Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-juncti on proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser mi croscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most sukapical plasma membrane at cell borders, while on the basolateral membrane both Cx 32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most suba pical plasma membrane and were colocalized with only Cx32-immunoreactive li nes. In freeze-fracture analysis, many small gap-junction plaques were obse rved within a well-developed tight-junction strand network. The fence funct ion of tight junctions in the cells, as examined by diffusion of labeled sp hingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or a nti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These r esults suggest that Cx32 gap junctions, but not those with Cx26, are closel y coordinated with the expression and function of tight junctions in hepato cytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression. (C) 2001 Academic Press.