Immunohistochemical Study on the Gap Junctional Protein in Tissues

Cells communicate with each other by gap junctions which are composed of channels that directly connect the interior of interacting cells. Such junctions are widely distributed in tissues of all animals and couple the cells both electrically and metabolically by allowing small molecules to pass from the cytoplasm of one cell into the cytoplasm of another. The presence of gap junctions can be demonstrated morphologically by electron microscopy and functionally by dye transfer, metabolic coupling and electric coupling methods. Howerer, the observation of gap junctions in tissues has been limited to electron microscopy, because the above functional methods are applicable only to the cultured cells. In this study, we developed an immunohistochemical method for visualization of gap junctions in tissues. Gap junctions were successfully isolated from rat liver plasma membranes by a combination of NaOH treatment, sonication and sucrose gradient centrifugation. The isolated gap junctions were morphologically intact, and they were comprised of a 26,000 protein on SDS-PAGE. Antibodies to gap junctional protein were raised in mice, and immunostaining was performed using the antibodies on various tissues including normal, preneoplastic and neoplastic tissues of rodents and humans. The antibodies reacted to the livers of all the species examined, and to rat kidney, stomach, pancreas and salivary glands. It was found that there was a significant difference in the number and size of gap junctions among the tissues. It was noteworthy that the number of gap junctions were markedly decreased in chemically-induced hyperplastic nodules and hepatocellular carcinomas in rats, indicating that cellular communication is reduced during carcinogenesis. The results of this study demonstrated that this immunohistochemical method is useful in quantitating gap junctions in tissues of various kinds of animals including humans.departmental bulletin pape