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PubMed2005

Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Blotting, Western↗
PubMed1994

Cell cycle of transdifferentiating supporting cells in the basilar papilla.

Mitosis of supporting cells has been shown to contribute to the cellular repopulation of the basilar papilla after acoustic trauma. In the present work we report data obtained with light and transmission electron microscopy after acoustic trauma in chicks. We report changes that occur in cell shape, surface morphology, intercellular junctions, nuclear shape and location, and cytoplasmic organization of supporting cells after trauma. The findings strongly suggest that supporting cells transdifferentiate and that the proliferative pattern is similar to interkinetic nuclear migration, as previously shown in the developing neural tube and basilar papilla. S-phase nuclei were positioned adjacent to the basement membrane, suggesting that interaction with the extracellular matrix may occur during the cell cycle. Supporting cells divided with the long axis of the spindle parallel to the reticular lamina and displayed no signs of intercellular communication during mitosis. This suggested to us that the fate of the progeny cells is determined prior to mitosis and that the progeny may be of identical phenotypic fate. Dividing cells had a smooth apical surface. The smooth surface may provide a marker to help identify dividing cells with scanning electron microscope analysis.

Animals↗
PubMed1996

Genomes with distinct function composition.

The functional composition of organisms can be analysed for the first time with the appearance of complete or sizeable parts of various genomes. We have reduced the problem of protein function classification to a simple scheme with three classes of protein function: energy-, information- and communication-associated proteins. Finer classification schemes can be easily mapped to the above three classes. To deal with the vast amount of information, a system for automatic function classification using database annotations has been developed. The system is able to classify correctly about 80% of the query sequences with annotations. Using this system, we can analyse samples from the genomes of the most represented species in sequence databases and compare their genomic composition. The similarities and differences for different taxonomic groups are strikingly intuitive. Viruses have the highest proportion of proteins involved in the control and expression of genetic information. Bacteria have the highest proportion of their genes dedicated to the production of proteins associated with small molecule transformations and transport. Animals have a very large proportion of proteins associated with intra- and intercellular communication and other regulatory processes. In general, the proportion of communication-related proteins increases during evolution, indicating trends that led to the emergence of the eukaryotic cell and later the transition from unicellular to multicellular organisms.

Animals↗
PubMed2003

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane.

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3-4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12.6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42.7 and 52.3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35.16%) in comparison to HLEC cultured on plastic (68.4%), as well as, HPCEC cultured either on AM or plastic (61% and 79.3%, respectively; p<0.001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52.9%; p<0.05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

Adult↗
PubMed2003

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed approximately 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Animals↗
PubMed2004

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin (Cx) protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of Cx40 and Cx43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Animals↗
PubMed1991

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes.

Several cell types participate in the formation of vitreoretinal traction membranes in proliferative intraocular disorders. The communication between these cells involves hormones, growth factors, and the interaction with extracellular matrix molecules. We have previously demonstrated a partial colocalisation of two potent mediators of cell attachment, fibronectin and vitronectin, in periretinal membranes from patients with proliferative vitreoretinopathy (PVR). We found a similar pattern of vitronectin and fibronectin deposition in proliferative diabetic retinopathy (PDR) (n = 6). Now we show the expression of the corresponding cell surface receptors, integrins, for fibronectin and vitronectin by proliferating cells in 22 periretinal membranes, including traumatic (n = 8) and idiopathic (n = 8) PVR as well as PDR membranes (n = 6). Integrins are membrane receptors for extracellular matrix macromolecules which are involved in such basic biological phenomena as embryogenesis and metastasis. Future studies on the pathogenesis of vitreoretinal proliferation will have to focus on the initiation, maintenance, and regulation of this intercellular communication network involving attachment proteins and integrins.

Diabetic Retinopathy↗
PubMed1999

Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.

In epidermis, it has been suggested, intercellular communication through gap junctions is important in coordinating cell behavior. The connexins, may facilitate selective assembly or permeability of gap junctions, influencing the distribution of metabolites between cells. Using immunohistochemistry, we have compared the distribution of connexins 26 and 43 with that of proliferating cells (Ki67 labeling) in normal epidermis, hyperplastic epidermis (tape-stripped epidermis, psoriatic lesions, and viral warts), and vaginal and buccal epithelia. Connexin 43 was abundant in spinous layers of all epidermal specimens and in vaginal and buccal epithelia. Connexin 26 was absent from the interfollicular and interductal epidermis of normal hair-bearing skin, and nonlesional psoriatic epidermis but present at very low levels in plantar epidermis. Connexin 26 was prominent in lesional psoriatic epidermis and viral warts and in vaginal and buccal epithelia. In three independent experiments connexin 26 appeared in a patchy intercellular distribution in the basal epidermis within 24 h of tape stripping, proceeding to more extensive distribution in basal and suprabasal layers by 48 h. The increase in connexin 26 preceded that in cell proliferation. In vaginal epithelium, buccal epithelium, and viral warts connexin 26 was restricted mainly to suprabasal, nonproliferating cells. In psoriatic lesional epidermis connexin 26 was also located mainly in suprabasal, nonproliferating cells. Connexin 26 was present in a patchy distribution in the basal layer of psoriatic lesional epidermis, but double labeling for connexin 26 and Ki67 showed that many connexin 26 positive basal cells were nonproliferative, suggesting that connexin 26 may be related to differentiation rather than to proliferation. These observations would be consistent with a role for connexin 26 containing gap junctions during both early and later stages of keratinocyte differentiation in hyperplastic epidermis and in vaginal and buccal epithelia.

