HEPATOCYTE GAP-JUNCTIONS ARE PERMEABLE TO THE 2ND MESSENGER, INOSITOL 1,4,5-TRISPHOSPHATE, AND TO CALCIUM-IONS
HEPATOCYTE GAP-JUNCTIONS ARE PERMEABLE TO THE 2ND MESSENGER, INOSITOL 1,4,5-TRISPHOSPHATE, AND TO CALCIUM-IONS
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DOI：
10.1073/pnas.86.8.2708
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发表时间：
1989-04-01
影响因子：
11.1
通讯作者：
BENNETT, MVL
中科院分区：
文献类型：
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作者：
SAEZ, JC;CONNOR, JA;BENNETT, MVL
肝细胞通过缝隙连接很好地结合在一起，这允许小分子在细胞之间扩散。尽管许多组织中的缝隙连接对比cAMP更大的分子具有渗透性，并且在一些制剂中缝隙连接通过cAMP本身，但几乎没有直接证据支持其他第二信使物种的渗透。可能是最小的第二信使，可能会穿过缝隙连接，但这个问题很复杂，因为当细胞内游离钙浓度[Ca~(2+)]i升高到微摩尔水平或更高时，缝隙连接通道就会关闭。肌醇1，4，5-三磷酸(InsP3)是引起细胞内钙释放的第二信使，也可能通过这一机制降低细胞的通透性。我们在这里报告了新鲜分离的成对或小簇大鼠肝细胞中钙离子和InsP3跨界流的证据。用钙离子指示剂Fura-2直接监测钙离子的跨连接扩散或通过局部钙离子释放来检测InsP3的通过。当游离酸通过细胞间时，注射Fura-2。注射InsP3或CaCl2后，注射细胞内的[Ca~(2+)]_i立即升高(峰值1微米)，接触细胞的细胞内[Ca~(2+)]_i迅速升高(几秒钟内)。InsP3诱导的胞浆内游离钙浓度最初升高幅度较大，与钙离子的简单扩散不一致。在耦合单元中，增加最大的区域不一定在接触区附近。相反，当细胞浸泡在正常的Ca~(2+)中时，注射CaCl2引起的[Cu2+]i升高总是比注射InsP3时更为弥漫，并且在注射CaCl2的细胞偶联细胞中，最早和最大的升高发生在细胞接触区域。这种分布的差异表明，注射的InsP3(或一种活性代谢物，但不是Ca~(2+))在细胞间扩散，导致细胞内存储的Ca~(2+)局部释放。Ca~(2+)注入诱导细胞内[Ca~(2+)]i升高，提示相邻细胞内[Ca~(2+)]_i的变化是由于注入细胞的跨界面扩散所致，而不是从细胞外液中摄取。然而，在无钙生理盐水中，[Ca~(2+)]i分布不均匀，表明钙释放机制参与了观察到的变化。在用解偶联剂辛醇(500微米)处理后，相邻细胞内的[Ca~(2+)]i没有增加，而辛醇本身不改变[Ca~(2+)]i。这些数据提供了第二信使Ca~(2+)和InsP3可以通过缝隙连接从细胞传递到细胞的证据，这一过程可能在组织功能中起重要作用。
Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca2+, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca2+ concentration, [Ca2+]i, is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP3), a second messenger that can evoke Ca2+ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca2+ and InsP3 in freshly isolated pairs or small clusters of rat hepatocytes. The Ca2+ indicator fura-2 was used to monitor transjuctional diffusion of Ca2+ directly or to detect passage of InsP3 by localized Ca2+ release. Fura-2 injected as the free acid passed between cells. Injection of InsP3 or CaCl2 immediately increased [Ca2+]i in the injected cell (peak values < 1 .mu.M), and [Ca2+]i increased rapidly in contacting cells (within seconds). The initial rise in [Ca2+]i induced by InsP3 was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca2+. In the coupled cells the regions of greatest increases were not necessarily near the contact zone. In contrast, the rise induced in [Cu2+]i by CaCl2 injection when cells were bathed in normal Ca2+ was always more diffuse than with InsP3 injection, and in cells coupled to a cell injected with CaCl2 the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP3 (or an active metabolite, but not Ca2+) diffused between cells to cause localized release of Ca2+ from intracellular stores. Ca2+ injection induced a rise in [Ca2+]i in coupled cells even when cells were maintained in Ca2+-free saline, suggesting that changes in [Ca2+]i seen in adjacent cells were due to transjunctional diffusion from the injected cell and not to uptake from the extracellular solution. However, in Ca2+-free saline, [Ca2+]i distribution was nonuniform, indicating that Ca2+-releasing mechanisms contribute to the observed changes. No increase in [Ca2+]i was seen in adjacent cells when Ca2+ was injected after treatment with the uncoupling agent octanol (500 .mu.M), which itself did not change [Ca2+]i. These data provide evidence that the second messengers Ca2+ and InsP3 can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.