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PubMed1988

The arthropod gap junction and pseudo-gap junction: isolation and preliminary biochemical analysis.

The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with greater than or equal to 20 mM NaOH or greater than or equal to 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16 Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergent-alkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).

Animals↗
PubMed1997

Cx43 gap junction gene expression and gap junctional communication in mouse neural crest cells.

Although gap junctions are not known to be important in mediating cell-cell interactions amongst migratory cells, our studies showed that the connexin 43 (Cx43) gap junction gene is widely expressed in mouse neural crest cell lineages. Using in situ hybridization analysis, Cx43 expression was detected in presumptive neural crest cells emerging from the neural folds of the early postimplantation embryo. Neural crest expression of the Cx43 gap junction gene was also indicated by the analysis of transgenic mice containing a lacZ reporter construct driven by the Cx43 promoter. In neural tube explant cultures of these transgenic mice, lacZ expression was observed in the emerging neural crest outgrowths. Whole mount X-gal staining of these transgenic embryos at various stages of development showed lacZ expression in neural crest cells distributed along the entire craniocaudal axis, with expression found in both cranial and trunk neural crest cells contributing to a wide range of embryonic tissues. This included presumptive cardiac neural crest cells localized in the heart. In light of the widespread expression of Cx43 in neural crest cell lineages, dye injection studies, were carried out to determine if neural crest cells are functionally coupled via gap junctions. Such studies revealed extensive dye coupling among presumptive neural crest cells, thus demonstrating that these migratory cells are indeed gap junctional communication competent. In total, these observations suggest that gap junctions may play a role in mouse neural crest development. This possibility is particularly intriguing given the recent finding that the Cx43 knockout mice die of defects associated with the outflow tract [Reaume et al., 1995], a region of the heart in which neural crest cells are required for normal development.

Animals↗
PubMed1995

Facilitation of gap-junctional communication and gap-junction formation in mammalian cells by inhibition of glycosylation.

The effect of inhibition of glycosylation on basal and cAMP-induced homologous and heterologous gap-junctional communication was studied in various normal and transformed cell lines that express mRNA and protein for the gap-junction-forming gene, connexin43. In communication-incompetent Morris hepatoma cells, inhibition of glycosylation alone did not induce junctional communication, but enhanced cAMP-induced junctional communication severalfold. This enhancement correlated with the presence of more gap junctions at the membrane appositions, but not with an increase in connexin43 mRNA or protein in these cells. In several other normal and transformed cell lines, inhibition of glycosylation enhanced both basal as well as cAMP-induced junctional communication. Furthermore, both basal and cAMP-induced heterologous junctional communication between nontransformed RL-CL9 and several other nontransformed and transformed cells was also enhanced when glycosylation was inhibited. Our data suggest that the formation of gap junctions between cells of the same type or different types is subject to local constraints imposed by the oligosaccharide moieties of the glycoproteins of the plasma membranes of the gap-junction-forming cells and that inhibition of glycosylation abrogates such constraints. Our findings thus suggest a new basis for the communication deficiency observed in several types of transformed cells and between transformed and normal cells.

Animals↗
PubMed2001

Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture.

Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18alpha-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La(3+) also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.

Adenosine Triphosphate↗
PubMed1999

Internalization of gap junctions in benign familial pemphigus (Hailey-Hailey disease) and keratosis follicularis (Darier's disease).

