| Proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important contributor to the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension. Proliferation inhibition and apoptosis induction of PASMCs may be one of the effective methods to reduce pulmonary vascular remodeling induced by chronic hypoxia. Recent studies have demonstrated that the expression of Na~+/H~+ exchanger-1 (NHE-1) in the membrane of PASMCs is elevated remarkably under hypoxia condition and NHE-1 regulation of intracellular pH may play a permissive role in PASMCs proliferation. Besides, some studies also showed that the NHE-1 specific inhibitors suppressed the PASMCs proliferation and reduced pulmonary vascular remodeling induced by chronic hypoxia. As we well known, intracellular acidification can induce apoptosis of many kinds of cells. So,we hypothesized that NHE-1 inhibition would induce PASMCs apoptosis by decreasing intracellular pH. This study was therefore undertaken to examine the effects of inhibition of NHE-1 expression on apoptosis and expression of apoptosis-associated genes in PASMCs of rats under normoxic or hypoxia conditions by transfecting NHE-1 ribozyme genes into them.Methods1. Based on the sequence and secondary structure of NHE-1 mRNA, two hammerhead ribozymes were designed, synthesized, and subcloned into the multiple clone sites BamHI/EcoRI in retroviral vector pLXSN respectively.2. After PASMCs were primary cultured, the recombinant retroviral vectors were transferred into the PASMCs with FuGENE 6 in vitro. The effects of ribozymes on expression of NHE-1 mRNA were detected by sqRT-PCR.3. The PASMCs transfected with recombinant retroviral vectors and the control cells were cultured under normoxic and hypoxia (<1% O2) conditions, respectively. pHi values in these cells were determined with fluorescent probe BCECF-AM, and cell cycle was measured by flow cytometric DNA analysis. We also observed cell apoptosis with electronmicroscopy and terminal deoxynucleotidyl transferase-medited dUTP nick end labeling (TUNEL) respectively. 4. Intracellular Ca2+ concentration ([Ca2+]i) of cells was measured with fluorescence dye Fura-2/AM. Expression of mRNA and proteins of fas, fasL, bcl-2 and bax in cells were detected by sqRT-PCR and immunohistochemically, respectively. Results1.DNA sequencing verified the successful construction of recombinant retroviral vectors of hammerhead ribozymes.2. The positive cell clones transfected with recombinant vectors were obtained after being screened with 100μg/ml G418. Expression of Neo gene in G418 resistant cells by RT-PCR and PCR suggested the successful transfection of recombinant vectors. In contrast to normal PASMCs and cells transfected with pLXSN, NHE-1 mRNA expression decreased significantly in the cells transfected with recombinant vectors of ribozymes.3. Under normoxic conditions and in contrast to normal PASMCs and cells transfected with pLXSN, pHi value in recombinant vectors transfected cells decreased significantly. Flow cytometric DNA analysis showed that cells in G0/G1 phase increased and cells in G2/M phase decreased significantly in recombinant vectors transfected cells. Meanwhile, apoptosis rates of theses cells increased significantly and cell apoptosis in theses cells was observed by electron microscopy.4. Under hypoxia conditions and in contrast to normal PASMCs and cells transfected with pLXSN, pHi values and G2/M phase cells didn't increase and G0/G1 phase cells didn't decrease significantly in recombinant vectors transfected cells. But apoptosis rates in these cells increased significantly and continuously. Electron microscopy showed swelling of mitochondria and expanding of endoplasmic reticulum which appeared earlier in recombinant vectors transfected cells, and cell edema in recombinant vectors transfected cells exposed for 24 hours to hypoxia.5. Under normoxic conditions and in contrast to normal PASMCs and cells transfected with pLXSN, [Ca2+]i values and mRNA expression and protein expres...
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