Cell Differentiation↗
PubMed2005

Impaired cerebellar functions in mutant mice lacking DNER.

DNER is a transmembrane protein carrying extracellular EGF repeats and is strongly expressed in Purkinje cells (PCs) in the cerebellum. Current study indicated that DNER functions as a new Notch ligand and mediates the functional communication via cell-cell interaction. By producing and analyzing knockout mice lacking DNER, we demonstrate its essential roles in functional and morphological maturation of the cerebellum. The knockout mice exhibited motor discoordination in the fixed bar and rota-rod tests. The cerebellum from the knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization. Histochemical and electrophysiological analyses detected that PCs retained multiple innervations from climbing fibers (CFs) in the mutant cerebellum. Synaptic transmission from parallel fibers (PFs) or CFs to PCs was apparently normal, while glutamate clearance at the PF-PC synapses was significantly impaired in the mutant mice. Moreover, the protein level of GLAST, the glutamate transporter predominantly expressed in Bergmann glia (BG), was reduced in the mutant cerebellum. Our results indicate that DNER takes part in stimulation of BG maturation via intercellular communication and is essential for precise cerebellar development.

Animals↗
PubMed2002

Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid.

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor-sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C-expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.

8,11,14-Eicosatrienoic Acid↗
PubMed2005

Gap-junction communication between subtypes of direction-selective ganglion cells in the developing retina.

The On-Off direction-selective ganglion cells (DSGCs) in the rabbit retina comprise four distinct subtypes that respond preferentially to image motion in four orthogonal directions; each subtype forms a regular territorial array, which is overlapped by the other three arrays. In this study, ganglion cells in the developing retina were injected with Neurobiotin, a gap-junction-permeable tracer, and the DSGCs were identified by their characteristic type 1 bistratified (BiS1) morphology. The complex patterns of tracer coupling shown by the BiS1 ganglion cells changed systematically during the course of postnatal development. BiS1 cells appear to be coupled together around the time of birth, but, over the next 10 days, BiS1 cells decouple from each other, leading to the mature pattern in which only one subtype is coupled. At about postnatal day 5, before the ganglion cells become visually responsive, each of the BiS1 cells commonly showed tracer coupling both to a regular array of neighboring BiS1 cells, presumably destined to be DSGCs of the same subtype, and to a regular array of overlapping BiS1 cells, presumably destined to be DSGCs of a different subtype. The gap-junction intercellular communication between subtypes of DSGCs with different preferred directions may play an important role in the differentiation of their synaptic connectivity, with respect to either the inputs that DSGCs receive from retinal interneurons or the outputs that DSGCs make to geniculate neurons.

Animals↗
PubMed2000

Human connexin40 gap junction channels are modulated by cAMP.

OBJECTIVE: Gap junction channels provide for direct electrical coupling between cells, and play an important role in homeostasis and electrical coupling. One of the proteins that form gap junctions, Connexin40 (Cx40), shows restricted expression in the body, and is found in blood vessels and in the atrium and conduction system of the heart. We have investigated whether gap junction channels formed of Cx40 are modulated by protein-kinase-A-mediated phosphorylation. METHODS: A communication-deficient human hepatoma cell line (SKHep1) was stably transfected with human Cx40 cDNA and the properties of Cx40 gap junctions channels and their modulation by cAMP were analyzed using immunocytochemistry, Western blotting, dual patch clamp, and dye coupling. RESULTS: Administration of 1 mM 8-Br-cAMP resulted in a mobility shift of Cx40 protein on western blot and increased macroscopic gap junctional conductance between cell pairs by 46.2 +/- 12.0% (mean +/- S.E.M., n = 8). Under control conditions, single channel experiments revealed three single channel conductances around 30, 80 and 120 pS. When cAMP was added, channel conductances of 46 and 120 pS were observed. In monolayers, cAMP also increased the permeability of Cx40 gap junction channels for Lucifer Yellow by 58%. CONCLUSIONS: Macroscopic conductance and permeability of Cx40 gap junctions is strongly increased by cAMP and may play a role in the regulation of intercellular communication in the heart and vasculature.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Power frequency magnetic field exposure and gap junctional communication in Clone 9 cells.

