Department of Pharmacology, School of Pharmaceutical Sciences, Central South University; Changsha, Hunan, China.
Am J Hypertens. 2013 Dec;26(12):1437-44. doi: 10.1093/ajh/hpt119. Epub 2013 Jul 17.
Dimethylarginine dimethylaminohydrolases 1 (DDAH1) is the major enzyme responsible for inactivation of asymmetric dimethylarginine (ADMA). This study seeks to clarify the correlations between mRNA expression levels of DDAH1 transcript variants and the relationship with ADMA metabolizing activity in human.
The mRNA expression levels of DDAH1 transcript variants in primarily cultured human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs) from healthy control subjects and patients suffering from both acute ischemic stroke (AIS) and acute myocardial infarction (AMI) were determined by real-time polymerase chain reaction. ADMA metabolizing activity of the cell lysates from HUVECs was determined by enzyme-linked immunosorbent assay.
A novel DDAH1 transcript variant DDAH1-V3 was identified. DDAH1-V3 mRNA expression correlated significantly with that of both -V2 (R = 0.811; P = 0.000008) and -V1 (R = 0.454; P = 0.04) in HUVECs. In PBMCs from healthy subjects, significant correlation was observed only between DDAH1-V2 and -V3 (R = 0.571; P = 0.001; n = 36). Delta threshold cycle (DCT) values for both DDAH1-V2 and -V3 transcripts were increased significantly in PBMCs from AIS patients (P < 0.05, respectively). In PBMCs from patients suffering from both AIS and AMI, positive pairwise correlations between mRNA levels of DDAH1 transcripts were also observed as analyzed by partial correlation analysis (P < 0.05, respectively). However, only mRNA expression level of the DDAH1-V1 transcript correlated significantly with intracellular ADMA metabolizing activity in HUVECs (R = 0.805; P=0.002).
This study demonstrated that although there are positive correlations between mRNA expression levels of DDAH1 transcript variants, only the DDAH1-V1 transcript is responsible for ADMA metabolism, and transcript specific primers are recommended to determine DDAH1 mRNA expression.
二甲基精氨酸二甲胺水解酶 1（DDAH1）是负责使非对称二甲基精氨酸（ADMA）失活的主要酶。本研究旨在阐明 DDAH1 转录变体的 mRNA 表达水平与人类 ADMA 代谢活性之间的相关性。
通过实时聚合酶链反应测定健康对照组和急性缺血性脑卒中（AIS）和急性心肌梗死（AMI）患者外周血单核细胞（PBMC）中主要培养的人脐静脉内皮细胞（HUVEC）中 DDAH1 转录变体的 mRNA 表达水平。通过酶联免疫吸附试验测定 HUVEC 细胞裂解物中的 ADMA 代谢活性。
鉴定出一种新的 DDAH1 转录变体 DDAH1-V3。在 HUVEC 中，DDAH1-V3mRNA 表达与 -V2（R = 0.811；P = 0.000008）和 -V1（R = 0.454；P = 0.04）的表达显著相关。在健康受试者的 PBMC 中，仅在 DDAH1-V2 和 -V3 之间观察到显著相关性（R = 0.571；P = 0.001；n = 36）。AIS 患者 PBMC 中 DDAH1-V2 和 -V3 转录物的 delta 循环阈值（DCT）值均显著升高（P < 0.05，分别）。通过偏相关分析（P < 0.05，分别），还观察到 AIS 和 AMI 患者 PBMC 中 DDAH1 转录物的 mRNA 水平之间存在正的两两相关性。然而，只有 HUVEC 中 DDAH1-V1 转录物的 mRNA 表达水平与细胞内 ADMA 代谢活性显著相关（R = 0.805；P=0.002）。
本研究表明，尽管 DDAH1 转录变体的 mRNA 表达水平之间存在正相关，但只有 DDAH1-V1 转录物负责 ADMA 代谢，建议使用转录物特异性引物来确定 DDAH1 mRNA 表达。
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