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PubMed1990

Time delays in propagation of cardiac action potential.

Recent work has suggested that discrete time delays could occur between cardiac cells under physiological conditions. To investigate the existence of such time delays, monolayers of 10-day chick-embryo ventricular myocytes were grown in cell culture on arrays of extracellular microelectrodes. The most closely spaced recording electrodes were on 20-microns centers. Some pairs of electrodes recorded from different points on the same cell, whereas other pairs straddled intercellular junctions. The preparation was electrically paced to obtain a repeatable propagation pathway. Arrival times of activation at each microelectrode were measured. After control recordings, the preparation was exposed to medium containing 20 mM NH4Cl for 15 min and then returned to normal medium. This intervention produces transient intracellular acidification that decreases intercellular coupling. After acidification, the average (multicellular) conduction velocity decreased to about two-thirds of the control value. Propagation velocity measured between most electrode pairs decreased proportionately. However, disproportionately long propagation delays of up to 410 microseconds were observed between some pairs of electrodes in various experiments. The delays recovered as pH returned to normal. The localized long delays were thought to be due to decreased intercellular coupling at gap junctions.

Action Potentials↗
PubMed2000

Intercellular communication and cell proliferation in precision-cut rat liver slices: effect of medium composition and DDT.

Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1'-bis(p-chlorophenyl)-2, 2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2'-deoxyuridine (BrdU) incorporation. In addition, the effect of different culture media (RPMI and WME) on control and DDT influenced Cx32 expression and cell proliferation was determined. Cx32 expression in control precision-cut liver slices was maintained during 8 h of culturing, but decreased after prolonged culturing. Control cell proliferation was significantly higher when WME was used as culture medium than when RPMI was used. In slices treated with DDT Cx32 expression was decreased. In slices cultured in RPMI medium, this decrease preceded a dose-dependent increase in cell proliferation. These results show the usefulness of precision-cut liver slices in studying cellular proliferation and intercellular communication.

Animals↗
PubMed2002

Gap junctions and connexin expression in the normal and pathological central nervous system.

Gap junctions are widely expressed in the various cell types of the central nervous system. These specialized membrane intercellular junctions provide the morphological support for direct electrical and biochemical communication between adjacent cells. This intercellular coupling is controlled by neurotransmitters and other endogenous compounds produced and released in basal as well as in pathological situations. Changes in the expression and the function of connexins are associated with number of brain pathologies and lesions suggesting that they could contribute to the expansion of brain damages. The purpose of this review is to summarize data presently available concerning gap junctions and the expression and function of connexins in different cell types of the central nervous system and to present their physiopathological relevance in three major brain dysfunctions: inflammation, epilepsy and ischemia.

Animals↗
PubMed1994

Gap junction alterations in the failing heart.

Electrical and mechanical integration between myocytes is mediated by three types of intercellular junction, the fascia adherens, desmosome and gap junction. Gap junctions are responsible for electrical coupling, and consist of clusters of plasma membrane channels that directly link the cytoplasmic compartments of neighbouring cells. Each channel consists of two hemichannels (connexons; one from each plasma membrane) aligned across the narrow extracellular gap, and each hemichannel is constructed from six connexin molecules. Using specific anticonnexin43 antibodies for immunofluorescence localization in combination with confocal laser scanning microscopy, alterations in the expression of connexin43 gap junctions have been investigated in chronic ischaemic heart disease and heart failure due to ischaemic cardiomyopathy. Two major alterations are apparent: (1) disturbance in the spatial distribution of gap junctions at the border zone of healed infarcts, and (2) reduction in the quantity of immunodetectable connexin43 in regions of normal gap junction distribution distant from infarct scars. These changes are likely to contribute to electromechanical dysfunction in ischaemic heart disease and heart failure, and appear to form part of a wider pattern of altered expression of different connexin types in the diseased heart.

Connexin 43↗
PubMed1979

The effect of hyperglycemia on brain capillary permeability in the lizard, Anolis carolinensis. A freeze-fracture analysis of blood-brain barrier pathology.

