HEPATIC-EFFECTS OF ENDOTHELIN - RECEPTOR CHARACTERIZATION AND ENDOTHELIN-INDUCED SIGNAL TRANSDUCTION IN HEPATOCYTES
HEPATIC-EFFECTS OF ENDOTHELIN - RECEPTOR CHARACTERIZATION AND ENDOTHELIN-INDUCED SIGNAL TRANSDUCTION IN HEPATOCYTES
复制标题
DOI：
10.1042/bj2870897
复制
发表时间：
1992-11-01
影响因子：
4.1
通讯作者：
OLSON, MS
中科院分区：
文献类型：
--
作者：
GANDHI, CR;BEHAL, RH;OLSON, MS
内皮素是一种有效的血管活性肽，最初从血管内皮细胞中分离出来，在灌注的大鼠肝脏中发挥糖原分解和血管收缩作用。在本文中，我们证明了大鼠肝细胞上内皮素-1 (ET-1) 的高亲和力结合位点。在37℃孵育后，ET-1与肝细胞的结合以时间依赖性方式发生，在3至6小时之间达到最大，随后下降；在此温度下，ET-1迅速内化，内化的配体在所有时间点都超过表面结合的配体。当结合测定在 4°C 下进行时，I-125-ET-1 与肝细胞的结合速率要慢得多；在此温度下，肝细胞中 ET-1 的隔离也显着减少。 ET-1 在刺激肝细胞中的磷酸肌醇代谢方面极为有效，在 ET-1 浓度低至 0.1 pM 时会显着激活该信号转导过程，EC50 为 1 pM。 ET-1 的作用通过百日咳毒素敏感的 G 蛋白耦合。霍乱毒素不影响 ET-1 介导的磷酸肌醇代谢，并且两种毒素都不影响 I-125-ET-1 与肝细胞的关联。将细胞暴露于 I-125-ET-1 并交联后，对肝细胞膜进行 PAGE，结果显示三种主要蛋白质的标记，表观分子质量分别为 32、49 和 72 kDa。这些蛋白质的 I-125-ET-1 标记均被未标记的 ET-1 抑制，而未标记的 ET-3 仅抑制 32 和 49 kDa 蛋白质的标记。 I-125-ET-3 标记了 49 kDa 蛋白质，并且该标记被未标记的 ET-1 和 ET-3 抑制。这些受体中的每一个似乎都有功能，因为 ET-1 和 ET-3 都刺激肝细胞中的磷酸肌醇代谢。当肝细胞与同源配体一起孵育时，ET-1 结合的下调和 ET-1 诱导的磷酸肌醇代谢的脱敏发生。下调后，ET-1受体通过延长孵育而恢复到肝细胞表面，尽管即使在ET-1结合能力完全恢复后，ET-1刺激的磷酸肌醇反应仍然受到抑制。这些结果证明肝细胞上存在多种 ET-1 高亲和力受体，并且该肽通过磷酸肌醇信号转导途径对肝实质细胞产生直接作用。
Endothelin, a potent vasoactive peptide originally isolated from the vascular endothelial cells, exerts glycogenolytic and vasoconstrictive actions in the perfused rat liver. In this paper we demonstrate high-affinity binding sites for endothelin-1 (ET-1) on rat hepatocytes. Upon incubation at 37-degrees-C, association of ET-1 with hepatocytes occurred in a time-dependent manner, was maximal between 3 and 6 h, and subsequently declined; at this temperature ET-1 was rapidly internalized with the internalized ligand exceeding the surface-bound ligand at all time points. The rate of association of I-125-ET-1 with hepatocytes was much slower when the binding assay was performed at 4-degrees-C; sequestration of ET-1 in hepatocytes was also substantially reduced at this temperature. ET-1 was extremely potent in stimulating phosphoinositide metabolism in hepatocytes, with significant activation of this signal transduction process occurring at ET-1 concentrations as low as 0.1 pM, with an EC50 of 1 pM. The effect of ET-1 was coupled via a pertussis toxin-sensitive G-protein. Cholera toxin did not affect ET-1-mediated phosphoinositide metabolism and neither toxin influenced the association of I-125-ET-1 with hepatocytes. PAGE of hepatocyte membranes following exposure of the cells to I-125-ET-1 and cross-linking revealed labelling of three major proteins with apparent molecular masses of 32, 49 and 72 kDa. I-125-ET-1 labelling of each of these proteins was inhibited by unlabelled ET-1, whereas unlabelled ET-3 inhibited the labelling of only the 32 and 49 kDa proteins. I-125-ET-3 labelled the 49 kDa protein and this labelling was inhibited by both unlabelled ET-1 and ET-3. Each of these receptors appears to be functional, since both ET-1 and ET-3 stimulated phosphoinositide metabolism in hepatocytes. Down-regulation of ET-1 association and desensitization of ET-1-induced phosphoinositide metabolism occurred upon incubation of hepatocytes with the homologous ligand. Following down-regulation, the ET-1 receptor was restored to the surface of the hepatocyte by prolonged incubation, although the ET-1-stimulated phosphoinositide response remained inhibited even after complete recovery of the ET-I association capability. These results demonstrate the presence of multiple high-affinity receptors for ET-1 on hepatocytes and the direct action of this peptide on hepatic parenchymal cells via the phosphoinositide signal transduction pathway.