Effects of chronic hypoxia on intracellular calcium concentration in pulmonary arterial smooth muscle cells

AIM:To study the effect of chronic hypoxia (CH) on the intracellular calcium (［²⁺］_i) in pulmonary artery smooth muscle cells (PASMCs) and the role of L-type calcium channel and calcium store. METHODS:The rat chronic hypoxia model was set up and intervene the PASMCs with normal PSS, calcium-free PSS, nifedipine, and heparine respectively. The resting ［Ca²⁺］_i was determined with the Fura-2/AM calcium imaging technique. RESULTS:(1) The ［Ca²⁺］_i in CH group in normal PSS was higher than that in control group in normal PSS (P<0.05). The ［Ca²⁺］_i in CH group in normal PSS was higher than that in calcium-free PSS (P<0.05). (2) No obvious change of ［Ca²⁺］_i before and after application of nifedipine in PASMCs of CH groups was observed. (3) No difference of ［Ca²⁺］_i before and after application of heparine in PASMCs of CH groups was detected. CONCLUSION:Chronic hypoxia increased the ［Ca²⁺］_i in PASMCs. Chronic hypoxia induced increase in ［Ca²⁺］_i may relate to the influx of extracellular calcium, but not due to the L-type calcium channel or the IP3R modulation only.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

Effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia

AIM: To investigate the effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) from rats exposed to chronic hypoxia. METHODS: (1) Wistar rats were randomly divided into control group (group A) and chronic hypoxia group (group B). Then group B received hypoxia 8 hours per day for 4 consecutive weeks. (2) Single PASMC was obtained via acute enzyme separation method. (3) Conventional whole-cell patch clamp technique was used to record resting membrane potential (Em) and ion currents of voltage-gated potassium channel. The changes of ion currents of voltage-gated potassium channel before and after applying cGMP (1 mmol/L), an agonist of protein kinase G (PKG), and cGMP plus H-8 (1 mmol/L), an inhibitor of PKG were compared between two groups. RESULTS: The Em of group B were significantly lower than that of group A. The ion currents of voltage-gated potassium channel in group A and group B were all significantly inhibited by cGMP [control group: from (118.0±5.0) pA/pF to (89.9±16.5) pA/pF, n=6, P＜0.05;chronic hypoxia group: from (81.0±5.0) pA/pF to (56.8±9.1) pA/pF, n=6, P＜0.05]and these effects were reversed by H-8 [control group: from (119.2±10.3) pA/pF to (117.8±9.1) pA/pF, n=6, P＞0.05;chronic hypoxia group: from (96.8±6.2)　pA/pF to (98.0±2.2)　pA/pF, n=6, P＞0.05]. CONCLUSIONS: The currents of voltage-gated potassium channel was inhibited by chronic hypoxic. The inhibitory effect of cGMP on currents of voltage-gated potassium channel in PASMCs from both normal and chronic hypoxic rats may be probably through the phosphorylation of voltage-gated potassium channel.     

Studies of potassium channel in pulmonary artery smooth muscle cells of pulmonary hypertension rats

AIM：In this work,we investigated the difference of membrane capacitance,membrane current,current density and I-V curves between smooth muscle cells isolated from pulmonary hypertension rat (PHR) or normotensive rat (NTR) pulmonary arteries.METHODS：Thirty young male Sprague-Dawley rats,aged 8-9 weeks,were used.Body weight was (200±20)g at the start of experiments.These rats were placed into a normobaric chamber for 6 h·day-1,6 day·week-1 for 4 weeks.Hypoxic exposure was accomplished by ventilation with room air and N2 resulting in a constant O2 concentration of (10±0.5)%.Whole cell recordings were made from smooth muscle cells freshly isolated from pulmonary arteries derived from PHR or NTR.RESULTS：The membrane capacitance of pulmonary hypertension rats was larger than that in SD rats;but membrane current and current density were lower than those in SD rats (P<0.05).The I-V curves of pulmonary hypertension rat were downward shift compared with that in SD rat.Iptakalim hydrochloride 10 μmol·L-1 significantly increased potassium currents.CONCLUSIONS：Membrane capacitance,membrane current,membrane potential are decreased,I-V curves was shift downward,compared with NTR.Iptakalim hydrochloride significantly increased NTR and PHR potassium currents.     