Hereditary skin disorders involving acantholysis, such as Hailey-Hailey disease and Darier's disease, have been genetically linked to distinct chromosomal parts which do not code for known structural proteins. Such evidence suggests that the genomic abnormalities underlying these dermatoses may concern functional/regulatory mechanisms of keratinocyte cohesion. Epidermal communication junctions (gap junctions) are responsible for direct coupling of cells and, thus, co-ordinate the behaviour of keratinocytes within the tissue. Consequently, they remain one of the potential, and poorly studied, elements in the pathogenesis of hereditary acantholytic diseases. We have investigated the distribution and fate of gap junctions during non-immune acantholysis, using fine immunolocalization methods at the light and electron microscopic levels. Our results demonstrate normal expression of epidermal gap junction proteins, connexins 26 and 43, in non-lesional skin of Hailey-Hailey and Darier's diseases. The gap junctions were not primarily dismantled during acantholysis, typical of both of the studied dermatoses, but underwent internalization and subsequent cytoplasmic dispersion in the portions of cells which were no longer attached to the rest of the tissue. In Darier's disease, perifollicular acantholysis did not specifically concern epithelium of appendages coexpressing connexin 26 in addition to connexin 43, further indicating that the observed changes in gap junction localization were secondary to the loss of cell-cell contact. We demonstrated that the sequence of changes was identical in both diseases and that the previously described putative differences were apparently related to the degree of acantholysis present in the studied biopsies. The fate of the junctional structures and proteins, documented in the present study, is most probably a form of recycling process also used by normal keratinocytes during organogenesis and tissue differentiation.

Acantholysis↗
PubMed2001

The origin of annular junctions: a mechanism of gap junction internalization.

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3-6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

Animals↗
PubMed2000

Early migratory rat neural crest cells express functional gap junctions: evidence that neural crest cell survival requires gap junction function.

Gap junctions mediate crucial intercellular interactions during development. This study provides evidence that early migrating rat neural crest cells assemble functional gap junctions, as demonstrated by dye transfer following microinjection of single cells, which were phenotypically identified as neural crest cells by their expression of the low- affinity nerve growth factor receptor. An immunohistochemical analysis using connexin- specific antibodies revealed that migrating rat neural crest cells express the gap junction constituents connexins 43 (Cx 43) and Cx 46. We tested the hypothesis that gap junctions play an important role during early neural crest cell development by perturbing their function in migrating neural crest cells. Our data show that markedly decreasing gap junction communication between these neural crest cells in vitro with either 18alpha-glycyrrhetinic acid or anandamide decreases their survival, whereas oleamide, a less effective blocker of connexon function, had quantitatively less effect on neural crest cell death. This cell death was associated with the occurrence of DNA nicking as detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) procedure, suggesting cell death via apoptosis. The effect of 18alpha-glycyrrhetinic acid and anandamide on neural crest cell survival was reversible and was not mimicked by the structurally related compounds glycyrrhizic acid and palmitoylethanolamide, respectively, which do not uncouple cells. These results indicate that gap junctions are necessary for the survival of spinal neural crest cells.

1-Octanol↗
PubMed1983

Comparative analysis of the gap junction protein from rat heart and liver: is there a tissue specificity of gap junctions?

Gap junctions have been isolated from both rat heart and liver, tissues where junctions are typical in appearance and physiology. The purity of the fractions obtained was monitored by electron microscopy (thin-sectioning and negative staining) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The myocardial gap junctions are comprised of a single polypeptide of Mr 28,000, apparently derived from a protein of Mr 30,000. Hepatic gap junctions are also comprised of a single native protein of Mr 28,000 as previously reported. Exhaustive trypsin digestion of the isolated junctions cleaves both of these proteins similarly, while leaving their characteristic junctional lattice structures intact. However, comparison of heart and liver junctional proteins by two-dimensional peptide mapping of tryptic and alpha-chymotryptic fragments, followed by high pressure liquid chromatography, reveals no homology between these proteins.

Animals↗
PubMed1992

Molecular biology and genetics of gap junction channels.

