Monoclonal antibody ECCD-1 inhibits intercellular communication in teratocarcinoma PCC3 cells

The monoclonal antibody ECCD-1 recognizing a certain class of cell surface proteins inhibits the Ca²⁺-dependent cell-to-cell adhesion in teratocarcinoma stem cells. In this paper, we studied the effect of ECCD-1 on cell-to-cell communication in PCC3 cells by measuring the transfer of lucifer yellow between cells. To this aim, PCC3 cells were cultured in the presence of ECCD-1 for various periods, and then the fluorescent dye was injected into a cell located in the center of cell colonies, followed by counting number of cells to which the dye was transferred. The results showed that ECCD-1 inhibits the dye transfer between cells, suggesting that the Ca²⁺-dependent cell-to-cell adhesion system (CDS) is essential for the functions of gap junction.     

Rapid automated multichannel microspectrofluorometry : A new method for studies on the cell-to-cell transfer of molecules

A new microchemical method is described to study the transfer of molecules between neighboring cells: rapid multichannel microfluorometry. The cell-to-cell transfer rates of metabolites or fluorescent tracers (microinjected by a microelectrophoretic or piezoelectric technique) are followed at pace with the in situ velocities of such processes via multisite topographic monitoring of fluorescence in injected cell and neighbors (continuously before, during and after microinjection). Furthermore, the sensitivity of the method allows the kinetic study of cell-to-cell transfer using metabolites (e.g., glucose-6-P) which are not fluorescent themselves, but which elicit associated changes in the redox state of endogenous fluorochromes, i.e. NAD(P) NAD(P)H transients.Since the cell-to-cell transfer of chemicals is known to proceed in various cells and tissues via intercellular junctions, the above technique was applied to a known coupled system (e.g. Chironomus salivary gland) and an uncoupled system (e.g. L cells). The cell-to-cell transfer of fluorescein was observable kinetically in Chironomus salivary glands, liver, Chang liver and Chang conjunctiva cultures. Equilibration of the dye between injected cell and neighbor was completed within a few hundred msec to a few sec, corresponding to an intercellular half total flux per unit concentration difference (g_f) of ˜2–10 × 10⁴ μm³/min. Such transfer was practically basent or a rare occurrence in L cells. In NCTC 8739 and L cells, the cell-to-cell transfer of glucose-6-P seemed to occur more frequently than that of fluorescein, suggesting the possibility that glucose-6-P (or a catabolite) may move through non-junctional regions of the cell membrane. Thus multichannel microfluorometry makes possible the study of tracer or metabolite transfer, without the need for multiple implantation of electrodes and with more kinetic detail than obtainable by other tracer techniques.     

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

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

Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.     

Gap junction intercellular communication during lymphocyte transendothelial migration

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.     

Endothelial cell motility is compatible with junctional integrity

Confluent endothelial cells in culture are generally regarded as a model of resting endothelium in blood vessels (i.e., forming junctions at points of cell-cell contact, losing ability to proliferate in response to growth factors, and remaining stationary). However, incompatibility between junctional integrity and endothelial cell motility remains uncertain. The aim of this study was to determine whether endothelial cells (in colonies generated from differentiating embryonic stem cells in contact with OP9 stromal cell layer) have a resting endothelial phenotype (i.e., lack motility). Time-lapse analyses showed that though endothelial cells were connected to each other through adherens junctions and tight junctions, they were moving continuously within the colonies. Endothelial cell movement was accompanied by formation of lamellipodia, which transiently accumulated green fluorescent protein-tagged beta-actin and p41-Arc (a subunit of the actin-related protein 2/3 complex) at their anterior tips, suggesting that the movement is an active behavior of endothelial cells. Endothelial cell-specific expression of yellow fluorescent protein-tagged vascular endothelial-cadherin and claudin-5 revealed that adherens junctions and tight junctions persisted during endothelial cell migration. Furthermore, intercellular junctions underwent dynamic remodeling at the leading edge of moving endothelial cells. These results suggest that endothelial cells can remain highly motile without losing intercellular junctions.     

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.     

Mechanosensitivity and intercellular communication in HOBIT osteoblastic cells: a possible role for gap junction hemichannels

Mechanically induced intercellular Ca2+ signalling was investigated in differentiated HOBIT osteoblastic cells. HOBIT cells express connexin43 clustered at the cell-to-cell boundary and display functional intercellular coupling assessed by intercellular transfer of Lucifer yellow. Mechanical stimulation of single cells, besides leading to an intracellular Ca2+ rise, induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. Suramin and apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signalling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18alpha-glycyrrhetinic acid.     

Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration

Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single-cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.     

Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III

Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout pigs were produced with transgenic pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the transgenic pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO pig fetuses were also used for NT to produce cloned KO pigs. Two healthy clone pigs were born. These pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original transgenic pigs and less than a half levels of the alphaGal epitopes existed in wild-type pig cells.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Development of intercellular communication during the epithelial reorganization of a renal cell line (LLC-PK1)

Junctional permeability determinations after microinjection of the fluorescent tracer, Lucifer Yellow CH, show that the cells in confluent monolayers of the renal epithelial cell lines LLC-PK1 and A6 are interconnected by intercellular junctions. This cell-to-cell communication network permits the fluorescent dye to diffuse from the microinjected cell into multiple adjacent neighboring cells. Cell-to-cell diffusion of the fluorescent dye was not observed at pH 6.0. Full recovery occurred, however, when the pH of the extracellular medium was adjusted to 7.4. To provide a sensitive index of the averaged efficacy of junctional communication, we measured the number of cells that survived ouabain treatment in a 50% mixture of wild and ouabain-resistant mutant LLC-PK1 cells. Electron probe microanalysis in uncoupled cells showed that ouabain treatment produced two populations of cells, with totally different intracellular Na+ and K+ content. Under this condition, only 50% of the population survived after 48 h of treatment. When ouabain treatment was initiated 24 h after plating, however, 100% survival was observed, and the cells contained uniform intracellular Na+ and K+ concentration. This finding is consistent with the theory that this protective effect is mediated through the presence of the functional communicating intercellular junctions. When ouabain was applied at different times after plating, full protection is reached by 2 h. The early development of cell-to-cell communication, which precedes the development of the occluding junctions and several transport systems by several hours, is consistent with the involvement of the intercellular junctions in the synchronization of the polarization process.     

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.     

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.     

Reactive oxygen species and the regulation of hyperproliferation in a colonial hydroid

Colonies of Podocoryna carnea circulate gastrovascular fluid among polyps via tubelike stolons. At polyp-stolon junctions, mitochondrion-rich cells in part regulate this gastrovascular flow. During competition, colonies hyperproliferate nematocytes and stolons; nematocysts are discharged until one colony is killed. Hyperproliferation then ceases, and normal growth resumes. Here, competing colonies were treated with azide, which inhibits respiration and upregulates reactive oxygen species (ROS). After the cessation of competition, azide-treated colonies continued to hyperproliferate. In azide-treated competing colonies, however, mitochondrion-rich cells were found to produce similar amounts of ROS as those in untreated competing colonies. Subsequent experiments showed that both azide treatment and competition diminished the lumen widths at polyp-stolon junctions, where mitochondrion-rich cells are found. In competing colonies, these diminished widths may also diminish the metabolic demand on these cells, causing mitochondria to enter the resting state and emit more ROS. Indeed, results with two fluorescent probes show that mitochondrion-rich cells in competing colonies produce more ROS than those in noncompeting colonies. In sum, these results suggest that competition perturbs the usual activity of mitochondrion-rich cells, altering their redox state and increasing ROS formation. Via uncharacterized pathways, these ROS may contribute to hyperproliferation.     

ROCK and Dia have opposing effects on adherens junctions downstream of Rho

Adherens junctions (AJs) are crucial for maintaining the integrity of epithelial tissues and are often disrupted during tumour progression. Rho family proteins have been shown to regulate adherens junctions. We find that activation of the effector kinase ROCK and acto-myosin contraction disrupts AJs downstream of Rho. In contrast, signalling through the Rho effector Dia1 is required to ensure a dynamically stable interface between cells and the maintenance of adherens junction complexes. The ability of Dia1 to regulate the actin network is crucial for the localization of adherens junction components to the cell periphery.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Calcium-induced assembly of adherens junctions in keratinocytes

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.     

Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek's Disease Virus

Marek's Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-to-cell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.     

Modulation of metabolic communication through gap junction channels by transjunctional voltage; synergistic and antagonistic effects of gating and ionophoresis

Gap junction (GJ) channels assembled from connexin (Cx) proteins provide a structural basis for direct electrical and metabolic cell-cell communication. Here, we focus on gating and permeability properties of Cx43/Cx45 heterotypic GJs exhibiting asymmetries of both voltage-gating and transjunctional flux (J(j)) of fluorescent dyes depending on transjunctional voltage (V(j)). Relatively small differences in the resting potential of communicating cells can substantially reduce or enhance this flux at relative negativity or positivity on Cx45 side, respectively. Similarly, series of V(j) pulses resembling bursts of action potentials (APs) reduce J(j) when APs initiate in the cell expressing Cx43 and increase J(j) when APs initiate in the cell expressing Cx45. J(j) of charged fluorescent dyes is affected by ionophoresis and V(j)-gating and the asymmetry of J(j)-V(j) dependence in heterotypic GJs is enhanced or reduced when ionophoresis and V(j)-gating work in a synergistic or antagonistic manner, respectively. Modulation of cell-to-cell transfer of metabolites and signaling molecules by V(j) may occur in excitable as well as non-excitable tissues and may be more expressed in the border between normal and pathological regions where intercellular gradients of membrane potential and concentration of ions are substantially altered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.