| Pulmonary hypertension (PH) is associated with profound vascular remodeling andalterations in Ca2+homeostasis in pulmonary arterial smooth muscle cells(PASMCs).Previous studies show that canonical transient receptor potential1(TRPC1) is themajor constituent of store-operated Ca2+channel(SOCC) and TRPC genes areupregulated and store-operated Ca2+entry(SOCE) is augmented in PASMCs ofchronic hypoxic rats and patients of pulmonary arterial hypertension(PAH).Here wereported the involvement of TRPC and SOCE, and the contribution of stromalinteraction molecule(STIM)-Orai-TRPC-calcineurin(CaN)-nuclear factor of activatedT cells (NAFT) signaling pathway in the development of monocrotaline(MCT)-induced PAH. We further examined the reverse effect of cyclosporin A(CosA) inMCT-induced PAH for searching new theraputic target of PAH.1. Enhanced store-operated Ca2+entry and TRPC channel expression in MCT-induced PAH ratsObjective: To discuss the function and the pathologic physiology significance ofTRPC/SOCE signaling pathway in the development of MCT-induced PAH.Methods: PAH was induced in SD rats by a single intraperitoneal injection of MCTat a dose of60mg/kg. We measured:①mean right ventricular systolic pressure(mRVSP), mean right ventricular pressure(mRVP), right ventricular mass index(RVMI), histological staining and morphometry;②examine the expression of TRPCmRNA and protein, the timecourse of the upregulated TRPC mRNA expression byreal time RT-PCR and Western blot;③cyclopiazonic acid(CPA)-induced contractionin pulmonary arteries(PAs) and the timecourse curve, Endothelin-1(ET-1)-inducedPAs contraction and the inhibition of ET-1response caused by various SOCE blockers;④CPA and ET-1-induced Ca2+influx in pulmonary artery smooth musclecells(PASMCs), and the inhibition of ET-1-induced PASMCs Ca2+response caused byGd3+and La3+.Results: In comparison to the CON,①mRVSP, mRVP and RVMI were markedlyelevated in MCT; in MCT rats, the thickness of pulmonary vascular smooth muscleswas increased, inner diameter of PAs was diminished;②the expression of TRPC1/TRPC4mRNA and protein were significantly increased;③in MCT rats, applicationof CPA to deplete Ca2+stores caused dramatic increase in PAs contraction and in Ca2+influx of PASMCs(P<0.01), suggested that MCT upregualted SOCE;④theupregulation of TRPC1mRNA was higher than that of TRPC4, and the TRPC1mRNA expression peak appeared earlier than that of TRPC4; Importanly, the timecourse of increase in TRPC1expression corresponded to that of CPA-inducedcontraction, indicated that TRPC1plays an important role in SOCE;⑤MCTsignificantly potentiated the effect of ET-1on vasoconstriction in PAs; the ET-1-induced contraction and Ca2+response in MCT group were more susceptible to theinhibition caused by Gd3+, La3+, and SK&F-96365, as well as the general CRACinhibitor BTP-2.Conclusion: MCT-induced PAH is associated with increased TRPC1/4expressionand SOCE, which are involved in the enhanced vascular reactivity to ET-1; Theupregulation of TRPC1mRNA was higher than that of TRPC4, and the TRPC1mRNA expression peak appeared earlier than that of TRPC4; Importanly, the timecourse of increase in TRPC1expression corresponded to that of CPA-inducedcontraction, and support the hypothesis that TRPC1-dependent SOCE is an importantpathway for the development of PAH/PH.2. Alteration of TRPC signaling pathway of pulmonary artery in MCT-inducedPAH ratsObjective: To investigate the significance of STIM-Orai-TRPC-CaN-NAFT signalingpathway in the development of MCT-induced PAH.Methods: The mRNA levels of various genes in this singnaling pathway wereexamined using quantitative real-time RT-PCR. According to the changes in gene expression, the timecourse curve of mRNA expression and the proteins were furtherexamined and time course of change in mRVP was examined as well. Analyse thepotential relationship among mRVP, CPA-induced PAs contraction and geneexpression.Results:①In the upstream of TRPC, STIM2, Orai1/Orai2mRNA expression wereincreased significantly in PA of MCT-treated rats. STIM1and Orai3mRNA did nothave much change in both groups. The results of Western blot were consistent withthat of real-time RT-PCR.