Gap junctional communication between cells provides a mechanism for the movement of molecular information between cells via the unit of gap junction structure and function, the gap junctional channel. In the past five years, there has been rapid progress in identifying and characterizing a multigene family that is responsible for producing the gap junction polypeptides that are responsible for generating gap junctional channel oligomers between cells. The products of these genes have been referred to as connexins, and the multigene family can be categorized into two classes at present, the alpha class and the beta class. Members of these two classes can be distinguished on the basis of their primary sequence and overall predicted topological organization. The gap junction genes map to different chromosomes in both mice and humans, and these genes are utilized on a cell specific basis. Furthermore, these genes can be developmentally regulated, and multiple genes can be co-expressed simultaneously by the same cell type. Efforts to understand the precise structure-function relationship of the products of these different genes is now being approached by utilizing various expression systems. Criteria that can be used as a basis for determining membership in the multigene family is presented and discussed, as well as the rationale for using a nomenclature system for the gap junction multigene family that is based on genetic and structural relationships rather than the molecular size of the deduced protein products.

Amino Acid Sequence↗
PubMed1983

Differences between liver gap junction protein and lens MIP 26 from rat: implications for tissue specificity of gap junctions.

Liver gap junctions and gap-junction-like structures from eye lenses are each comprised of a single major protein (Mr 28,000 and 26,000, respectively). These proteins display different two-dimensional peptide fingerprints, distinct amino acid compositions, nonhomologous N-terminal amino acid sequences and different sensitivities to proteases when part of the intact junction. However, the junctional protein of each tissue is well conserved between species, as demonstrated previously for lens and now for liver in several mammalian species. The possiblity of tissue-specific gap junction proteins is discussed in the light of data suggesting that rat heart gap junctions are comprised of yet a third protein.

Amino Acid Sequence↗
PubMed2004

Selective permeability of gap junction channels.

Gap junctions mediate the transfer of small cytoplasmic molecules between adjacent cells. A family of gap junction proteins exist that form channels with unique properties, and differ in their ability to mediate the transfer of specific molecules. Mutations in a number of individual gap junction proteins, called connexins, cause specific human diseases. Therefore, it is important to understand how gap junctions selectively move molecules between cells. Rules that dictate the ability of a molecule to travel through gap junction channels are complex. In addition to molecular weight and size, the ability of a solute to transverse these channels depends on its net charge, shape, and interactions with specific connexins that constitute gap junctions in particular cells. This review presents some data and interpretations pertaining to mechanisms that govern the differential transfer of signals through gap junction channels.

Animals↗
PubMed2000

Electron crystallographic methods for investigating gap junction structure.

Gap junctions are clusters of closely packed intercellular membrane channels embedded in the plasma membranes of two adjoining cells. The central pore of the membrane channels serves as a conduit between cell cytoplasms for molecules less than 1000 Da in size. Advances in the purification of gap junctions and electron cryocrystallography and computer reconstruction techniques have produced new insights into the intercellular channel structure. Methods are described here for the purification of gap junction membranes, biochemical treatments to produce hemichannel layers ("split junctions"), assessment of the purity of gap junction preparations, electron cryomicroscopy, image processing and reconstruction, three-dimensional visualization, and interpretation. The critical step in electron crystallographic structure determination remains the isolation of crystalline material in sufficient and pure quantities for recording of electron microscope images. Along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the quality of gap junction purification is assessed using electron microscopy of negatively stained preparations. Electron microscopy is also used to assess the crystallinity of the purified gap junctions and split junctions. Electron cryocrystallography is a powerful technique for high-resolution structural characterization. Image processing is used to combine and enhance two-dimensional images. Electron crystallographic analysis is used to generate a three-dimensional structure from a set of electron micrographs. This three-dimensional information is extracted from a set of images recorded after tilting the specimen in the electron microscope stage and recombined using Fourier analysis techniques analogous to those used in X-ray crystallography. Computer modeling of the three-dimensional gap junction structures is a useful tool for analyzing hemichannel docking.

Animals↗
PubMed1996

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Alcohols↗
PubMed2002

Sharing signals: connecting lung epithelial cells with gap junction channels.

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.

Animals↗
PubMed1994

Comparison of glucocorticoid-mediated changes in the expression and function of rat hepatocyte gap junctional proteins.

