Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer

Summary Vascular endothelial cultures, derived from large vessels, retain many of the characteristics of theirin vivo counterparts. However, the observed reduction in size and complexity of intercellular gap and tight junctions in these cultured cells (Larson, D.M., and Sheridan, J.D., 1982,J. Cell Biol. 92:183) suggests that important functions, thought to be mediated by these structures, may be alteredin vitro. In our continuing studies on intercellular communication in vessel wall cells, we have quantitated the extent of junctional transfer of small molecular tracers (the fluorescent dye Lucifer Yellow CH and tritiated uridine nucleotides) in confluent cultures of calf aortic (BAEC) and umbilical vein (BVEC) endothelium. Both BAEC and BVEC show extensive (and quantitatively equivalent) dye and nucleotide transfer. As an analogue of intimal endothelium, we have also tested dye transfer in freshly isolated sheets of endothelium. Transfer in BAEC and BVEC sheets was more rapid, extensive and homogeneous than in the cultured cells, implying a reduction in molecular coupling as endothelium adapts to culture conditions. In addition, we have documented heterocellular nucleotide transfer between cultured endothelium and vascular smooth muscle cells, of particular interest considering the prevalence of myo-endothelial junctionsin vivo. These data yield further information on junctional transfer in cultured vascular endothelium and have broad implications for the functional integration of the vessel wall in the physiology and pathophysiology of the vasculature.     

Functional Effects of Casein Kinase I-Catalyzed Phosphorylation on Lens Cell-to-Cell Coupling

The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49 was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease [Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568–2564]. In 9-day old lens cultures, in which gap junctions are composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49, the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens. Received: 31 July 2000/Revised: 12 January 2001     

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and alpha-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

Summary The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×10⁴ cells/cm², where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells).Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Junctional transfer in cultured vascular endothelium: I. Electrical coupling

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.     

Induction of intercellular communications in epithelial cell cultures

A popular criterion of cell-cell communication in tissue cultures is dye coupling: the ability of the injected fluorescent dye of low molecular weight to be transferred from one cell to another. We report about a new factor which induces cell-to-cell dye coupling in previously uncoupled epithelial sheets. Paradoxically it is the standard fluorescent microscopy itself (that is, blue light of 320- to 480-nm wavelength) which induces rapid morphological alterations of cell culture followed by the transfer of fluorescent dye from one cell to another. Thus monitoring cell-cell dye coupling by fluorescent microscopy may itself induce the dye coupling in previously uncoupled epithelial cells.     

Measurement of gap junctional communication by fluorescence activated cell sorting

Summary Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.     

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.     

Gap junction formation between normal and reaggregated endoderm cells ofXenopus laevis neurulae

Summary Using freeze-fracture electron microscopy and fluorescent dye injection we have analysed the contacts between cells of the deeper endoderm taken from neurulae ofXenopus laevis. Endodermal cells in situ have large 1.5 m diameter gap junctions composed of 8 nm P-face particles and corresponding E-face pits. Beside gap junctions, particle aggregates typical of desmosomal plaques are present but there are no tight junctions. The dissociation of endoderm into single cells involves profound structural alterations in the surface membrane including the complete disappearance of junctional structures among them gap junctions. The reaggregation of endoderm cells leads to the restoration of the surface membrane IMP (Intra Membrane Particle) pattern and, after ca. 30 min, to the establishment of functional pathways allowing for the intercellular transfer of fluorescent dye. Concomitantly gap junctions reappear. The observation that the dissociation and reaggregation of endodermal cells involves IMP alterations which go beyond the cell junctions themselves is discussed as an adaptation of the plasma membrane to changing environmental conditions.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Development of Sertoli cell junctions in vitro--a freeze-fracture study

Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo.     

Contacts between pigmented retina epithelial cells in culture.

The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

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.     

CELL CONTACTS IN THE MOUSE MAMMARY GLAND : I. Normal Gland in Postnatal Development and the Secretory Cycle

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.     

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.     

Primary culture of capillary endothelium from rat brain

To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.     

Development of sertoli cell junctions in vitro—A freeze-fracture study

Summary Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo. This study was supported by Grant 801D185 (Dr. Posalaky) from the Medical Education and Research Foundation, St. Paul-Ramsey Hospital.