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

camp依赖性蛋白激酶、蛋白激酶C （PKC）、钙调素/ ca++依赖性蛋白激酶和几种酪氨酸激酶的激动剂和/或拮抗剂调节各种细胞系统中的间隙连接通讯。Takeda等人（1987）已经确定一个主要的肝间隙连接蛋白，连接蛋白32，在体外被PKC磷酸化。他们还发现，在完整的肝细胞中加入PKC的激活剂phorbol酯（PMA），会迅速增加连接蛋白32丝氨酸残基的磷酸化。在这个项目中，我们通过冷冻断裂电镜证明，在完整肝细胞中添加PMA会导致间隙连接区域明显减少（在20分钟内）。此外，用两种针对2型和3型PKC的单克隆抗体在电镜下对PKC进行免疫定位，发现这两个表位都与PMA处理的肝细胞的间隙连接膜有关。我们的研究结果表明，PMA诱导的连接蛋白32磷酸化导致间隙连接的破坏。事实上，我们在PMA处理的肝细胞中检测到间隙连接膜和环形间隙连接的内在化，这是间隙连接破裂的一个连续步骤。

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.