Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis. IMAGES:

内皮素（Endothelin, ET）是一种由21个氨基酸构成的强效血管收缩肽，最初从培养的血管内皮细胞上清液中被分离获取。为深入解析其生物合成途径，我们在爪蟾卵母细胞中表达了编码人ET-1 212氨基酸前体（前内皮素原-1，preproET-1）的合成RNA。通过抗ET-1血清结合放射免疫分析法（radioimmunoassay, RIA）对细胞匀浆及卵母细胞孵育培养基进行检测，结果显示：注射preproET-1合成RNA的卵母细胞中可检测到ET-1样免疫反应性，空白对照卵母细胞则无此信号；且培养基中的免疫反应性水平显著高于细胞匀浆。当在经莫能菌素处理的卵母细胞中表达preproET-1时，免疫活性物质的分泌量出现显著下降，这提示分泌过程由高尔基复合体（Golgi complex）所介导。卵母细胞孵育培养基中的ET-1样免疫活性物质经反相高效液相色谱（reverse-phase HPLC）分离后得到两个主要峰，其保留时间分别与人源大内皮素-1（big ET-1）和ET-1的保留时间一致。表达合成preproET-1 RNA的卵母细胞孵育培养基可诱导兔腔静脉产生典型的血管收缩反应，这与通过ET-1样免疫反应性含量所预测的生物活性相吻合。上述研究结果表明，ET的成熟通路广泛存在于不同类型的细胞中，且爪蟾卵母细胞可作为研究ET-1合成细胞生物学特性的有效实验工具。IMAGES: