Cryoelectron Microscopy and Cytochemistry of Gap Junctions

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In this project, we have demonstrated the following cytochemical aspects of gap junctions.1. Enzyme cytochemistry : Ca^<++>-ATPase activity is localized on the cytoplasmic side of gap junctional membranes.2. Cytochemical demonstration of charge on gap junctions : Cytoplasmic side of gap junctions shows the high density distribution of anionic sites.3. Immunocytochemistry of Protein Kinase C (PKC) on gap junctions : The phorbol ester, 12-O-Tetradecanoyl Phorbol-13-Acetate (TPA), one of the most potent tumor-promoting agents, is known to inhibit metabolic cooperation between cultured cells. In addition, TPA is also known to activate protein kinase C both in vivo and in vitro. In this project we have demonstrated that TPA-treatment causes a translocation of cytosolic PKC to the gap junction. Our results imply that PKC have a crucial role in TPA-induced inhibition of metabolic cooperation.4. Immunocytochemistry of gap junction protein (connexin 32) : We have studied the intracellular distribution of connexin 32 in newly born rat hepatocytes. Immunoelectron microscopy demonstrated that the mouse monoclonal antibody against connexin 32 binds directly to the adherent junction region as well as the gap junction in 6 day-old rat hepatocytes. Our findings imply that the adherent junction mediates an early adhesion event between hepatocytes that is a prerequisite for the assembly of connexin 32 of gap junctions.