Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D _eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D _eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P _j ) an estimate of the cytoplasmic diffusion coefficient (D _cyt = 0.7 × 10⁻⁶ cm²/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P _j of 31 × 10⁻⁵ cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P _j of 4.4 × 10⁻⁶ cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. Received: 1 December 1998/Revised: 22 February 1999     

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.     

Intercellular coupling among interstitial cells of Cajal in the guinea pig small intestine

A major difficulty in the investigation of interstitial cells of Cajal (ICC) is in identifying these cells within intact, living gastrointestinal tissues. To overcome this difficulty we developed a method to visualize ICC in the myenteric plexus region (ICC-MP) of the guinea pig ileum. Cells were identified with Nomarski optics and were injected with the fluorescent dye Lucifer yellow. The identity of the cells as ICC was verified by immunohistochemical labeling for the protein c-Kit. Using the dye coupling method we found that 24.4% (93/381) of ICC-MP were coupled to 1-21 other ICC. Octanol reduced dye coupling incidence among ICC-MP to 2% (1/49). Raising the pH in the medium to 7.8-7.9 increased the dye-coupling incidence to 86% (37/43, P<0.001). Lowering the pH to 6.4-6.8 had the opposite effect (coupling incidence 1/44). These findings demonstrate that ICC are mutually connected by channels, apparently gap junctions, that can allow the passage of both electrical currents and small molecules. As it was modulated by pH, it is likely that ICC coupling is under physiological control.     

Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106-01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch-clamp technique. These studies suggested that Cx43 in ROS cells mediated cell-cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.     

Intercellular communication is cell cycle modulated during early Xenopus laevis development

We investigated intercellular communication during the seventh and tenth cell cycles of Xenopus laevis development using microinjection of Lucifer yellow and FITC-dextran as well as freeze-fracture electron microscopy. We found that gap junction-mediated dye coupling visualized using Lucifer yellow was strongly cell cycle modulated in the tenth cell cycle. Cytoplasmic bridge-mediated dye coupling visualized via FITC-dextran was also, of course, cell cycle modulated. The basis of cell cycle-modulated gap junctional coupling was investigated by measuring the abundance of morphologically detectable gap junctions through the tenth cell cycle. These proved to be six times more abundant at the beginning than at the end of this cell cycle.     

A catalytic antibody produces fluorescent tracers of gap junction communication in living cells

The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with a k(cat) of 1.7 x 10(-5) s(-1) and a catalytic proficiency (k(cat)/k(uncat)K(m)) of 1.4 x 10(10) m(-1) (K(m) = 7 microm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.     

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.     

Gap junctional communication and compaction during preimplantation stages of mouse development

The ability of gap junction antibodies to block dye transfer and electrical coupling was examined in the compacted 8-cell mouse zygote. In control zygotes, Lucifer yellow injected into 1 cell transferred to the rest of the embryo. When antibodies raised against the major protein extracted from gap junctions were co-injected with Lucifer yellow, dye transfer failed in 86% of the zygotes tested and electrical coupling was almost completely inhibited. Subsequently, the antibody-containing cells were extruded. When the antibodies were injected into 1 cell at the 2-cell stage, 82% of the zygotes divided normally to the 8-cell stage. Cells containing gap junction antibodies were uncompacted, but continued to divide. We conclude that these antibodies inhibit gap junctional communication in the early mouse zygote and that communication through gap junctions may be involved in the maintenance of compaction.     

Nogo-66 inhibits the dye-coupling of astrocytic gap junctions in vitro

Communication between astrocytes via the gap junction is crucial for maintaining homeostasis of the extra-neuronal microenvironment of the central nervous system. Dysfunction of astrocytic gap junctions is involved in many brain disorders. Our previous studies demonstrated a novel co-localization of Nogo-66 receptor at glial gap junctions in rat cerebellum and posterior pituitary. The present study was aimed at exploring whether Nogo-66 can modulate glial gap junctions in vitro. We confirmed the co-localization of Nogo-66 receptor with Cx43 in cultured astrocytes, and stimulated astrocytes with myelin extracts, or Nogo-66-Fc conditioned medium. Finally, we expressed and purified a functionally effective GST-Nogo-66 peptide. Lucifer yellow transfer assay was adopted to measure the gap junction permeability. The results showed that the spreading of Lucifer yellow was inhibited significantly by all three treatments as compared with their corresponding controls. Therefore, this study shows a novel inhibitory effect of Nogo-66 on the permeability of astrocytic gap junctions, suggesting a presumable role of Nogo-66 receptor in modulating the glial gap junction.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Lucifer yellow - an angel rather than the devil

