title: Correlations of DDAH1 transcript variants with human endothelial asymmetric dimethylarginine metabolizing activity.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacology, School of Pharmaceutical Sciences, Central South University; Changsha, Hunan, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Ji-Peng
authors.initials: JP
authors.lastname: Zhou
authors.forename: Da-Bin
authors.initials: DB
authors.lastname: Kuang
authors.forename: Mu-Peng
authors.initials: MP
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Xiong
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.forename: Jian
authors.initials: J
authors.lastname: Xia
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Bai
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Yang
authors.forename: Yuan-Jian
authors.initials: YJ
authors.lastname: Li
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Chen
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D001120:Arginine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014471:Umbilical Veins
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; C487735:dimethylarginine; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase
keywords: asymmetric dimethylarginine (ADMA); blood pressure; dimethylarginine dimethylaminohydrolase 1 (DDAH1); human umbilical vein endothelial cells (HUVECs); hypertension; peripheral blood mononuclear cells (PBMCs).