Cryoelectron Microscopy and Cytochemistry of Gap Junctions

在这个项目中，我们已经证明了以下细胞化学方面的间隙连接。酶细胞化学：Ca^++-ATP酶活性位于细胞间隙连接膜的胞质一侧.间隙连接上电荷的细胞化学显示：间隙连接的胞质侧显示高密度分布的阴离子位点.蛋白激酶C（PKC）在间隙连接上的免疫细胞化学：佛波酯，12-O-十四烷酰基佛波醇-13-乙酸酯（TPA），最有效的促肿瘤剂之一，已知抑制培养细胞之间的代谢合作。此外，还已知TPA在体内和体外均激活蛋白激酶C。在这个项目中，我们已经证明，TPA治疗引起胞浆PKC的移位到差距连接。我们的研究结果表明PKC在TPA诱导的代谢合作抑制中起着至关重要的作用.缝隙连接蛋白（connexin 32）的免疫细胞化学：我们研究了新生大鼠肝细胞内connexin 32的分布。免疫电镜显示，小鼠抗连接蛋白32的单克隆抗体直接结合到6日龄大鼠肝细胞的粘附连接区以及差距连接。我们的研究结果表明，粘附连接介导的肝细胞之间的早期粘附事件是间隙连接的连接蛋白32的组装的先决条件。

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.