| Pulmonary vascular remodeling is the major characteristic of hypoxia-induced pulmonary hypertension, mainly represented by over-proliferation of pulmonary artery smooth muscle cells (PASMCs). The stimulus for PASMCs proliferation is not fully understood, but it appears to involve release of growth factors and other vasoactive substances that are activators of Na+/H+ exchange. Increased Na+/H+ exchange with an intracellular alkalization is an early event in cell proliferation. This intracellular alkalization by stimulation of Na+/H+ exchange appears to play a permissive role in the PASMCs proliferation of vascular remodeling.Sodium hydrogen exchanger -1 (NHE-1) is expressed in virtually all of the eukaryotic membranes, where it most likely fulfills 'housekeeping' functions including mediating electroneutral exchange of one extracellular sodium ion for one intracellular hydrogen ion and modulating intracellular pH (pHi). The expression of NHE-1 in the membrane of PASMCs is elevated remarkably under hypoxia condition and the NHE-1 specific inhibitors can suppress the PASMCs proliferation. In this study, NHE-1 specific hammerhead ribozymes and antisense RNA were transfected into PASMCs in vitro. Then, the changes in NHE-1 mRNA expression, Na+/H+ exchange activity, pHi value and H-TdR incoporation in PASMCs were measured to identify the role of NHE-1 in PASMCs proliferation. Methods1. Based on the sequence and secondary structure of NHE-1 mRNA, twohammerhead ribozymes were designed and synthesized, and the DNAIIsequence encoding antisense RNA was obtained by PCR. They were subcloned into the multiple clone sites BamHI/EcoRI in retroviral vector pLXSN respectively.2. For endogenous expression of hammerhead ribozymes and antisense RNA, the recombinant retroviral vectors were transferred into the PASMCs with FuGENE 6 in vitro. The effects of ribozymes and antisense RNA on expression of NHE-1 mRNA were detected by RT-PCR.3. In the PASMCs transfected with recombinant retroviral vectors, pHi value was determined with fluorescent probe BCECF-AM and 22Na uptake was measured. We also analyzed the changes in the kinetics of Na+/H+ exchange, including pHi recovery rate and extracellular Na+-dependent pHi recovery rate after intracellular acid loading. H-TdR incorporation was performed to assess the proliferation of PASMCs.4. pHi value, 22Na uptake, Na+/H+ exchange kinetics and 3H-TdR incorporation were measured in PASMCs treated with 10% serum, PDGF and EGF.Results1. DNA sequencing and restriction endonuclease cleave electrophoresis of plasmid verified the successful construction of recombinant retroviral vectors of hammerhead ribozymes and antisense RNA. The positive cell clones transfected with recombinant vectors were obtained after being screened with 100 g/ml G418. Expression of Neo gene in G418 resistant cells by RT-PCR and PCR also suggested the successful transfection of recombinant vectors.2. In contrast to normal PASMCs and cells transfected with pLXSN, NHE-1 mRNA expression, pHi value, pHi recovery rate after intracellular acid loading, 22Na uptake and 3H-TdR incorporation decreased significantly in the transfected cells with recombinant vectors of ribozyms and antisense RNA.3. Treated with 10% serum, PDGF and EGF for 10 minutes, pHi valueincreased in recombinant vectors transfected cells, but the increased extent was remarkably lower than in the normal PASMCs. When the treating time prolonged to 24 hours, no change in pHi value was found in recombinant transfected cells. Treated with 10% serum, PDGF and EGF for 24 hours, pHi recovery rate, 22Na uptake and 3H-TdR incoporation were significantly lower in the recombinant transfected cells than in the normal PASMCs. But, 3H-TdR incorporation in the recombinant transfected cells increased to some extent after treatment of 10% serum, PDGF and EGF. Conclusions1. We successfully constructed the recombinant retroviral vectors of NHE-1 specific hammerhead ribozymes and antisense RNA and transfect...
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