Angiotensin I is largely converted to angiotensin (1-7) and angiotensin (2-10) by isolated rat glomeruli.
Angiotensin I is largely converted to angiotensin (1-7) and angiotensin (2-10) by isolated rat glomeruli.
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DOI：
10.1161/hypertensionaha.109.128819
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发表时间：
2009-05
期刊：
影响因子：
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通讯作者：
Janech MG
中科院分区：
文献类型：
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作者：
Velez JC;Ryan KJ;Harbeson CE;Bland AM;Budisavljevic MN;Arthur JM;Fitzgibbon WR;Raymond JR;Janech MG
肾小球内的肾素-血管紧张素系统（RAS）酶活性已检查先前使用肾小球裂解物和免疫为基础的测定。然而，肾小球提取物的制备损害了其解剖结构的完整性。此外，基于抗体的检测侧重于血管紧张素（ANG）-II检测，忽略了其他ANG-I衍生代谢物的产生，其中一些可能与ANG-II交叉反应。因此，我们的目的是使用MALDI-TOF质谱法（MS）作为分析方法来检查新鲜分离的完整肾小球中ANG-I的代谢。通过筛分分离雄性Sprague-Dawley大鼠的肾小球，并在Krebs缓冲液中在1 μM ANG-I存在下孵育15 - 90分钟，含或不含各种肽酶抑制剂。通过MALDI-TOF MS/MS或线性阱四极杆MS确认肽序列。使用定制的缬氨酸-13C，15 N标记的肽作为标准品定量峰。ANG-I裂解产生的最显著峰为899和1181 m/z，分别对应于ANG-1-7和ANG-2-10。还检测到ANG-II、ANG-1-9和ANG-3-10的较小峰。ANG-I的消失在氨肽酶-A或脑啡肽酶的抑制过程中显着减少。相反，卡托普利不改变ANG-I降解。此外，在同时抑制氨肽酶-A和脑啡肽酶，ANG-I的消失显着衰减相比，所有其他条件。这些结果表明，有显着的肾小球内转换ANG-I为ANG-2-10和ANG-1-7，分别由氨肽酶-A和脑啡肽酶介导。这些替代性ANG肽的形成对于平衡ANG-II的局部作用可能是至关重要的。这些酶活性的增强可能构成ANG-II介导的肾小球疾病的潜在治疗靶点。
Intraglomerular renin-angiotensin system (RAS) enzyme activities have been examined previously using glomerular lysates and immune-based assays. However, preparation of glomerular extracts compromises the integrity of their anatomic architecture. In addition, antibody-based assays focus on angiotensin (ANG)-II detection, ignoring the generation of other ANG-I-derived metabolites, some of which may cross-react with ANG-II. Therefore, our aim was to examine the metabolism of ANG-I in freshly isolated intact glomeruli using MALDI-TOF mass spectrometry (MS) as an analytical method. Glomeruli from male Sprague-Dawley rats were isolated by sieving and incubated in Krebs buffer in the presence of 1 μM ANG-I for 15 - 90 minutes, with or without various peptidase inhibitors. Peptide sequences were confirmed by MALDI-TOF MS/MS or linear-trap-quadrupole MS. Peaks were quantified using customized valine-13C.15N-labeled peptides as standards. The most prominent peaks resulting from ANG-I cleavage were 899 and 1181 m/z, corresponding to ANG-1-7 and ANG-2-10, respectively. Smaller peaks for ANG-II, ANG-1-9 and ANG-3-10 also were detected. The disappearance of ANG-I was significantly reduced during inhibition of aminopeptidase-A or neprilysin. In contrast, captopril did not alter ANG-I degradation. Furthermore, during simultaneous inhibition of aminopeptidase-A and neprilysin, the disappearance of ANG-I was markedly attenuated compared to all other conditions. These results suggest that there is prominent intraglomerular conversion of ANG-I to ANG-2-10 and ANG-1-7, mediated by aminopeptidase-A and neprilysin, respectively. Formation of these alternative ANG peptides may be critical to counterbalance the local actions of ANG-II. Enhancement of these enzymatic activities may constitute potential therapeutic targets for ANG-II mediated glomerular diseases.