Gap junctional intercellular communication (GJIC) is often modulated by chemical carcinogens and during carcinogenesis, in part, through changes in gap junction mRNA levels. However, the mechanisms by which gap junction mRNA levels are altered in either normal or cancer cells are largely unknown. Since glucocorticoids are potent modulators of gene expression and stability, we have investigated the effects of these hormones on GJIC and gap junction mRNA expression in rat hepatocytes cultured in three different media. Addition of dexamethasone to cultures of rat hepatocytes resulted in a maintenance of GJIC and both major liver gap junctional mRNAs, connexin (Cx)26 and Cx32, at levels above those in hepatocytes cultured in glucocorticoid-free media. In addition, hepatocytes cultured without dexamethasone for 24 h could be induced to communicate and increase Cx mRNA levels by the addition of dexamethasone to their medium. These media-independent changes in GJIC and gap junction mRNA levels by dexamethasone warrant further investigations into their mechanisms of action and the potential therapeutic value of glucocorticoids in the treatment of cancer.

Animals↗
PubMed1988

Reconstitution of cardiac gap junction channeling activity into liposomes: a functional assay for gap junctions.

Cardiac gap junctions were reconstituted into liposomes. To determine if reconstitution resulted in membrane channel formation, we developed an assay for channel function that used a liposome-entrapped peroxidase to detect entry of a substrate into the liposome. The data demonstrate, for the first time, that reconstituted gap junctions from heart are capable of channel-forming activity in artificial membranes.

Animals↗
PubMed2003

Transfer of biologically important molecules between cells through gap junction channels.

Gap junctions are unique intercellular channels that connect the cytoplasm of adjacent cells. They are the only channels that mediate the direct cytoplasmic exchange of small hydrophilic molecules between cells--a process called gap junctional communication. Formed by a family of integral membrane proteins called connexins, gap junctions are dynamic multifunctional complexes that are essential for healthy vertebrate development and physiology. Defects in connexin proteins, and, therefore, in gap junctional communication, are associated with a large variety of pathologies in humans and experimental animals. Thus, knowledge of the molecules that pass through gap junction channels is extremely important. However, aside from some notable cases, the repertoire of biologically important transjunctional molecules remains relatively unexplored. Indeed, the study of the intercellular transfer of endogenous molecules presents formidable challenges. Here we review developments in identifying biologically relevant molecules that pass between cells through gap junction channels.

Animals↗
PubMed2003

Nitric oxide enhances de novo formation of endothelial gap junctions.

OBJECTIVE: Gap junctions (formed by connexins, Cx) are important for functional coordination of cells in the vascular wall. However, little is known about their physiological regulation in this tissue. We examined the effects of nitric oxide (NO), an important mediator of vasomotion, wound healing and angiogenesis, on the formation of gap junctions in endothelial cells (human umbilical vein endothelial cells, HUVEC). METHODS: Flow cytometry was used to determine dye transfer through newly formed gap junctions between acutely coincubated HUVECs. Parallel experiments in wild-type HeLa cells (no connexins) and transfected HeLa cells exclusively expressing Cx43, Cx40 or Cx37 were performed to determine the specific role of Cx subtypes. The intracellular distribution of Cx40 was examined after fractionation with triton by Western blotting. Intracellular levels of cGMP and cAMP were measured by radioimmunoassay. RESULTS: The NO donor SNAP (1 microM) enhanced gap-junctional coupling in HUVECs by about 40%. This was associated with an enhanced incorporation of Cx40 into the membrane. Both effects were restricted to Cx40 as analyzed in experiments with Cx-selective HeLa cells. The NO-induced increase in cell coupling was elicited by a corresponding rise of cGMP, which secondarily increased intracellular cAMP levels. The latter was an integral part of the signal cascade, since the protein kinase A (PKA) inhibitor H89 blocked the SNAP-induced incorporation of Cx40 into the plasma membrane. CONCLUSIONS: We conclude that NO is a potent modulator of gap-junctional coupling in endothelial cells. It enhances de novo formation of endothelial gap junctions by increasing incorporation of Cx40 into the plasma membrane due to PKA activation.

Biological Transport↗