title: Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.
abstract: OBJECTIVE
  To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB.
  
  
  METHODS
  HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence.
  
  
  RESULTS
  0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment.
  
  
  CONCLUSIONS
  Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.
authors.affiliation: Department of Geriatric-Cardiovascular Diseases, The First Affiliated Hospital of Medical College in Xi'an Jiaotong University Xi'an 710061, China.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Ren
authors.affiliation: Department of Cardiology, The Second Affiliated Hospital of Medical College in Xi'an Jiaotong University Xi'an 710004, China.
authors.forename: Ming-Juan
authors.initials: MJ
authors.lastname: Zhang
authors.affiliation: Department of Geriatric-Cardiovascular Diseases, The First Affiliated Hospital of Medical College in Xi'an Jiaotong University Xi'an 710061, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Geriatric-Cardiovascular Diseases, The First Affiliated Hospital of Medical College in Xi'an Jiaotong University Xi'an 710061, China.
authors.forename: Ruo-Wen
authors.initials: RW
authors.lastname: Huang
keywords: NF-κB; Ouabain; endothelium; α subunit