Connexin43 signaling contributes to spontaneous apoptosis in cultures of primary hepatocytes.

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.