OBJECTIVETo investigate whether the cellular gap junctional communication(GJIC) down-regulation in alveolar epithelial cells (CCL-64 cells) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supernatant is related with the phosphorylation states of connexin 43(Cx43) protein.

METHODWestern-blot analysis was used to identify phosphorylated Cx43 species.

RESULTSWestern-blot analyses of SiO2- and phorbol 12-myristate 13-acetate(TPA)-treated CCL-64 cells showed the same phosphorylation states of Cx43 as the control group. There were no Cx43 protein in nucleus of CCL-64 cells.

CONCLUSIONThe inhibition on GJIC induced by SiO2 and TPA in CCL-64 cells may not be brought about by altering the phosphorylation states of Cx43.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; metabolism ; Gap Junctions ; drug effects ; Lung ; drug effects ; metabolism ; Mink ; Phosphorylation ; Silicon Dioxide ; toxicity ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo investigate whether cellular gap-junctional communication(GJIC) down-regulation and the internalization of connexin 43(Cx43) in Chinese hamster lung fibroblasts (CHL) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supermatant is related with the phosphorylation states of Cx43 protein.

METHODSWestern-blot analysis was used to identify phosphorylated Cx43 species.

RESULTSSamples from membrane protein, total protein and nucleoprotein in CHL cells with 50-500 micrograms/ml doses of silica-stimulated PAM supernatants showed NP, P1, P2, P3 four immunoreactive bands of Cx43 protein by contrast with the control group and 0 microgram/ml SiO2 group. And with the dose of SiO2 increased, the increment of the levels of P2 and P3 was observed. Moreover, the groups treated with SiO2 and protein kinase C inhibitor, Palmitoyl-DL-Camitine chloride (PMC), simutaneously showed reduced level of P2 and P3, as compared with the groups treated with SiO2 only.

CONCLUSIONThe inhibition of GJIC and the internalization of Cx43 by SiO2 in CHL cells may relate to the changes of phosphorylation states of Cx43, and its mechanism may be similar to that of phorbol 12-myristate 13-acetate (TPA), i.e. via PKC activation pathway.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; metabolism ; Cricetinae ; Gap Junctions ; drug effects ; Lung ; drug effects ; metabolism ; Phosphorylation ; Silicon Dioxide ; toxicity

OBJECTIVETo explore the effect of silica dioxide(SiO2) on proliferation and downregulation of gap junctional intercellular communication (GJIC) in pulmonary alveolar epithelial cells (CCL-64 cells).

METHODSThe pulmonary alveolar macrophages(PAMs) were incubated in the serum-free RPMI 1640 containing the various concentration of SiO2 for 24 hours. The supernatants were prepared and added 5% (V/V) into 2% (V/V) NBS RPMI 1640 to stimulate the proliferation of CCL-64 cells for 24 hours. A set of "blank control", run in parallel, contained RPMI 1640 + 2% (V/V) NBS alone. The proliferation of CCL-64 cells was detected using MTT assay(to show as the absorbency, A570nm). GJIC function was measured using the fluorescence redistribution after photobleaching(FRAP) assay [to express as the transfer rate of the fluorescence, K (x 10(-3)/s)], with a laser scanning confocal microscope(LSCM, Leica TCS SP).

RESULTSThe silica-exposed PAM supernatants could induce both the proliferation(F = 9.679, P < 0.01) and downregulation of GJIC(F = 20.587, P < 0.01) of CCL-64 cells. In the range of 50-500 micrograms/ml SiO2 concentrations, the proliferation (A570nm values) and GJIC(the transfer rate, K) were fitted well in a dose-dependent manner(proliferation: r = 0.891, P < 0.05; GJIC: r = -0.943, P < 0.05).

CONCLUSIONBy way of stimulating the PAM, SiO2 could inhibit GJIC function in lung alveolar epithelial cells, and induce epithelial cell proliferation. In the pathogenesis of silicosis, the downregulation of GJIC of the pulmonary epithelial cells may play an important role in silica-mediated alveolar epithelial cell injury.

