[Role of bcl-2 and bax expression in apoptosis of hypoxic pulmonary artery smooth muscle cells of rats induced by Na+/H+ exchanger isoform-1 inhibition in vitro].
OBJECTIVE
To investigate the role of bcl-2 and bax expression in apoptosis of hypoxic pulmonary artery smooth muscle cells (PASMCs) of rats induced by Na+/H+ exchanger isoform-1 (NHE-1) inhibition in vitro.


METHODS
Cultured rat PASMCs transfected with NHE-1 ribozyme gene (PRZ cell), cultured rat PASMCs transfected only with pLXSN vector (PX cell) and non-transfected rat PASMCs (PA cell) were obtained in our previous studies. The cells were cultured under low oxygen tension (<1% O2) for 0, 2, 6, 12, 24 and 48 hours respectively. Then cell apoptosis was examined with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Intracellular Ca2+([Ca2+]i) of cells was measured using fluorescence dye Fura-2/AM. The changes in mRNA expression of bcl-2 and bax in cells were assessed by semi quantitative reverse transcription-polymerase chain reaction (sqRT-PCR). The expression of Bcl-2 protein and Bax protein of cells was detected immunohistochemically.


RESULTS
In contrast to normal PASMCs and cells transfected with pLXSN, the apoptosis rates in recombinant vectors transfected cells increased significantly and continuously. [Ca2+]i values were also increased in these cells, but the increase extent was remarkably lower than in the normal PASMCs (all P<0.01). The expression of bax mRNA and protein of cells transfected with recombinant vectors was increased significantly compared with cells of other two groups. Meanwhile, the expression of bcl-2 mRNA and protein of these cells were decreased significantly.


CONCLUSIONS
The NHE-1 specific hammerhead ribozymes induce apoptosis of PASMCs under low oxygen tension by inhibiting the increase in intracellular Ca2+ concentration, increasing bax expression and decreasing bcl-2 expression.
Lu
Jun-Yu
JY
Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Yao
Wei
W
Qian
Gui-Sheng
GS
Yang
Xiao-Jing
XJ
Chen
Wei-Zhong
WZ
Li
Shu-Ping
SP
D000818:Animals; D017209:Apoptosis; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D051028:bcl-2-Associated X Protein