Regulation of cardiovascular signaling by kinins and products of similar converting enzyme systems - Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism
Regulation of cardiovascular signaling by kinins and products of similar converting enzyme systems - Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism
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DOI：
10.1152/ajpheart.01116.2002
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发表时间：
2003-06-01
影响因子：
4.8
通讯作者：
Lew, RA
中科院分区：
文献类型：
--
作者：
Norman, MU;Reeve, SB;Lew, RA
与之密切相关的金属内肽酶EC 3.4.24.15(EP24.15；硫胺寡肽酶)和24.16(EP24.16；神经降压素)在体外可切割许多血管活性多肽，如缓激肽和神经降压素。我们之前已经证明，EP24.15和EP24.16(26)的抑制剂可以增强对缓激肽的降压反应，这表明在体内缓激肽代谢中有一种或两种酶起作用。在这项研究中，我们使用了能够区分EP24.15和EP24.16的选择性抑制剂来确定它们在培养的内皮细胞(转化的人脐静脉内皮细胞杂交系EA.hy926或绵羊主动脉内皮细胞)中的活性。使用特定的猝灭荧光底物[7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-D-Lys(2，4-dinitrophenyl)]，以及多肽底物缓激肽和神经降压素(通过高效液相色谱-质谱仪检测)来评估内肽酶的活性。我们的结果表明，这两种肽酶都存在于内皮细胞中；然而，EP24.16对胞液和膜准备以及完整细胞的底物切割的贡献明显高于EP24.15。这些发现，再加上之前在体内的观察，表明血管内皮细胞中的EP24.16活性可能在循环中缓激肽和/或其他多肽的降解中发挥重要作用。
The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-D-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.