Research Unit, Biomedical Research Foundation, Prince of Asturias University Hospital, Alcalá de Henares, Madrid, Spain; Nephrology Section, Biomedical Research Foundation, Prince of Asturias University Hospital, Alcalá de Henares, Madrid, Spain.
Pharmacol Res. 2013 Oct;76:106-18. doi: 10.1016/j.phrs.2013.07.006. Epub 2013 Aug 2.
Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.
虽然钙敏感受体激动剂是为了阻断甲状旁腺激素的合成而开发的，但一些报告表明，它们也可能通过未知机制降低血压。钙敏感受体激动剂诱导的内皮血管活性因子合成的变化可能与此有关。我们用钙敏感受体激动剂 R-568 处理 Wistar 大鼠，用尾套式血压计记录收缩压（SBP），用免疫组化和 Western blot 评估组织中内皮型一氧化氮合酶（eNOS）和内皮素转换酶 1（ECE-1）的含量，用 ELISA 法测量循环内皮素-1（ET-1）水平。R-568 降低了 SBP 和循环 ET-1 水平，但 eNOS 表达没有变化。相比之下，R-568 增加了肺和血管中 ECE-1 的含量。为了分析所涉及的机制，我们研究了 R-568 对人内皮细胞的影响。R-568 既不改变 eNOS 蛋白含量，也不改变前原 ET-1 mRNA 的表达，但增加了 ECE-1 蛋白含量，减少了 ET-1 的合成和 ECE-1 的活性。ECE-1 活性的抑制作用非常强，类似于经典的 ECE 抑制剂磷氨霉素，添加外源性锌可恢复酶活性。此外，与对照组细胞相比，用 R-568 处理的细胞中免疫沉淀的 ECE 中的锌含量减少了 3 倍。总之，R-568 通过从酶中排出锌来抑制 ECE，随后降低酶活性并减少循环 ET-1 水平，这可能是 R-568 处理的大鼠 SBP 降低的原因。这种下降部分会被 ECE-1 本身合成的增加以及调节 SBP 的其他体内平衡机制所补偿。