The anatomical basis of the blood-brain barrier in the American chameleon, Anolis carolinensis, is the system of tight intercellular junctions that occurs between apposed endothelial cells of brain capillaries. Under normal physiological conditions, capillaries in the brain cortex of these animals remain sealed by interendothelial zonulae occludentes and, consequently, escape of exogenous tracer proteins such as horseradish peroxidase (HRP) into the extracellular compartment of the central nervous system is prevented. Systemic injection of 2.7 mg of D-glucose into chameleons results in increased brain capillary permeability, as evidenced by escape of HRP or Trypan blue into the intercellular spaces of central neuropil. Freeze-fracture analysis of brain capillary endothelia of glucose-hyperglycemic lizards revealed no alteration of the ridge and groove construction of endothelial tight junctions, indicating that although the blood-brain interface becomes leaky during severe hyperglycemia, the capillary zonulae occludentes are not affected. Evidence obtained in this study strongly supports the notion that the increased capillary permeability is the result of amplified transendothelial transport. The effect is manifest as and facilitated by the formation of chains of pinocytotic vesicles derived from the luminal surface of the endothelial cells, which fuse to create open trans-endothelial conduits. It is likely that formation of open channels that traverse brain capillary endothelial cells, as a response to hyperglycemia, could allow temporarily unrestricted passage of a wide range of molecules, some potentially toxic, into the CNS extracellular milieu. This is the first report to unequivocally document with freeze-cleave techniques, that abnormally elevated levels of blood sugar can affect blood-brain interface permeability. This finding suggests that similar consequences may be expected to result from diabetic hyperglycemia in humans.

Animals↗
PubMed2001

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells.

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.

Animals↗
PubMed1998

Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium.

Killing of unirradiated cells by medium from cultures of irradiated cells implies the release of a cytotoxic substance by the irradiated cells. The finding of the gamma-ray-induced cytotoxic effect exclusively in epithelial cells and not in fibroblasts suggested that tissue architecture or cell communication might be important. Normal human keratinocytes and fibroblasts and radiosensitive carcinoma cells were irradiated as single cells, microcolonies of three or four cells, or confluent monolayers. The medium was removed and filtered, and cultures which had never been irradiated were seeded at cloning densities and treated with the medium from the irradiated cells. It was found that the degree of cell-cell contact had no effect on the ability of medium from irradiated epithelial cell cultures to reduce the clonogenic survival of unirradiated cells. Cell density was the only important factor. Inhibition of gap junction intercellular communication using the tumor promoter phorbol myristate acid (PMA), which closes gap junctions, increased killing by the bystander effect when the PMA was added to epithelial cells prior to irradiation. Rescue of epithelial cells exposed to the medium from the irradiated cells was not possible even after only 30 min exposure. This suggests that a signal transduction mechanism may control death or survival by the bystander effect rather than by release of a factor which is directly cytotoxic.

Apoptosis↗
PubMed1997

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells.

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Animals↗
PubMed1970

Low-resistance junctions between cancer cells in various solid tumors.

Electrical coupling, which reveals the presence of specialized low-resistance intercellular junctions, has now been found in four types of tumors, Sarcoma 180, Novikoff hepatoma, and Morris hepatomas 3924-A and 7777. Coupled cancer cells were distinguished from coupled normal cells by intracellular marking techniques. Although the evidence suggests that coupling may be extensive in some cases, it is not possible to say that the coupling was normal. In particular, the results do not exclude less obvious defects in the specialized junctions, such as abnormal distribution or decreased permeability to molecules other than small inorganic ions. The results are discussed in relation to previous studies of coupling in Novikoff hepatomas and in cultures of S1801 and II cell lines.

Animals↗
PubMed2004

Specific PKC isoforms regulate blastocoel formation during mouse preimplantation development.

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (delta, theta, iota/lambda, zeta) PKC isoforms and PKCmicro/PKD1 showed partial colocalisation with the tight junction marker ZO-1alpha+ in TE and all four PKCs (delta, theta, iota/lambda, zeta) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKCdelta and zeta activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase alpha1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase.

Animals↗
PubMed2002

Retinoids inhibit squamous cell carcinoma growth and intercellular communication.

