The Effects Of Gap Junction On Cadmium-induced Apoptosis In BRL 3A Cells

Gap junctions (GJs) mediate exchange of material and information between adjacent cells. GJs play important roles in the control of homeostatic balance in cell differentiation, proliferation, transformation and apoptosis. Cadmium (Cd) is a cytotoxic industrial and environmental pollutant, and Cd is known to induce apoptosis in a variety of cells. To investigate the effects of Cd on gap junctional intercellular communication (GJIC) between BRL 3A cell and the effects of gap junction on cadmium-induced apoptosis in BRL 3A cells, BRL 3A cells were treated with CdAC2 (0,2.5,10 ?mol/L) for 12 h or cells were treated with Cd (10 ?mol/L), or with GA (5 ?mol/L) for 9 h, alone or taken together. RTCA was used to determine the cell Index. The GJIC between BRL 3A cells was detected by the scrape loading dye transfer assay. The distribution of Cx43 was observed using confocal laser scanning microscopy. Nuclear morphology was analyzed by Hoechst 33258 staining, intracellular free Ca2+ concentration ([Ca2+]1) and cell apoptosis were measured by flow cytometry (FCM). The Cx32, Cx43, p-Cx43, Bax, Bcl-2, caspase-3, ERK, JNK and p38 were measured by western blotting.In results:? BRL 3A cells were treated with CdAC2 (0,2.5,10 ?mol/L) for 12 h, CI value was decreased in a concentration-dependent manner (P<0.05 or P<0.01), the GJIC was inhibited (P<0.01) between adjacent cells, Cx43 immunofluorescent staining was distributed mostly in the intracellular region rather than the membrane, Cx32 level, Cx43 level and Cx43 phosphorylation were decreased (P<0.05 or P<0.01). ? BRL 3A cells were treated with CdAC2 (10 ?mol/L) for 9 h, Cd inhibited GJIC obviously, Cd did not change expression levels of Cx32 (P>0.05), decreased expression levels of Cx43 (P<0.05), increased expression levels of p-Cx43 and [Ca2+]i (P<0.01), GA has no significant change on the effect that Cd had on the expression level of Cx32, Cx43 and p-Cx43 (P>0.05), but can enhanced the release of [Ca2+], (P<0.01). ? The BRL 3A cells exposed to CdAC2(10 ?mol/L) for 9 h, showed typical morphological changes of apoptosis with chromatin condensation and nuclear fragmentation and the apoptosis rate was increased. GA enhanced morphological changes of nucleus, apoptosis rate, Bax/Bcl-2 ratio and cleaced caspase-3/procaspase-3 ratio (P<0.05 or P<0.01) in BRL 3A cells caused by Cd. The phosphorylation levels of ERK, JNK and p38 were apparently increased by Cd (P<0.01). GA enhanced significantly the phosphorylation levels of ERK and p38 activation induced by Cd (P<0.01), but had no significant effect on phosphorylation levels of JNK activation (P>0.05).In conclusion, Cd has toxic effect on BRL 3A cells. Cd inhibits GJIC between cells, which may be mediated by aberrant distribution of Cx43, change the expression of Cx32, Cx43 and the phosphorylation of Cx43. GA enhanced Cd-induced cytotoxicity, and promoted Cd-induced apoptosis, probably involving expression levels of apoptosis-related proteins regulation, the ERK and p38 signaling pathways in BRL 3 A cells.