Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec, QC G1V 4G2, Canada.
Peptides. 2010 Aug;31(8):1546-54. doi: 10.1016/j.peptides.2010.04.027. Epub 2010 May 7.
Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [(3)H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K(i) 525 nM) among known substrates to displace [(3)H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-alpha repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24h (approximately 12-fold dynamic range by [(3)H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-alpha was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [(3)H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes.
血管紧张素转换酶（ACE）是一种药物靶点，也是一种有效的缓激肽（BK）失活外肽酶。我们利用最近描述的[(3)H]依那普利拉结合测定法，来定量测定在已知或新型 ACE 表达调节剂刺激下完整的人脐静脉内皮细胞（HUVEC）中 ACE 表达的全动态范围。此外，还使用相同的测定法确定了一组生理底物对 ACE 的亲和力。在已知的底物中，BK 对 ACE 具有最高的亲和力（K(i)为 525 nM），可置换[(3)H]依那普利拉与 ACE 的结合。肿瘤坏死因子（TNF）-α抑制 HUVEC 中 ACE 的表达，而佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）在 24 小时内上调其表达（通过[(3)H]依那普利拉结合测定，约为 12 倍动态范围，通过 ACE 免疫印迹证实）。在同时刺激 PMA 和细胞因子的细胞中，ACE 表达处于中间水平。相比之下，高葡萄糖、胰岛素或表皮生长因子（EGF）均不能影响 ACE 的表达。TNF-α的作用可被依那西普、IKK2 抑制剂 TPCA-1 或 p38 抑制剂减弱，而 PMA 的作用可被对佛波醇酯和钙敏感的 PKC 同工型抑制剂减弱。短期 PKC 和 MEK1 依赖性 c-Fos 表达增加与 PMA 诱导的 ACE 上调相关性最好。应用于 HUVEC 的[(3)H]依那普利拉结合测定法支持 ACE 是一种特别活跃的激肽酶，内皮 ACE 表达是动态和特异性调节的。这在炎症性疾病和糖尿病中具有潜在的重要性。