Inhibitory effect of Salidroside on the proliferation of rabbit pulmonary artery smooth muscle cells under hypoxia

AIM: To investigate the effect of Salidroside on the proliferation, DNA synthesis, intracellular Ca²⁺ content of rabbit PASMC (pulmonary artery smooth muscle cells) under hypoxia. METHODS: Techniques of cell culture, MTT test, [³H][³H][³H]-TdR incorporation, fluo-3 and confocal laser scanning microscopy were used. RESULTS: The A value of MTT and [³H][³H]-TdR incorporation of PASMC increased significantly by 62% (P＜0.05) and 138% (P＜0.01) after 24 h hypoxia. Salidroside (32×10^-5 mol/L) inhibited the action of hypoxia on the proliferation of PASMC, the A value of MTT and [³H][³H]-TdR incorporation declined significantly by 29% (P＜0.05) and 37% (P＜0.01) compared with hypoxia group. A calcium channel blocker, verapamil could also inhibit the accelerative effect of hypoxia on the proliferation of PASMC. The intracelluler Ca²⁺ content of PASMC raised markedly under hypoxia, but the effect of hypoxia on the intracelluler Ca²⁺ content could be inhibited by Salidroside. CONCLUSION: Salidroside inhibited the proliferation, DNA synthesis of PASMC induced by hypoxia. The inhibitory action of Salidroside on the increase in intracellular Ca²⁺ concentration under hypoxia might be one of the mechenisms.     

Effect of BQ123 on the voltage-gated K+ current of pulmonary artery smooth muscle cells of rats exposed to chronic hypoxia

AIM:To study the effect of BQ123 on voltage-gated K+ current in pulmonary artery smooth muscle cells (PASMCs) from chronic hypoxic rats. METHODS:Twelve age and body weight matched Wistar rats were randomly divided into control and chronic hypoxic group. Single PASMCs were obtained with acute enzyme (collagnaseⅠ plus papain) dispersing method. Using the whole cell patch-clamp technique in freshly isolated PASMCs from normorxic and hypoxic rats, the effects of ET-1 and BQ123, a selective ETA receptor antagonist, on voltage-gated K+ current were recorded. RESULTS:(1) ET-1 (10-8 mol·L-1) caused inhibition of K+ current in PASMCs from normoxic and hypoxic rats. The effect of ET-1 on K+ current in PASMCs from hypoxic rats was greater than that from normoxic rats ［+50 mV, percent inhibition were (71.04±6.58)% and (60.21±5.32)%, respectively, P<0.01, n=6］. (2) In normoxic PASMCs, neither BQ123 alone produced influence on the IKV (P>0.05, n=5), nor ETA receptor blockade had change of ET-1 mediated IKV inhibition. (3) In chronic hypoxic PASMCs, BQ123 significantly reduced the effect of ET-1 mediated IKV inhibition, from (28.49±6.69) pA/pF to (74.19±9.74) pA/pF at +50 mV (P<0.01, n=6). CONCLUSION:In normoxic condition, the effect of ET-1 on IKV of PASMCs is not mediated by BQ123, a selective ETA receptor antagonist. During exposure to chronic hypoxia, the inhibition of ET-1 on IKV of PASMCs is partly mediated by BQ123, namely, ETA receptor mediates the effect of ET-1 on IKV of chronic hypoxic PASMCs.     

Effects of aging and hypoxia on morphology of cultured rat pulmonary arterial smooth muscle cells

AIM:To observe the effects of aging and hypoxia on morphology of cultured rat pulmonary arterial smooth muscle cells (PASMCs). METHODS:The cells were divided into four groups: young and normoxic group (A group), aging and normoxic group (B group), young and hypoxia group (C group), aging and hypoxia group (D group). Afterwards, the different morphological variation was observed by means of optical microscope, immune histochemistry and immune fluorescence. RESULTS:Huge differences in morphological characters in PASMCs in hypoxia and in normoxic were observed, particularly, the difference was clearly shown in F-actin concentration and array in the cytolymph. Compared with normoxic group, the concentration of SM-α-actin in hypoxic PASMCs group decreased sharply. CONCLUSION:Aging and hypoxia lead to morphological change in PASMCs. Both factors stimulate the phenotypic modulation in PASMCs, but the phenotypic modulation effect is more apparent in the condition of hypoxia.     

