Institute of Pharmacology & Toxicology, University of Bonn, Bonn, Germany.
Br J Pharmacol. 2013 Jan;168(2):471-87. doi: 10.1111/j.1476-5381.2012.02190.x.
Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts.
MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [(3) H]-thymidine was determined as measure of proliferation and that of [(3) H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot.
ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-β caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin.
hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.
由于内皮素（ET）可能作为促纤维化介质发挥作用，因此研究了人肺成纤维细胞中 ET 同工型的表达和释放、其受体以及潜在的促纤维化 ET 作用。
培养 MRC-5 和原代人肺成纤维细胞（phLFb）。通过 qPCR 测定前 ET 同工型的表达，通过 ELISA 测定 ET-1 的释放。通过实时测量动态质量重分布（DMR）分析 ET 受体功能。[(3) H]-胸苷掺入被确定为增殖的测量，[(3) H]-脯氨酸掺入被确定为胶原合成的测量。通过 Western blot 测定磷酸化 p42/44 MAP 激酶。
ET-1 是人肺成纤维细胞（hLF）中的主要 ET，TGF-β 进一步选择性地持续上调 ET-1，导致细胞外 ET-1 积累增加。hLFb 表达编码 ET-A 和 ET-B 受体的 mRNA。在蛋白质水平上证实了两种受体的表达。ET-1 诱导明显的 DMR 信号，该作用涉及 ET-A 和 ET-B 受体。ET-1 对 hLFb 增殖和胶原合成的刺激作用仅通过 ET-A 受体介导。ET-1 再次通过 ET-A 受体，迅速激活 ERK MAPK，这被证明是 ET-1 诱导的胶原合成中的关键细胞信号。与相应的毒蕈碱激动剂的作用相反，ET-1 诱导的 ERK 激活和胶原合成对百日咳毒素的敏感性较低。
hLFb 具有构建功能性自分泌/旁分泌内皮素能系统所需的所有元素，该系统似乎驱动促纤维化气道和肺重塑过程，其中只有 ET-A 而不是 ET-B 受体似乎具有重要意义。
