CHANGES IN CELLULAR-DISTRIBUTION OF CONNEXIN-32 AND CONNEXIN-26 DURING FORMATION OF GAP-JUNCTIONS IN PRIMARY CULTURES OF RAT HEPATOCYTES

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26), Previously, we reported that both C x32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes, The reappearing gap junctions were accompani ed by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular commu nication (GJIC) for more than 4 weeks. In the present study, we examin ed the cellular location of the gap junction proteins and the structur es in the hepatocytes cultured in our system, using confocal laser mic roscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis, In immunoelectr on microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of th em were larger than those in liver sections in vivo. Freeze-fracture a nalysis also showed that the size of gap junction plaques increased an d that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not onl y the synthesis of Ox proteins but also the size of the gap junction p laques was increased markedly. In the adluminal lateral membrane of th e cells, Cx32-immunoreactive lines were observed and many small gap ju nction plaques were closely associated with a more developed tight jun ction network. In the basal region of the cells, small Cx32- and Cx26- immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cyt oplasm, These results indicated the formation and degradation of gap j unctions in the cultured hepatocytes. (C) 1996 Academic Press, Inc.