Effects of levcromakalim on pulmonary arterial endothelial cells and smooth muscle cells exposed to hypoxia

AIM:To explore the effects of levcromakalim(Lev) on pulmonary arterial endothelial cells (PAEC) and smooth muscle cells (PASMC) exposed to hypoxia and the mechanisms involved.METHODS:The effects of Lev on [Ca²⁺]_i, and expression of PKC_α, eNOS, iNOS and PDGF-B mRNA and protein levels were observed. The nitrite (NO₂^-) and entothelin-1(ET-1) concentrations in supernatant in cultured PAEC and PASMC were measured. The proliferation and apoptosis of PASMC was also detected.RESULTS:When PASMC were exposed to hypoxia, Lev reduced concentration of ET-1 in cultured cell supernatant, lowed the expression of PKC_α, iNOS and PDGF-B both at mRNA and protein levels, decreased [Ca²⁺]_i concentration, increased proliferation and promoted the apoptosis in PASMC. However, in the presence of Lev, the [Ca²⁺]_i concentration was not changed in the hypoxic PAEC. The NO₂^- concentration in cultured cell supernant and expression of eNOS at mRNA and protein levels in hypoxic PASMC and PAEC were also unchanged. The downregulated ET-1 activity in PASMC and PAEC and proliferation in PASMC involved in the inhibition of PKC_α signaling pathway.CONCLUSIONS:Lev reduce some disadvatage effect of hypoxia on PASMC and PAEC. The mechanism of Lev action may partly involve in the downregulation of PKC_α signaling functions.     

Effect of chronic hypoxia on [Ca2+]iin pulmonary artery endothelial cells and smooth muscle cells under acute hypoxia

AIM AND METHODS: Using Ca²⁺-sensitive fluorescent probe Fura-2,we measured the changes of [Ca²⁺]_iin cultured rat pulmonary artery endothelial cells (PAEC) and porcine pulmonary artery smooth muscle cells (PASMC) from normoxic (NC group) or chronic hypoxic group (CH group) when they were exposed to acute hypoxia. RESULTS: The increase in [Ca²⁺]_iin 6th passage of PASMC caused by acute hypoxia in CH group was significantly lower than that in the same passage of NC group (P<0.05).On the contrary, in PAEC, the acute hypoxia induced increase in _i, which was significantly higher in 5th passage of CH group than that in NC group (P<0.05). CONCLUSION: The decrease of the elevation of [Ca²⁺]_icaused by acute hypoxia in PASMC of CH group indicated that it functioned to lower the constrictive response to hypoxia.The intensive increase in [Ca²⁺]_icaused by acute hypoxia in PAEC of CH group might lead to more relaxing factors derived from PAEC,which results in a decrease in HPV.     

Effects of Rho kinase on p-Smad1 nuclear translocation in rat pulmonary artery smooth muscle cells

AIM: To explore the mechanism of bone morphogenetic protein (BMP) and Rho kinase signal pathways on the proliferation of pulmonary artery smooth muscle cells. METHODS: Pulmonary smooth muscle cells were isolated from the rat distal pulmonary artery and cultured. BMP and Rho kinase pathways were activated by BMP-2 and platelet-derived growth factor BB(PDGF-BB),respectively. Rho kinase inhibitor Y-27632 and MEK inhibitor U0126 were also used. Immunofluorescent staining was applied to observe p-Smad1 distribution across the nucleus, and the cells with positive p-Smad1 nuclear accumulation were counted and the nuclear translocation rate was calculated. The total p-Smad1 and its distribution across the nucleus were quantitatively determined by Western blotting. The cell proliferation was analyzed by CCK-8 assay. RESULTS: Exposure to BMP-2 significantly increased both the total amount of p-Smad1 and its nuclear accumulation in pulmonary smooth muscle cells. Pretreatment with PDGF-BB significantly decreased the nuclear accumulation of p-Smad1 induced by BMP-2 without decrease of total p-Smad1. However, pretreatment with Y-27632 or U0126 reversed the inhibitory effect of PDGF-BB on p-Smad1 nuclear accumulation. BMP-2 significantly inhibited the cell proliferation, but PDGF-BB blocked the effect of BMP-2 and significantly increased the cell proliferation. After pretreated with Y-27632 or U0126, the PDGF-BB-activated cell proliferation was suppressed.CONCLUSION: PDGF-BB-activated Rho kinase inhibits BMP-2-induced p-Smad1 nuclear translocation via MEK/ERK1/2, and increases pulmonary artery smooth muscle cell proliferation.     

