Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors
Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors
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DOI：
10.1242/jcs.01345
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发表时间：
2004-09-15
影响因子：
4
通讯作者：
Goldenberg, RCS
中科院分区：
文献类型：
--
作者：
Fortes, FSA;Pecora, IL;Goldenberg, RCS
缝隙连接是连接蛋白形成的通道，在大多数细胞类型的细胞间通讯中起重要作用。在免疫系统中，特别是在巨噬细胞中，连接蛋白的表达和功能性间隙连接的建立仍然是有争议的问题。巨噬细胞表达P2 X（7）受体，一旦被细胞外ATP的结合激活，导致可渗透高达900 Da的分子的跨膜孔的打开。有证据表明，在不同的细胞系统中，间隙连接和P2受体之间存在相互作用。因此，我们使用ATP敏感和ATP不敏感的J774.G8巨噬细胞系来研究这种相互作用。为了研究J774-巨噬细胞样细胞中的连接通讯，我们通过显微注射荧光黄来评估细胞间的通讯。ATP敏感的J774细胞（ATP-s细胞）的汇合培养物是偶联的，而ATP不敏感的J774细胞（ATP-i细胞）基本上是解偶联的，所述ATP不敏感的J774细胞通过将J774细胞过度暴露于细胞外ATP直到它们不显示ATP诱导的透化现象而衍生。Western-blot和逆转录聚合酶链反应分析显示，ATP-s和ATP-i细胞表达连接蛋白43（Cx43），而只有ATP-s细胞表达P2 X（7）受体。因此，在全细胞膜片钳记录下，ATP-1细胞未显示任何可检测的ATP诱导电流。使用免疫荧光显微镜，Cx43的反应被发现在细胞表面和区域的细胞-细胞接触的ATP-S细胞，而在ATP-i细胞，Cx43的免疫反应只存在于胞质隔室。使用共聚焦显微镜，它表明，在ATP-s细胞以及在腹腔巨噬细胞，Cx43和P2 X（7）受体共定位于ATP-s细胞和腹腔巨噬细胞的膜。
Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.