Segmental differentiations of cell junctions in the vascular endothelium. Arteries and veins

A systematic survey of endothelial junctions in elastic (aorta) and muscular (mesenteric) arteries and in medium (renal and mesenteric) and large (cava inferior) size veins has been carried out in the rat using freeze-cleaved preparations. The arterial endothelium is provided with a complex of occluding and communicating junctions (gap junctions) comparable to, though less elaborate than, that described in arterioles. The particles of the occluding junctions behave like "single unit" particles and have the tendency to remain on B faces upon membrane cleavage. In the venous endothelium the junctions take the form of long occluding junctions with few associated communicating junctions (maculae communicantes). As in arterial endothelium, the junctional particles appear preferentially on B faces in cleaved preparations. These structures, although continuous over long distances, are interrupted focally by areas in which the junctional elements are similar to those found in venules: the ridges and grooves are short, discontinuous, randomly distributed along the general line of cell contact, and often particle-free. In muscular arteries two unusual types of junctions are encountered. Both are disposed in loops over short distances along the perimeter of the cell. One type appears to be a strectched-out version of the usual combination of occluding and communcating junctions of the arterial endothelium (this type is also occasionally encountered in the venous endothelium). The other type is reminiscent of the septate junctions found in the epithelia of invertebrates but the apparent similarity remains to be checked by further work.     

A vertebrate-like blood--brain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.     

Open junctions in the endothelium of the postcapillary venules of the diaphragm

We have previously established that approximately 30% of the endothelial junctions in the pericytic venules of the mouse diaphragm are open to a gap of approximately 30--60 A, and are fully permeated by hemeundecapeptide (H11P) (mol diam approximately 20 A). To estimate the size limit for molecules that can permeate these junctions, we have administered graded tracers intravenously and studied their behavior at the level of pericytic venules in bipolar microvascular fields (BMFs) in the mouse diaphragm. Horseradish peroxidase (HRP) (mol diam approximately 50 A) permeated only approximately 50% of the open junctions of the venular endothelium. Outflow through venular junctions appeared to be modest since the tracer remained restricted to the perivenular spaces. Hemoglobin (Hb, mol diam 64 x 55 x 50 A) permeated only a few (less than 5%), and ferritin (mol diam 110 A), practically none, of the endothelial junctions of the pericytic venules. The findings suggest that under normal conditions the size limit for permeant molecules for open venular junctions is approximately 60 A. Replicas of freeze-fracture preparations from appropriate regions in BMF showed that the intercellular junctions of the venular endothelium have the same organization as previously described for the corresponding segments of the microvasculature in the omentum and mesentery: discontinuous creases or grooves either free of or marked by few intramembrane particles only. Administration of histamine (topically or systemically) and 5-hydroxytryptamine (5-HT) (topically) resulted in typical focal separations of the endothelial junctions and intramural deposits of large tracer particles (carbon black) in the postcapillary venules.     

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.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

In vivo assembly of tight junctions in fetal rat liver

Examination of glutaraldehyde-fixed, freeze-fractured livers from 14-15-day rat fetuses provided the basis for the following observations. Membrane particles align in otherwise poorly particulated areas of the presumptive pericanalicular plasma membrane (A face), frequently forming a discontinuous "honey-comb" network joining small particle islands. Even at this early stage, contiguous B-fracture faces contain furrows, rather than rows of pits, distinguishing the linear particle aggregates on the A face as developing tight junctions rather than gap junctions. Short segments of these linear arrays merge with smooth ridges clearly identifiable as segments of discontinuous tight junctions. With the continuing confluence of particulate and smooth ridge segments, mature tight junctions become fully appreciable. We conclude that tight junctions form de novo by the alignment and fusion of separate particles into beaded ridges which, in turn, become confluent and are transformed into continuous smooth ones. At 21 days of fetal life, most of the images of assembly have disappeared, and the liver reveals well-formed bile canaliculi sealed by mature tight junctions.     

