Inhibition of metabolic cooperation in Chinese hamster V79 cells by various organic solvents and simple compounds

Gap-junctional intercellular communication is a biological process implicated in the regulation of cell proliferation and differentiation. Metabolic cooperation between 6-thioguanine-sensitive and resistant Chinese hamster cells, in vitro, has been used as a means to detect chemicals which can inhibit this form of intercellular communication. To further characterize this in vitro system as a potential screening assay for potential teratogens, tumor promoters and reproductive toxicants, a series of common solvents as well as other chemicals representing eight different functional groups, i.e., alcohols with straight or side chains, glycols, ketones, esters, ethers, phenols, aldehydes, amines and amino compounds and oxygen-heterocyclic compounds, were tested for their ability to inhibit colony-formation and to inhibit metabolic cooperation. A wide range of effects were observed which suggested a structure/activity relationship between a chemical's ability to inhibit gap junction-mediated intercellular communication and the cytotoxicity of a chemical. Possible mechanisms affecting the modulation of gap junctional communication by these chemicals are discussed.Abbreviations: Hypoxanthine guanine, phosphoribosyltranferase, HG-PRT; 6-thioguanine, 6-TG.On leave from: Beijing Municipal Research Institute of Environmental Protection, Beijing, People's Republic of China     

Gap junction remodeling and cardiac arrhythmogenesis: cause or coincidence?

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins and, in in vitro expression systems, gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isoforms. Spatially defined patterns of expression of three connexin isoforms - connexin43, connexin40 and connexin45 - form the cell-to-cell conduction pathways responsible for the orderly spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a common feature of human heart disease conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions and/or quantitative alterations in connexin expression, notably reduced ventricular connexin43 levels. The idea that such changes may contribute to the development of a pro-arrhythmic substrate in the diseased heart has gained ground over the last decade. Recent studies using transgenic mice models have raised new opportunities to explore the significance of gap junction remodeling in the diseased heart.     

Rat myometrial smooth muscle cells show high levels of gap junctional communication under a variety of culture conditions

Summary Gap junctional communciation was examined in rat myometrial smooth muscle cells cultured under a variety of conditions. As a functional measure of gap junctional communication, donor cells were microinjected with the fluorescent dye, Lucifer yellow, and the transfer of dye from donor cells to primary neighbor cells was monitored by fluorescence microscopy. In a myometrial smooth muscle cell line established from midgestation (Day 10) rats, high levels of dye transfer, in excess of 90%, were observed in primary cultures and at Passages 1 and 10. A slight decrease in dye transfer to 75% was observed at Passage 5. Similarly, high levels of dye transfer were observed in a smooth muscle cell line established from the myometrium of a late-gestation (Day 19) rat under subconfluent as well as confluent culture conditions. Myometrial smooth muscle cell cultures established from sexually immature 19-day-old rats also exhibited high levels of dye transfer in primary cultures and at Passage 10. Treatment of primary myometrial smooth muscle cell cultures derived from immature 19-day-old rats with 17β-estradiol (50 ng/ml) and 4-pregnen-3,20-dione (150 ng/ml) for 48 h in vitro had no significant effect on the high levels of dye transfer. Thus, extensive dye transfer was observed in the rat myometrial smooth muscle cells under all culture conditions examined, regardless of sexual maturity or gestational stage of the animal, in vitro hormone treatment, or cell density.     

Physiological levels of melatonin enhance gap junction communication in primary cultures of mouse hepatocytes

Gap junction communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. Melatonin, a hormone secreted by the pineal gland, has putative oncostatic properties. Intercellular communication through gap junctions was assessed by microinjecting Lucifer yellow fluorescent dye into primary hepatocytes and visualizing the spread of the dye to adjacent neighboring cells using phase contrast/fluorescent microscopy. Treatment of primary hepatocyte cultures with a physiological range of melatonin concentrations for 24 h prior to microinjection resulted in significant enhancement in intercellular communication at 0.2 and 0.4 nmol/L but not at lower (0.1 nmol/L) or higher (0.8 or 1.0 nmol/L) concentrations. A time-dependent study showed that the changes in intercellular communication began 10 h after melatonin treatment and reached a maximum at 12 h of treatment. This nonlinear, functional gap junction response to melatonin occurred in the physiological concentration range detected in blood of mammals during nightly releases of the hormone by the pineal gland. These melatonin levels may affect the ability of gap junction communication to exert cell growth control in vivo. The uneven decline between individuals in nocturnal release of melatonin that occurs with age could identify potentially sensitive subpopulations susceptible to developing pathologies involving alterations in biological processes dependent on gap junction communication. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pannexins,distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ‐forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co‐expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.     

Degradation of Connexin43 Gap Junctions Involves both the Proteasome and the Lysosome

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1–3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin–proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.     

