Cytochemical study of protein kinase C and gap junctions in various cells treated with phorbol ester.

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Agonists and/or antagonists of cAMP-dependent protein kinase, protein kinase C (PKC), calmodulin/Ca++-dependent protein kinase, and several tyrosine kinases modulate gap junctional communication in various cell systems. Takeda et al. (1987) have determined that a major liver gap junction protein, connexin 32, is phosphorylated in vitro by PKC. They also showed that the addition of phorbol ester (PMA), an activator of PKC, to intact hepatocytes rapidly increased connexin 32 phosphorylation at serine residues. In this project, we have demonstrated that the addition of PMA to intact hepatocytes leads to marked decreases in gap junction areas (within 20 min.) by freeze-fracture electron microscopy. Furthermore, immuno-localization of PKC by electron microscopy with two monoclonal antibodies against type 2 and type 3 PKCs showed that both epitopes are associated with the gap junctional membranes in PMA treated hepatocytes. Our results suggest that PMA induced phosphorylation of connexin 32 causes breakdown of the gap junction. In fact, we detected internalization of the gap junctional membrane and annular gap junctions, which is one sequential step in the breakdown of the gap junction, in PMA treated hepatocytes.