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

金鱼精巢支持细胞间连接和血睾屏障

Freeze-fracture and etching technique combined with thin sectioning and lanthanum impregnation has been used for the study of Sertoli cell junctions and the blood-testis barrier formation in goldfish testis with lobular organization. Some observations and results are first given in this paper. The results of experiments can be summarized as the following: 1). Sertoli cell junctions are compound junctions of tight junctions, desmosomes and gap junctions. Tight junctions usually appear as parallel or network like ridges on the P face and fine grooves on the E face at the freeze-etching replicas. Desmosomes and gap junctions often are located between or nearby the ridges of tight junctions. In addition, endoplasmic reticulum cristae near the junction area can also be observed. 2). The number, area and density of each individual junction vary with the development and differentiation stages of germinal cells in the cyst. 3). Tight junctions can be observed at any stage during germinal cell differentiation through the period of spermatogenesis and spermiogenesis. However, they appear morphologically different as type I and type II. 4). Lanthanum can partially penetrate into the intercellular spaces of spermatogonium and early primary spermatocyte but can't penetrate after the stage of late primary spermatocyte. 5). The blood-testis barrier formation starts at the stage of pachytene spermatocytes. The formation of the blood-testis barrier is the result of the development of the tight junction from type I to type II.     

A new occludens-like junction linking endothelial cells of small capillaries (probably venules) of rat jejunum.

Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new type of junction linking endothelial cells. This new junction reveals tight junctions (zonulae occludentes) in that the adjacent plasma membranes are held closely together along lines of attachment organized in the form of a loose, but frequently discontinuous network. In contrast to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows lack any particulate components. Images of cross-fractured focal points of attachment suggest that the external leaflets of adjacent membranes are closely apposed but not actually fused, as is the case with zonulae occludentes. It appears that this new type of endothelial junction is characteristic of small venules. Thus we propose that it be termed small venule endothelial junction.     

The ultrastructure of the rat primary decidual zone

The rat primary decidual zone (PDZ) is a transitory, avascular region of transformed fibroblasts surrounding the implanting embryo. Tracer studies have indicated that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight of the tracer. To clarify the morphological basis of the permeability barrier, we have studied the ultrastructure of the PDZ with particular emphasis on the intercellular features and cellular junctions. The cells of the PDZ were large and tightly packed; their apposed membranes showed extensive interdigitations in some regions, but elsewhere they were relatively straight. Tight junctions, gap junctions, and desmosomelike junctions were observed between decidual cells. The tight junctions usually consisted of one or two points of membrane fusion, and they were oriented both parallel and perpendicular to the long axis of the PDZ. These junctions were frequently associated with gap junctions. Scattered pockets of dilated extracellular space between decidual cells contained collagen fibrils and an amorphous, dense material. These extracellular components were also sequestered by the decidual cells in deep invaginations of the cell surface that were continuous with the extracellular space. Decidual cells also exhibited flangelike processes that penetrated the basal laminae of the adjacent epithelium and capillary endothelium. Our present observations indicate that decidual cells are connected by tight junctions, and a previous study demonstrated that macromolecules up to 40 kDa readily cross the PDZ; hence, the tight junctions appear to be discontinuous. We suggest that the structures restricting the movement of large macromolecules (66 kDa and larger) across the PDZ from blood vessels to the embryo may include discontinuous tight junctions, membrane interdigitations, and amorphous intercellular material.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.     

Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules

Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of approximately 0.05 to 0.15 micrometer Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy. The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values. A similar distribution was obtained after perfusion of H1 or H2 receptor agonists coupled to ferritin (2-pyridylethylamine- ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively). The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H1 and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H2 receptors.     

Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

Studies on the juxtaglomerular apparatus

The vascular pole of the juxtaglomerular apparatus in Tupaia belangeri was studied with special reference to the intercellular contacts of the periendothelial cells and the endothelium of the vas afferens. The periendothelial cells of the vascular pole of the glomerulum are connected by numerous gap junctions; and the granulated epithelial cells are suggested to form a functional unit. Probably there is a continuity of this system throughout the entire vascular pole including (1) all granulated cells, (2) all lacis cells, (3) the mesangium cells and (4) the adjacent smooth muscle cells of the vas afferens and vas efferens. Analysis of the endothelial junctions shows a zonular arrangement of tight junctions indicating a rather tight blood-tissue barrier next to the glomerular vascular pole; The ultrastructure of the different cell types of the vas afferens is also described, emphasizing the granulated epithelial cells and their innervation.     

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.     

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK)

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.