that granulosa-lutein cells of the pregnant mouse and rat ovaries are connected by gap junctions and septate-like zones of contact. Lutein cell gap junctions enlarge and become partially internalized by the end of the first week of gestation. Expansion of the gap junction domain appears to be due initially to intercalation of particles along borders of small gap junctions devoid of smaller non-junctional particles. The number of gap junction lined processes appearing at the cell border increases concomitantly with hypertrophy of the lutein cell during the second week of pregnancy. Strands of particulate or grooved membrane emanate from the margin of larger gap junctions undergoing interiorization. Most large gap junctions are intimately associated with elements of the smooth endoplasmic reticulum. Spherical gap junctional profiles assume a deeper location in the lutein cell and may form concentric arrays by term while true surface gap junctions appear to fragment in the post-partum corpus luteum. The modifications observed are interpreted with respect to biogenesis of the gap junction and the hormonal control of lutein cell function.