Effects of docosahexaenoic acid on large-conductance calcium-activated potassium channels in rat pulmonary artery smooth muscle cells

AIM：To investigate the effects of docosahexaenoic acid (DHA) on large-conductance calcium-activated potassium channels (BKCa) in rat pulmonary artery smooth muscle cells (PASMCs)．METHODS：BKCa currents in individual PASMCs were recorded by patch-clamp technique in whole-cell configuration．Calcium sparks in PASMCs caused by DHA were recorded by confocal microscopy. RESULTS：DHA activated BKCa . BKCa current densities were (30.5±6.5)pA/pF,(59.4±5.8)pA/pF, (87.2±4.3)pA/pF and (117.3±7.1) pA/pF (P<0.01) with the addition of DHA at concentrations of 0, 0.1, 1 and 10 μmol/L, respectively. Hypoxia inhibited BKCa currents in PASMCs, but this inhibition was reversed by DHA (10 μmol/L). DHA (10 μmol/L) induced an increase in ［Ca2+］i with a maximal increase rate of (71.9±4.1)%. CONCLUSION：DHA activates BKCa in rat PASMCs, leading to the vasodilation of pulmonary arteries.     

An approach to calcium signal transmission between L-type calcium channel and sarcoplasmic reticulum of atrial myocytes during atrial fibrillation

AIM: To inquire into the Ca2+ signal transmission from L-type calcium channel to the sarcoplasma reticulum in atrial fibrillation. METHODS: Ten adult cross-bred dogs were used in the experiment. Five dogs underwent continuous rapid atrial pacing (500±20 beats/min) for twenty-four weeks to create persistent atrial fibrillation. Another group of size-matched dogs (n=5) without pacemaker implantation was used as a control group. Canine atrial myocytes were isolated by enzymatic dissociation. The Ca2+ cytosolic transient in atrial myocytes was analyzed by confocal imaging after loading myocytes with the acetoxymethyl ester of fluo-3 (Fluo-3/AM). Ca2+ signal transmission from L-type Ca2+ channels in the plasma membrane to the sarcoplasma reticular IP3R and RyR in atrial myocytes during atria fibrillation were measured. RESULTS: (1) Ca2+ signal transmission from L-type calcium channel to IP3R in the sarcoplasma reticulum:intracellular Ca2+concentration was slightly increased in two groups after blocking T-type calcium channel and RyR, but showed no statistic significance (P＞0.05) between them. (2) Ca2+ signal transmission from L-type calcium channel to RyR in the sarcoplasma reticulum: intracellular Ca2+concentration was risen (1.5576±0.1989) in control groups after blocking T-type calcium channel and IP3R, and no significance was observed (P＞0.05) compared with that in atrial fibrillation group (1.5372±0.2952). CONCLUSIONS: Ca2+ signal transmission possibly exists from L-type calcium channel to RyR and IP3R in the sarcoplasma reticulum, but it does not play an important role in intracellular Ca2+-overload and abnormal Ca2+ signal transmission during atrial fibrillation.     

Role of NHE-1 in the proliferation and apoptosis of pulmonary artery smooth muscle cell in rats

AIM: To evaluate the role of Na⁺/H⁺ exchanger-1(NHE-1)in the proliferation and apoptosis of pulmonary artery smooth muscle cells in rats. METHODS: Twenty Wistar rats were equally randomized into the control group and 3-week hypoxic group. Intracellular pH (pHi) of the smooth muscle was determined with fluorescence measurement of the pH-sensitive dye BCECF-AM and the expression of NHE-1 mRNA was detected with RT-PCR. The primary culture of pulmonary artery smooth muscle cell in vitro was performed. In situ cell death detection kit (TUNEL) was used to study the effect of specific NHE-1 inhibitor, dimethyl amiloride (DMA), on the apoptosis of muscle cells which had intracellular acidification. RESULTS: pHi value and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell were significantly higher respectively in the hypoxic group than those in the control group (P＜0.01). DMA elevated the apoptotic ratio significantly. The effect was enhanced when DMA concentration was augmented and the time was prolonged. CONCLUSION: With the function of adjusting pHi, NHE-1 may play an important role in the proliferation and apoptosis of pulmonary artery smooth muscle cells.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Effects of three kinds of calcium activator on the proliferation of cultured vascular smooth muscle cells in rats

AIM: To investigate the effects of intracellular free calcium ([Ca²⁺]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP₃) and ryanodine (RY). MAPK activity was measured by [γ-³²P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [³H]-Leucine ([³H]-Leu) and [³H]-Thymidine ([³H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP₃ and RY significantly increased [Ca²⁺]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [³H]-Leu and [³H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca²⁺]i concentration but independent to its origin.     

