Burghardt R C, Barhoumi R, Sewall T C, Bowen J A
Department of Veterinary Anatomy and Public Health, Texas A & M University, College Station 77843, USA.
J Membr Biol. 1995 Dec;148(3):243-53. doi: 10.1007/BF00235042.
The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mM 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mM 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1-3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mM 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mM octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3-5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 microM monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mM 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.
在几种表达不同水平缝隙连接蛋白连接蛋白43（Cx43）的细胞类型中，研究了环磷酸腺苷（cAMP）对缝隙连接介导的细胞间通讯的快速作用，这些细胞类型包括永生化大鼠肝细胞和颗粒细胞、牛冠状动脉小静脉内皮细胞、原代大鼠子宫肌层细胞和马子宫上皮细胞。在亚汇合培养物中，在存在或不存在1.0 mM 1-辛醇（一种通过关闭缝隙连接通道使细胞解偶联的试剂）的情况下，通过光漂白后荧光恢复测定法监测由8-溴-cAMP诱导的连接通讯变化的功能分析。单独用1.0 mM 8-溴-cAMP处理的通讯细胞在1-3分钟内（取决于细胞类型）荧光恢复百分比显著增加，并且连接通讯在长达24小时内仍显著升高。向用1.0 mM辛醇解偶联1分钟的培养细胞中添加1.0 mM 8-溴-cAMP，在3-5分钟内开始显示缝隙连接通透性部分恢复。对随后进行间接免疫荧光以监测Cx43分布的培养物进行相同处理。细胞连接通透性的变化与免疫反应性Cx43分布的变化相关。用10 microM莫能菌素处理2小时的细胞通讯速率降低，同时伴随着囊泡状细胞质Cx43染色增加和连接斑点状表面染色减少。与单独用莫能菌素处理的培养物相比，向这些培养物中添加1.0 mM 8-溴-cAMP对通讯速率或Cx43分布没有影响。这些数据表明，环磷酸腺苷对Cx43缝隙连接的作用是通过增加Cx43向质膜缝隙连接斑的运输和/或组装来促进缝隙连接通透性的增加。
