J Vet Sci.  2016 Mar;17(1):63-70. 10.4142/jvs.2016.17.1.63.

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells

• Hu D
• Zou H
• Han T
• Xie J
• Dai N
• Zhuo L
• Gu J
• Bian J
• Yuan Y
• Liu X
• Liu Z

• KMID: 2363336
• DOI: http://doi.org/10.4142/jvs.2016.17.1.63

Abstract

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

Keyword

BRL 3A cells; apoptosis; cadmium; gap junction blockage

MeSH Terms

Animals
Apoptosis/*drug effects
Cadmium/*toxicity
Calcium/metabolism
Cell Communication/drug effects
Connexin 43/genetics
Enzyme Activation/drug effects
Gap Junctions/*drug effects
Gene Expression Regulation/drug effects
Hepatocytes/cytology/*drug effects
Rats
Signal Transduction/drug effects
Cadmium
Calcium
Connexin 43

Figure

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  Fig. 1 Effects of 18β-glycyrrhizic acid (GA) on cadmium (Cd)-induced cytotoxicity in BRL 3A rat liver cells. (A) BRL 3A cells treated with different Cd concentrations (0, 2.5, 5, 10 and 20 µM) showed dose-dependent decreases in cell index (CI) values. (B) Treatment of BRL 3A cells with Cd (10 µM) and GA (5 µM) for 9 h, either alone or together, revealed that GA enhanced Cd-inhibited CI values. Data were normalized at the time that the growth medium was replaced.

• 

  Fig. 2 Effects of Cd and GA on gap junctional intercellular communication (GJIC), the expression of connexin 43 (Cx43) and p-Cx43, and the release of intracellular free Ca2+ concentration ([Ca2+]i). (A) Effect of GA on Cd-induced down-regulation of GJIC in BRL 3A cells. GJIC was assessed using the scrape-loading/dye transfer method under an inverted fluorescence microscope. (B) Western blot analysis was performed using Cx43 and p-Cx43 antibodies. All experiments were performed in triplicate. (C) Blots for Cx43 and p-Cx43 were semi-quantified using Image Lab software. The values show the ratio of Cx43/β-actin and the ratio of p-Cx43/β-actin. Data are expressed as the means ± SD (n = 3). (D) [Ca2+]i was detected with Fluo4/AM by flow cytometry. LY, Lucifer yellow; RD, rhodamine-labeled dextran. *p < 0.05 or **p < 0.01 vs. control group, #p < 0.05 or ##p < 0.01 vs. Cd-treated group.

• 

  Fig. 3 Effects of GA on Cd-induced apoptosis in BRL 3A cells. (A) Morphological features of BRL 3A cells were visualized by Hoechst 33258 staining. White arrows indicate apoptotic BRL 3A cells showing nuclear condensation. (B) BRL 3A cells were stained with annexin V/PI, and percentages of apoptotic cells were determined by flow cytometry. (C) Western blotting was performed using Bax, Bcl-2 and caspase-3 antibodies. All experiments were performed in triplicate. (D) Blots for Bax, Bcl-2 and caspase-3 were semi-quantified using Image Lab software. Values show the ratio of Bax/Bcl-2 and the ratio of cleaved caspase-3/procaspase-3. (E) Δψm was detected with JC-1 staining by flow cytometry. **p < 0.01 vs. control group, ##p < 0.01 vs. Cd-treated group.

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  Fig. 4 Effects of Cd and GA on MAPK pathways. (A) ERK, JNK and p38 expression and phosphorylation were detected by western blot analysis with corresponding antibodies. All experiments were performed in triplicate. (B) Blots for ERK, JNK and p38 phosphorylation were semi-quantified using Image Lab software. Data are expressed as the means ± SD (n = 3) vs. the control group.

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