BACKGROUND: Retinoids have been shown to inhibit the growth of squamous cell carcinoma and other malignancies. They have also been shown to alter gap junctional intercellular communication (GJIC) and the expression of connexins, the protein subunits of gap junctions. We report in this study that the alteration of GJIC by retinoids may be directly related to inhibitory effects on cell growth. MATERIALS AND METHODS: SCC-13 cells were treated with all-trans retinoic acid (tRA) and 13-cis retinoic acid (cRA) at 10(-7) and 10(-6) M concentrations in culture. No treatment and ethanol vehicle controls were included for each experiment. Serial cell counts of parallel cultures were performed to determine cell growth. The parachute technique was performed in combination with fluorescence activated cell sorting (FACS) analysis to determine GJIC. Northern and Western blot analysis were performed to assess connexin mRNA and protein expression. RESULTS: The growth rate was inhibited for cells treated with tRA (10(-6) M) (P < 0.05) and cRA (10(-6) M) (P = 0.068) vs. vehicle control. GJIC was significantly inhibited with both tRA (10(-7) and 10(-6) M) (P < 0.001) and cRA (10(-7) and 10(-6) M) (P < 0.001) at 24, 48, and 96 h as determined by FACS analysis. To correlate GJIC with cell growth, we studied the effect of glycyrrhetinic acid, a known inhibitor of GJIC. Glycyrrhetinic acid also significantly inhibited cell growth (P < 0.05) vs. control. Connexin 26 and connexin 43 mRNA and protein expression were not significantly altered after retinoid treatment. CONCLUSION: Retinoic acids inhibit both cell growth and GJIC in SCC-13 cells. Retinoids may inhibit cell growth through alteration of GJIC in SCC-13 cells.

Blotting, Northern↗
PubMed2004

[Antiadhesive molecule T-cadherin is an atypical low-density lipoprotein receptor in vascular cells].

Elevated serum LDL level, which results in cholesterol accumulation in vascular wall, is widely accepted as a risk factor in atherosclerosis development. Additionally to metabolic effects, LDL can produce hormone-like effects in a number of cells: activate second messenger systems, regulate gene expression and activate platelets and stimulate cell proliferation. The responses elicited by LDL are rapid, dose-dependent and capable of being saturated, indicating the involvement of specific receptor/binding sites in LDI-stimulated signal transduction. This LDL-binding protein was isolated from human aorta media and identified as T-cadherin. Cadherins are a superfamily of adhesion molecules that mediate Ca2+ -dependent cell-cell adhesion in embryogenesis and in adult organism's solid tissues. Intercellular junctions are formed as a result of interactions between extracellular domains of the neighboring cells' cadherins. Binding of the intercellular domain to the acting cytoskeleton ensures stability of cadherin-mediated adhesive junctions. T-cadherin is a unique member of calcium-dependent adherent proteins; in contrast to classical cadherins T-cadherin is anchored to the cell surface membranes via a glycosyl phosphatidyl inositol (GPI) moiety. Subcellular distribution of T-cadherin is restricted to lipid rafts on the cell membranes where it co-localizes with signal-transducing molecules. The function of T-cadherin has not yet been revealed. It was originally cloned from chicken embryo brain where the spatial-temporally restricted pattern of T-cadherin suggests its role as a negative guidance cue in tegulating the segmental organization of trunk neural crest migration and motor axon projections. Comparative study of the T-cadherin expression in human organs and tissues revealed that T-cadherin content was maximal in cardiovascular system. Its expression in VSMC depends on the cell phenotype and proliferate activity and increases in atherosclerotic lesion and restenosis. T-cadherin seems to play a key role in the regulation of the vascular cell phenotype, migration and growth. We hypothesize that T-cadherin is an anti-adhesive molecule which participates in intercellular interactions informing cells about their environment and regulating migration and proliferation of cells in vascular wall, while LDL interfere with the normal function of T-cadherin.

Animals↗
PubMed2003

WF-536 inhibits metastatic invasion by enhancing the host cell barrier and inhibiting tumour cell motility.

1. Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf-536, (+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf-536 in inhibition of tumour metastasis, we analysed the effect of Wf-536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems. 2. In a coculture system of human fibrosarcoma HT1080 cells plated on a monolayer of human ECV304 cells, Wf-536 (0.3-3 micromol/L) inhibited the paracellular infiltration of tumour cells. 3. Wf-536 (3-30 micromol/L) inhibited the invasion of tumour cells through the reconstituted basement membrane (Matrigel) layer. 4. Wf-536 (10-30 micromol/L) inhibited the migration of tumour cells. At 0.3-3 micromol/L, Wf-536 also restrained hepatocyte growth factor/scatter factor (HGF)-induced increases in paracellular permeability of the ECV304 cell layer. 5. These results suggest that Wf-536 suppresses tumour metastasis by both enhancing the barrier function of host cell layers and inhibiting tumour cell motility at the stage of host tissue penetration by metastatic tumour cells.

Basement Membrane↗
PubMed2000

Human hemangiosarcomas have a common polymorphism but no mutations in the connexin37 gene.

Gap junctional intercellular communication is often impaired in cancers, and the genes which encode the connexin gap junction proteins are considered to be tumor-suppressor genes. In this study, we analyzed the presence of mutations in the connexin 37 (Cx37) gene in 22 human hepatic angiosarcomas, 6 and 4 of which were associated with exposure to vinyl chloride and Thorotrast, respectively. The other 12 samples were from patients with no history of exposure to these 2 agents. In 9 samples, a proline (ACC) to serine (ACT) amino acid change in codon 319 was detected. However, DNA from non-tumorigenic tissue of the same patients also showed this amino acid change, suggesting that this is a polymorphism rather than a mutation. Subsequent analysis of 84 DNA samples from normal donors revealed the frequencies of Pro/Pro, Pro/Ser and Ser/Ser alleles to be 65.5%, 23.8% and 10.7%, respectively, while among the group of angiosarcoma patients the corresponding figures were 59.1%, 31.8% and 9. 1%, respectively. Thus, there was no correlation between the polymorphism at codon 319 and hepatic angiosarcoma occurrence. However, among the 6 cases of vinyl chloride-associated angiosarcoma, the percentages of the polymorphic alleles were 33.3%, 66.7% and 0%, respectively. While the number of samples was too small to allow us to conclude that the Ser319 allele in Cx37 predisposes to this rare type of human cancer, it may be noted that codon 319 is located at the cytoplasmic tail of Cx37, where most regulatory sequences reside, and that it could be a site of phosphorylation for some protein kinases, which may in turn affect the function of Cx37, including intercellular communication.

Carcinogens↗
PubMed1996

Connexin expression systems: to what extent do they reflect the situation in the animal?

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins in Xenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.

Animals↗
PubMed2000

Liver cell-specific transcriptional regulation of connexin32.

Gap junctional intercellular communication facilitates liver homeostasis and growth control in the liver. The major gap junction protein expressed by hepatocytes is connexin32 (Cx32) and non-parenchymal hepatic cells do not express this gene. We investigated the regulation of Cx32 transcription by trans-activating factors in liver cells. Transient transfection assays using deletions of the rat Cx32 promoter (nt -753 to -33) linked to the luciferase gene were performed in MH1C1 rat hepatoma cells that express endogenous Cx32 compared with WB-F344 rat liver epithelial cells that do not. The basal promoter element was located within nt -134 to -33 and was 1.4-fold more active in MH1C1 cells than WB-F344 cells whereas the entire promoter fragment (nt -754 to -33) was four-fold more active in MH1C1 cells. Specific nuclear protein-DNA complexes that bound to Sp1 consensus sites within the basal promoter were formed using nuclear extracts from both types of cells. Additional promoter sequences increased promoter activity more strongly in MH1C1 cells than WB-F344 cells and this was correlated with the binding of hepatocyte nuclear factor-1 (HNF-1) to two HNF-1 consensus sites centered at -187 and -736. Expression of HNF-1 and binding to these elements was only observed with MH1C1 cells. Other specific protein-DNA complexes were formed, however, that included YY-1- and NF-1-containing complexes, but these were not related to promoter activity. Dexamethasone increased Cx32 promoter activity and expression in MH1C1 cells, but had little effect in WB-F344 cells and did not alter protein-DNA complex formation. These data suggest that Sp1 is responsible for Cx32 promoter basal activity, that HNF-1 determines the cell-specific expression of Cx32, and that dexamethasone increases Cx32 expression through other mechanisms.

Animals↗
PubMed1996

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

Cell Communication↗
PubMed1997

A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

Animals↗