Alteration of cardiac sarcoplasmic reticulum function in vitamin D3-induced calcium overload rats

AIM:To study alterations of cardiac sarcoplasmic reticulum (SR) function in vitamin D₃-induced calcium overload rats. METHODS: The Ca-overload rat models were prepared by vitamin D₃ plus nicotine. Cardiac SR was seperated by centrifuging. The measurement of SR Ca²⁺uptake and Ca²⁺ release activities were preformed by the Millimore filtration technique. Specific SR -ryanodine binding capacity was measured by radioligand method. RESULTS: Compared with control,myocardial calcium content in calcium overload rats increased by 78%(P＜0.01), SR Ca²⁺ uptake and Ca²⁺ release activities decreased by 64% and 40% respectively(P＜0.01),and in the meantime ,the Ca²⁺-ATPase activity decreased by 65%(P＜0.01).Maxmum value for -ryanodine binding decreased by 51%(P＜0.01). CONCLUSION:The function of cardiac SR in calcium-overload rats was decreased.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Effect of diazoxide on change of H2O2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

AIM: To investigate the contribution of diazoxide,an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP),and mitochondrial membrane potential (ΔΨm) to change of H₂O₂ in rat pulmonary artery smooth muscle cells (PASMCs) and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia.METHODS: The rat PASMCs were isolated from fresh normal lung tissues and cultured,which were divided into 6 groups,as follows: ① control group;② diazoxide group;③ 5-HD group;④chronic hypoxia group;⑤ chronic hypoxia+diazoxide group;⑥ chronic hypoxia +5-HD group.The relative change in mitochondrial potential was detected with rhodamine fluorescence (R-123) technique.The level of H₂O₂ in rat PASMCs was detected with chemiluminescence method.The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.RESULTS: After exposed to diazoxide for 24 h,the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in normoxic rat PASMCs were significantly increased,and the apoptosis of rat PASMCs was significantly decreased as compared with control group (P<0.05).However,there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24 h.Chronic hypoxia or chronic hypoxia+diazoxide markedly increased the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in rat PASMCs,and also markedly decreased the apoptosis of rat PASMCs as compared with control group (P<0.05),and these changes were more significant in chronic hypoxia +diazoxide group than those in chronic hypoxia group (P<0.05).5-HD partly weakened the effect of hypoxia on the intensity of R-123 fluorescence,the level of H₂O₂,the A value and the apoptosis of rat PASMCs (P<0.05).Significant and positive correlations were found between the intracellular H₂O₂ and the R-123 fluorescence or the A value.Significant and negative correlation was found between the intracellular H₂O₂ and the apoptosis of rat PASMCs.CONCLUSION: The results suggest that the opening of MitoK_ATP followed by a depolarization of ΔΨm can contribute to the increase in the level of H₂O₂ in rat PASMCs and to the proliferation of rat PASMCs induced by hypoxia.This might be a mechanism of the development of hypoxic pulmonary hypertension.     

Model construction of rat coronary artery smooth muscle cell endoplasmic reticulum stress induced by thapsigargin

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.     

Effects of adipose tissue-derived mesenchymal stem cells on calcium channels of pulmonary artery in rats with pulmonary arterial hypertension induced by monocrotaline

AIM: To investigate the effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) on calcium channels of pulmonary artery in monocrotaline (MCT)-induced pulmonary hypertensive rats.METHODS: ADMSCs were isolated from adipose tissue by collagenase digestion. Twenty-four Sprague-Dawley rats were randomly divided into 3 groups: normal control (Ctr) group, pulmonary arterial hypertension (PAH) group and ADMSCs transplantation group. Mean pulmonary arterial pressure (MPAP) was measured by catheterization, and right ventricular hypertrophy index (RVHI) was calculated. The expression of voltage-gated calcium channel α1c subunit (CaVα1c), sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA-2a), inositol 1,4,5-triphosphate receptor 1(IP3R-1), transient receptor potential channel 1 (TRPC1) and TRPC6 at mRNA and protein levels in the pulmonary trunks was determined by RT-PCR and Western blotting, respectively.RESULTS: MPAP and RVHI were higher in PAH group than those in Ctr group, while those in ADMSCs group were significantly decreased as compared with PAH group. The expression of CaVα1c, TRPC1 and TRPC6 at mRNA and protein levels was obviously increased in PAH group as compared with Ctr group, while that in ADMSCs group was significantly decreased as compared with PAH group. Compared with Ctr group, the expression of SERCA-2a and IP3R-1 at mRNA and protein levels was obviously decreased in PAH group, while that in ADMSCs group was significantly increased as compared with PAH group.CONCLUSION: MPAP and RVHI are attenuated by ADMSCs in MCT-induced pulmonary hypertensive rats. The reduction of pulmonary arterial pressure by ADMSCs transplantation in MCT-induced pulmonary hypertensive rats may be related to the changes of calcium channels.