Department of Toxicology-Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, B-1090 Brussels, Belgium.
Toxicol Sci. 2012 Jan;125(1):175-86. doi: 10.1093/toxsci/kfr277. Epub 2011 Oct 14.
Primary hepatocyte cultures suffer from the progressive occurrence of dedifferentiation followed by spontaneous apoptosis. This is associated with modifications in the expression of connexins (Cxs), which are the building stones of hemichannels that in turn form gap junctions between neighboring cells. Specifically, a shift is observed from the adult hepatocellular Cx32 species toward the fetal Cx43 isoform. The current study was set up to investigate the role of Cx43 in spontaneous apoptosis taking place in primary hepatocyte cultures. For this purpose, freshly isolated adult rat hepatocytes were cultivated in conventional conditions for 4 days with daily monitoring of Cx expression, Cx localization, and gap junction channel and hemichannel functionality. Gap junction activity was low shortly after isolation, whereas the inverse was observed for hemichannel functionality. Both channel types displayed high activity near the end stages of the cultivation period. The Cx32-to-Cx43 switch became progressively manifested at the translational level. At the transcriptional level, a fivefold decrease in Cx32 messenger RNA abundance and a twofold increase in Cx43 expression were noticed within the first 24 h of cultivation. Throughout the cultivation period, Cx32 was mainly located at the plasma membrane surface, whereas Cx43 immunostaining was more diffuse. Application of three Cx43 inhibitors resulted in the downregulation of both hemichannel functionality and gap junction activity. This was paralleled by decreased expression and activity of caspase 3 as well as by reduced expression of Bid. Collectively, these data show that Cx43 signaling actively contributes to the occurrence of spontaneous apoptosis in cultures of primary hepatocytes.
原代肝细胞培养物会逐渐出现去分化，随后自发凋亡。这与连接蛋白（Connexins，Cxs）的表达变化有关，Cxs 是半通道的构建基石，而半通道又在相邻细胞之间形成间隙连接。具体而言，观察到从成人肝细胞 Cx32 物种向胎儿 Cx43 同工型的转变。本研究旨在探讨 Cx43 在原代肝细胞培养物中自发凋亡过程中的作用。为此，新鲜分离的成年大鼠肝细胞在常规条件下培养 4 天，每天监测 Cx 表达、Cx 定位以及间隙连接通道和半通道功能。分离后不久，间隙连接活性较低，而半通道功能则相反。两种通道类型在培养期末期都表现出高活性。Cx32 到 Cx43 的转换在翻译水平上逐渐表现出来。在转录水平上，在培养的前 24 小时内，Cx32 信使 RNA 丰度下降了五倍，Cx43 表达增加了两倍。在整个培养期间，Cx32 主要位于质膜表面，而 Cx43 免疫染色则更为弥散。应用三种 Cx43 抑制剂可下调半通道功能和间隙连接活性。这与 caspase 3 的表达和活性降低以及 Bid 的表达减少平行。综上所述，这些数据表明 Cx43 信号积极参与原代肝细胞培养物中自发凋亡的发生。