②In the downstream of TRPC, the expression of CaNBβ,NFATc3and NFATc4mRNA were upregulated in MCT-treated rats, but CaNAα/β/γ,CaNBα and NFATc1/2mRNA expression have no difference between CON and MCTrats.③Timecourse curve of mRNA expression in the upregulated gene shows thatSTIM2-Orai1-TRPC1-CaNBβ-NFATc3/NFATc4mRNA expression were increasedsignificantly in early stage(3days) after MCT injection, and then reached a plateau(5days), and sustained for3w.While mRVP reached the peak in2w after MCT injec-tion, the upregulation of gene expression was earlier than that of mRVP, suggestingthat MCT may induced PAH through upregulating STIM2-Orai1-TRPC1-CaNBβ-NFATc3/NFATc4.④Time course change of STIM2-Orai2-CaNBβ mRNA werehighly correlated with timecourse curve of CPA-induced PAs contraction; while thecoefficient of timecourse curve of TRPC1expression and CPA-induced PAs contrac-tion was0.716(P=0.071); time course change of TRPC1mRNA was strongly correlat-ed with that of STIM2, Orai1, CaNBβ, NFATc4; and time course change of CaNBβmRNA was strongly correlated with that of mRVP, STIM2, Orai1, TRPC1, NFATc4,indicating that TRPC1-CaNBβ may be the central link of this signaling pathway.Conclusion: Upregulation of STIM2-Orai1/Orai2-TRPC1/4-CaNBβ-NFATc3/NFATc4may contribute to the development of MCT-induced PAH. The upregulationof TRPC1and its related genes(TRPC1-Orai1-STIM2-CaNBβ-NFATc4) was earlierthan that of mRVP after MCT injection, TRPC1-CaNBβ may be the central link andinitiation factor, which caused the upregulation of gene expression in TRPC signalingpathway, enhancement of SOCE, and then finally raised mRVP in MCT-inducedPAH. 3. Preventive and therapeutical effects on molecular changes of TRPC signalingpathway by cyclosporin A in MCT-induced PAH ratsObjective: To further examine the contribution of STIM-Orai-TRPC-CaN-NFATsignaling pathway in the development of PAH, and find new therapeutic targets forpharmacotherapy of PAH.Methods: After the injection of MCT(see Part1), cyclosporin A(CosA) was intraperi-toneally injected at a dose of35mg/kg on alternate days to establish CosA-treatedMCT-induced PAH rat model. We measured:①mRVSP, mRVP, RVMI and histologi-cal staining and morphometry;②CPA-induced vasoconstriction;③ET-1-induceddose-dependent contraction in PAs;④the inhibition of ET-1-induced vasoconstrictionby Gd3+;⑤the mRNA and protein levels of STIM-Orai-TRPC-CaN-NFAT signalingpathway.Results: Compared with MCT-treated rats,①mRVSP, mRVP and RVMI weredramatically decreased in CosA-treated rats; In CosA group, hyperlasia of pulmonaryarteriolar smooth muscle was weakened, the ratio of luminal area over total area wasincreased notably, and the ratio of the wall thickness over artery radius was diminish-ed markedly;②after CosA treatment, CPA-induced vasoconstriction was reducedfrom MCT's74.0±17.8%to MCT+CosA's29.4±13.0%; and dose-dependent curvecaused by ET-1was blunted by CosA treatment, EC50was increased from MCT's0.95±0.38nM to MCT+CosA's3.26±0.38nM;③CosA treatment lowered Orai2-TRPC1/TRPC4-CaNBβ-NFATc3mRNA levels but not STIM2and NFATc4mRNA inPAs of MCT rats.Conclusion: CosA inhibits MCT-induced PAH, pulmonary artery proliferation andcardiac hypertrophy, and blunted TRPC1/4/SOCE-induced vasoconstriction and ET-1response by3w's treatment. CosA may prevent the development of PAH by inhibitingOrai2-TRPC1/4-CaNBβ-NFATc3transcriptional activity.In summary, TRPC/SOCE is an important signaling pathway in the development ofMCT-induced PAH. Combined with the results of previous studies in chronic hypoxicrats, our study first raise that TRPC-dependent SOCE is a common signaling pathway involved in the development of PAH. MCT induces severe PAH, pulmonary arteryproliferation and cardiac hypertrophy by upregulating transcriptional activity ofSTIM2-Orai1-TRPC1-CaNBβ-NFATc3/NFATc4and by enhancing TRPC/SOCE. Thissevere PAH may be blunted through CosA treatment by inhibiting Orai2-TRPC1/4-CaNBβ-NFATc3. These findings have important experimental consequences inproviding new therapeutic target to prevent PAH.
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