Exposure to a power-frequency magnetic field has been reported to produce a statistically significant inhibition of gap junctional communication (GJC) in Clone 9 cells that have been pre-stressed by treatment with low concentrations of chloral hydrate (CH) [C.F. Blackman, J.P. Blanchard, S.G. Benane, D.E. House, J.A. Elder, Double blind test of magnetic field effects on neurite outgrowth, Bioelectromagnetics, 19 (1998) 204-209]. This observation might provide mechanistic insight into the possible role of electromagnetic fields (EMFs) in the carcinogenic process, since cancer cells frequently show decreased or absent GJC, and tumor promoting chemicals have been observed to inhibit GJC. Magnetic field exposure conditions were 45 Hz, 23.8 microT rms + parallel DC 36.6 microT, for 30 min of exposure. The responses of Clone 9 cells to the GJC-inhibiting effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate and the chemical CH were evaluated and compared to reported results [S.G. Benane, C.F. Blackman, D.E. House, Effects of perchloroethylene and its metabolites on intercellular communication in Clone 9 rat liver cells, J. Toxicol. Environ. Health, 48 (1996) 427-437]. Before magnetic field exposure, cells were exposed for 24 h to either 3 (nine experiments) or 5 mM (11 experiments) CH to produce GJC of 67% or 50%, respectively, relative to unexposed controls. GJC was assessed microscopically using the scrape-loading technique and a blinded protocol. No statistically significant effect was observed due to magnetic field exposure with either CH concentration.

Animals↗
PubMed2002

Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells.

Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced upregulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide- and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries.

Acetylcholine↗
PubMed1999

Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

Animals↗
PubMed1996

Participation of gap-junctional cell communication on the adaptive response in human cells induced by low dose of X-rays.

To investigate the radioadaptive response of normal cells to low-dose radiation, we irradiated human embryonic (HE) cells and HeLa cells with low-dose X-rays and examined the changes in sensitivity to subsequent high-dose X-irradiation using the trypan blue dye-exclusion test. When HE cells were irradiated by 200 cGy, the growth ratio of the living cells 5 days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a conditioning irradiation of 10-20 cGy 4 h before the irradiation of 200 cGy, the relative growth ratios increased significantly to 45-53%, and a peak was reached at a conditioning dose of 13 cGy to the cells. This conditioning effect was not observed in LeLa cells. When the HE cells were suspended in a Ca2+ ion-free medium or TPA added to the medium while receiving the conditioning irradiation of 13 cGy, the effect of the conditioning dose was not observed. This indicates that normal cells show an adaptive response to low-dose radiation and become more radioresistant. These results suggest that gap junctional intercellular communication may play a role in radioadaptive responses in human cells.

Adaptation, Physiological↗
PubMed2003

Microglia activation influences dye coupling and Cx43 expression of the astrocytic network.

Under inflammatory conditions, activated microglia are capable of producing proinflammatory cytokines that are reported to influence cell-to-cell communication. The present study was performed to evaluate the influence of microglial activation on the coupling efficiency of the astroglial network. Primary astrocyte cultures of newborn rats were cocultured with either 5% (M5) or 30% (M30) microglia. Microglial activation (rounded phagocytotic phenotype) was investigated using the monoclonal anti-ED1 antibody, and immunofluorescence with a polyclonal anti-Cx43 antibody was used to study astroglial Cx43 expression and distribution. Functional coupling of astrocytes was evaluated by monitoring the transfer of microinjected Lucifer yellow into neighboring cells. The data obtained can be summarized as follows: astroglia/M30 cocultures contained significantly fewer resting microglia and significantly more activated microglia than the M5 cocultures; significantly reduced astroglial Cx43 staining was found in M30 cocultures concurrently with a reduced number of dye coupled astrocytes; and the positive correlation of percent activated microglia with reduced astroglial Cx43 expression was highly significant, indicating that the degree of intercellular communication in the astroglial network may be modulated by the activation of microglia under in vitro conditions.

Animals↗
PubMed1985

Intercellular junctions between human odontoblasts. A freeze-fracture study after demineralization.

Intercellular junctions in the odontoblastic layer have been studied with a freeze-fracture technique. Children's tooth germs were fixed, sliced and demineralized. Samples of the pulpodentinal border were routinely prepared for freeze-fracture. Three kinds of intercellular junctions were detected between human odontoblast cell bodies: gap junctions, desmosomes and tight junctions. Numerous gap junctions are responsible for intercellular communication at different levels of the cell bodies. Focal tight junctions, parallel to the axis of the cell, and desmosomes are sites of cell-to-cell adhesion between lateral plasma membranes. At the distal end of the cell bodies, junctional complexes consist of zonular tight junctions and gap junctions. These zonular tight junctions, never before described between odontoblasts, contribute to the pseudo-epithelial organization of the odontoblastic layer. They constitute a predentin-pulp barrier, the permeability of which must be studied to establish their role in relation to dentin formation.

Adolescent↗