Cell Communication ; drug effects ; Cell Proliferation ; drug effects ; Epithelial Cells ; drug effects ; Gap Junctions ; drug effects ; Pulmonary Alveoli ; drug effects ; Silicon Dioxide ; toxicity ; Silicosis ; etiology

OBJECTIVETo study the effect of supernatants from silicon dioxide(SiO2) stimulated pulmonary alveolar macrophages(PAM) on the localization of connexin 43(Cx43) so as to explore the inhibition level of SiO2 on alveolar epithelial cellular gap-junctional communication(GJIC).

METHODSThe supermatants from the primary cultured PAM were prepared, and then added 5% (v/v) SiO2 into 2% (v/v) NBS RPMI 1640 to stimulate the normal mink lung epithelial cell line CCL-64 for 24 hours. The localizations of Cx43 in CCL-64 were analyzed by indirect immunofluorescence histochemistry and laser confocal scanning microscopy(LCSM).

RESULTSThe normal cultured CCL-64 cells displayed bright membrane-associated Cx43 plaques labeling and formed dashes at regions of intercellular junction. Being exposed to supernatants from SiO2-stimulated PAM, the CCL-64 cells retained a relative low degree of Cx43 labeling at the cell periphery, localized in cytoplasm, and the individual spot, rather than plaques, were smaller compared to normal cultured cells. Along with the increase of the concentrations of SiO2, the cells displayed a different staining pattern, with clear cluster labeling aggregating towards the nucleus.

CONCLUSIONThe altered localization of the gap-junctional protein Cx43 in alveolar epithelial cells, mediated by SiO2, indicated that the internalization of Cx43 may contribute to the inhibition on GJIC in silica-induced lung epithelium injury.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; analysis ; Epithelial Cells ; chemistry ; drug effects ; Fluorescent Antibody Technique, Indirect ; Gap Junctions ; drug effects ; Microscopy, Confocal ; Mink ; Pulmonary Alveoli ; chemistry ; drug effects ; Silicon Dioxide ; toxicity

OBJECTIVETo study the effects of extremely low frequency magnetic fields(ELF MF) on the amount and localization of connexin 43(Cx43) gap-junction protein in the Chinese hamster lung(CHL) cells, and to explore the mechanism of ELF MF suppression on gap-junctional intercellular communication(GJIC).

METHODSThe cells were irradiated for 24 h with 50 Hz sinusoidal magnetic field at 0.8 mT without or with 12-O-tetrade-canoylphorbol-3-acetate(TPA), 5 ng/ml for 1 h. The localization of Cx43 proteins were performed by indirect immunofluorescence histochemical analysis and detected by confocal microscopy. The second experiment was conducted to examine the quantity of Cx43 proteins level in nuclei or cytoplasm and detected by Western blotting analysis.

RESULTSThe cells exposed to TPA for 1 h displayed less bright labelled spots in the regions of intercellular junction than the normal cells. Most of Cx43 labelled spots occurred in the cytoplasm and aggregated near the nuclei. At the same time, the amount of Cx43 protein in cytoplasm were increased[(2.03 +/- 0.89) in ELF group, (2.43 +/- 0.82) in TPA group] as compared to normal control(1.04 +/- 0.17) (P < 0.01).

CONCLUSIONInhibition on GJIC function by ELF MF alone or combined with TPA may be related with the shift of Cx43 from the regions of intercellular junction to the cytoplasm.

Animals ; Cell Communication ; radiation effects ; Connexin 43 ; biosynthesis ; Cricetinae ; Cricetulus ; Cytoplasm ; metabolism ; Electromagnetic Fields ; adverse effects ; Gap Junctions ; radiation effects ; Lung ; metabolism ; radiation effects ; Tetradecanoylphorbol Acetate ; pharmacology