Nickel-induced increases in gap junctional communication in the uterine cell line SK-UT-1

Summary Previous studies have suggested that gap junctions may have a role in various uterine functions, including parturition. Because nickel has been demonstrated to increase uterine contractility in vitro, the effect of nickel (II) chloride on gap junctional communication was assessed in a tumorigenic uterine cell line, SK-UT-1 (ATCC HTB 114). Cells were exposed in vitro to 25 and 50 μM NiCl₂ for 24 h or 100 μM NiCl₂ for 3, 12, and 24 h, then functional gap junctional communication was measured as the transfer of Lucifer yellow dye from microinjected donor cells to their primary neighbor cells. Dye transfer was significantly increased only in cell cultures exposed to 100 μM NiCl₂ for 24h, compared to untreated controls, lower doses, and shorter exposure periods. This response was inhibited by the simultaneous co-treatment of SK-UT-1 cells with magnesium by adding 100 μM MgSO₄ to the dosing medium. Possible mechanisms and implications for these findings are discussed.     

Biochemical and immunological approaches to the study of gap junctional communication

Summary Studies on gap junctions isolated from rat liver by a procedure that avoids exogenous proteolysis (Hertzberg, E. L.; Gilula, N. B.; J. Biol. Chem. 254: 2138–2147; 1979) are described. The original isolation procedure was modified to increase the yield and has been extended to the preparation of gap junctions from mouse and bovine liver. Peptide map studies showed that the 27,000-dalton polypeptides present in liver gap junction preparations from all three sources are homologous and are not derived from other polypeptides of higher molecular weight that are observed in cruder preparations. Similar studies with lens fiber junctions demonstrated no homology between liver and lens junction polypeptides. Antibodies to the lens junction polypeptide did not cross-react with the liver gap junction polypeptide, further supporting this conclusion. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. Research in the laboratory was supported by grants to Dr. Gilula from the National Institute of Health (HL 16507 and GM 24753).     

Concentration/response effect of 2,2′, 4,4′, 5,5′-hexabromobiphenyl on cell-cell communication in vitro: assessment by fluorescence redistribution after photobleaching (“FRAP”)

Inhibition of gap junction-mediated cell-cell communication might be a mechanism for several types of cellular dysfunctions, including tumor promotion. Although many different assays have been designed to measure gap junction-mediated intercellular communication, we applied a new technique, termed Fluorescence Redistribution After Photobleaching (FRAP), to assess the ability of a known tumor promoter, 2,2, 4,4, 5,5-hexabromobiphenyl (245-HBB), to inhibit cell-cell communication in a concentration-dependent manner. WB-F344 (rat epithelial) cells were plated at low density, exposed to noncytotoxic concentrations of 1, 5, or 20 µg 245-HBB/ml medium, and stained with 6-carboxyfluorescein diacetate. Single cells in pairs or clusters of touching cells in each exposure group were examined with FRAP. The results revealed an inverse correlation between the degree offluorescence redistribution in photobleached cells and the concentration of 245-HBB. Therefore, FRAP appears to be a sensitive and rapid technique for determining complete or partial inhibition of chemically induced intercellular communication in vitro. These results also provide further evidence for the ability of 245-HBB to inhibit gap junction-mediated cell-cell communication in a concentration-dependent manner.Abbreviations 6-CFDA 6-carboxyfluorescein diacetate - FRAP fluorescence redistribution after photobleaching - 245-HBB 2,2, 4,4, 5,5-hexabromobiphenyl Michigan Agricultural Experiment Station journal article No. 12531.     

Cell junctions and intercellular communication

Summary We have compared intercellular communication in normal and regenerating rat liver. Gap junctions are greatly reduced in size and numbers 29 to 35 hr after hepatectomy, but we still find some 90% of hepatocytes coupled by electrophysiological criteria. The spread of dyes such as carboxyfluorescein however is very limited in the regenerating organs as compared to the situation in the controls. We show how the apparent discrepancies between morphological and physiological data can be reconciled. We also present a summary of preliminary findings on the biosynthesis of gap junction protein and some of the conclusions one can draw from the sequence of 58 amino acids at the amino terminal of the protein. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. The original research described was supported by Grants GM 06965 and RR 07003 from the National Institute of Health, and funds from the North-west Area Foundation. David Meyer and Barbara Yancey were the recipients of NIH postdoctoral fellowships (NS 06240 and AM05700). This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

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.     

Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). In the present study, the role of[Ca²⁺]_i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca²⁺ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca²⁺]_i, and was not prevented by loading cells with the intracellular Ca²⁺ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca²⁺]_i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca²⁺]_i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca²⁺]_i, within physiological levels is not sufficient to abolish GJIC. However, Ca²⁺-dependent processes that are active at normal resting [Ca²⁺ _i appear to be required for the maintenance of GJIC.Abbreviations [Ca²⁺] cytosolic free Ca²⁺ concentration - GJIC gap junctional intercellular communication - SLDT scrape-loading/dye transfer assay - DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane - tBuBHQ 2,5-di(tert-butyl)-1,4-benzohydroquinone - LDH lactate dehydrogenase - ER endoplasmic reticulum - Fura-2 1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid - BAPTA bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid - Fura-2/AM and BAPTA/AM are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively     

The role of junctional communication in animal tissues

Summary Permeable intercellular junctions are a common feature of most animal tissues. These junctions allow the free exchange of small ions and molecules between all the cells in coupled populations. Such limited syncytial interaction contributes to the integration of individual cells into organized tissues. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.