The fluorescent dye Lucifer yellow (LY) was introduced in 1978, and has been extremely useful in studying cell structure and communications. This dye has been used mostly for labelling cells by intracellular injection from microelectrodes. This review describes the numerous applications of LY, with emphasis on the enteric nervous system and interstitial cells of Cajal. Of particular importance is the dye coupling method, which enables the detection of cell coupling by gap junctions.     

Gap junctional intercellular communication in cells isolated from urethane-induced tumors in A/J mice

Studies using normal or neoplastically transformed established mouse lung epithelial cell lines revealed a reduction in gap junctional, intercellular communication (GJIC) with transformation. To determine the stage in tumor development at which GJIC is interrupted, we used the well-established model of lung tumors induced in strain A/J mice by urethane. In this system, tumor development follows a well-characterized pattern; hyperplasias, adenomas, and carcinomas are manifested at approximately 8, 16, and 40 weeks after urethane treatment, respectively. GJIC levels were examined using a novel technique where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye, Lucifer yellow, causing dye penetration into cells growing on the conductive part of the slide. Migration of the dye through gap junctions to nonelectroporated cells growing on the nonconductive area is then microscopically observed under fluorescence illumination. Unexpectedly, primary cells cultured from urethane-induced tumors, even late stage carcinomas, possessed extensive GJIC immediately upon isolation. Upon passage for several months however, these cells lost GJIC. These results suggest that the molecular changes that lead to the formation of the tumor in vivo are not sufficient to interrupt gap junctions. Propagation of tumor cells in culture induces additional alterations that can lead to gap junction closure.     

Cultured astrocytes from a syncytium after maturation

The formation of functional gap junctions between astrocytes was investigated during differentiation of these cells in culture. Precursor cells of GFA (glial fibrillary acidic) protein-positive astrocytes were cultured in a chemically defined medium as a homogeneous population. These cells were rarely coupled to one neighbour, as revealed by electrical and dye coupling and never formed a large syncytium, as investigated by injection and spread of Lucifer Yellow. Differentiation with respect to GFA protein accumulation can be induced in these cells by culturing in horse serum-containing medium. The formation of functional junctions developed within 2 weeks in about 20% of the cells. Coupled cells formed a large syncytium. When the astrocytes were co-cultured with primary cerebellar cells (consisting predominantly of small neurons) after the switch to serum-containing medium the percentage of coupled astrocytes increased to about 65%. Again the coupled cells formed a large syncytium. Since no physical contact was possible between the astrocyte cultures and the primary cerebellar cells the stimulation of coupling had to be signalized by soluble factor(s).     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

Gap junctional communication during neuromuscular junction formation

We have tested whether gap junctions form between nerve and muscle during their initial contact, before establishing the chemical synapse. Embryonic Xenopus stage 18-20 myotomes and neural tubes were permeabilized with DMSO to load appropriate reagents, dissociated, and cocultured. When myotomes, loaded with Lucifer yellow, were cocultured with unlabeled neural tube cells, 23% of the neurons contained dye after 24 hr. Affinity-purified gap junction antibodies loaded into myocytes or neurons reduced neuronal labeling significantly to 5%. [3H]uridine nucleotide transfer was observed in both directions between myocytes and neurons. Again gap junction antibodies substantially reduced recipient label. In all cases preimmune IgGs did not reduce transfer. When acetylcholine receptor clustering was examined in cultures containing gap junction antibodies, no difference in the number of neuronally induced AChR clusters was observed. This suggests that the cluster-inducing signal between nerve and muscle does not pass through gap junctions.     

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current “two-cell model” of the stria vascularis.