Are there functional gap junctions or junctional hemichannels in macrophages-猫眼课题宝

巨噬细胞中是否存在功能性间隙连接或连接半通道

DOI：

10.1182/blood.v88.1.328.bloodjournal881328

发表时间：

1996

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

L. A. Alves;R. Coutinho;P. Persechini;D. Spray;W. Savino;A. D. Carvalho

免疫系统迁移细胞中功能缝隙连接的存在是一个有争议的问题。在本报告中，我们将重点放在一种特殊的细胞类型，即巨噬细胞，因为连接蛋白-43，一种形成缝隙连接的蛋白质，已经在腹膜巨噬细胞和巨噬细胞系(J774)中被描述，通过Northern和Western印迹分析。为了测试这些细胞类型是否表达功能性缝隙连接，我们通过细胞内注射荧光黄来检测染料偶联。我们观察到，未受刺激的巨噬细胞之间没有偶联，也没有与表达由连接蛋白-43形成的功能性缝隙连接的上皮细胞系形成功能性缝隙连接。之前被脂多糖或干扰素-伽马激活的巨噬细胞之间也没有检测到染料偶联。我们使用更灵敏的双全细胞膜片钳技术进一步检查了功能偶联的存在，并且再次没有发现巨噬细胞之间的电偶联，这与染料显微注射数据一致。我们还使用染料摄取试验和全细胞膜片钳技术研究了细胞外三磷酸腺苷(ATP)激活半间隙连接通道的可能性。关闭缝隙连接半通道的条件(暴露于辛醇和细胞内低pH)不会减少ATP诱导的荧光黄摄取，而预计增加半通道开放的条件既不影响ATP通透性(二丁酰腺苷一磷酸)，也不降低其通透性(零细胞外CA+2)。最后，在使用来自Conexin-43基因敲除小鼠的常驻巨噬细胞的实验中，我们观察到了ATP诱导的染料摄取。因此，我们的实验数据表明，巨噬细胞在体外不形成功能性缝隙连接，细胞外ATP激活的通透通路不是由半缝隙连接通道形成的。

The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin- 43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch- clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.