Expression of voltage-gated potassium channel gene of pulmonary artery smooth muscle cells in COPD merge chronic hypoxic patients

AIM: To study the change of expressions of Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv3.1 genes in pulmonary artery smooth muscle cells (PASMCs) on COPD merge chronic hypoxic patients. METHODS: Human lung tissue was collected from surgical patients. RT-PCR technique was used to study the expression of Kv1.2, Kv1.3, Kv1.5, Kv2.1 and Kv3.1 genes. PASMCs were divided into two groups: ① PASMCs from normal human pulmonary artery, pure COPD patients and COPD merger chronic hypoxic patients pulmonary artery； ② Cultured PASMCs exposed to continual chronic hypoxia or normoxia. RESULTS: ① The expression of Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv3.1 encoding genes were found in human PASMCs exposed to either normixa or chronic hypoxia. ② The expression of Kv1.2, Kv1.5, Kv2.1 genes in PASMCs exposed to chronic hypoxia were significantly decreased compared with control groups (P<0.05). ③ The expression of Kv1.3, Kv3.1 genes in PASMCs exposed to chronic hypoxia showed no significant change compared with control groups (P>0.05). ④ The expression of Kv1.2, Kv1.5, Kv2.1, Kv3.1 genes in pure COPD patients were significantly increased compared with control groups (P<0.05). CONCLUSIONS: ①The results suggested that Kv1.2, Kv1.5, Kv2.1 genes may be oxygen sensitive gene. Their expressions are affected by chronic hypoxia, which probably play an important role in human pulmonary artery hypertension. ② Kv1.3, Kv3.1 genes may not be oxygen sensitive gene and their expression are not affected by chronic hypoxia, which might play a secondary role in human pulmonary artery hypertension.     

Influences of chronic hypoxia on the gene expression of Kv1.3,Kv2.1 and Kv3.1 induced by acute hypoxia in PASMC of rats

AIM and METHODS: Total RNA was extracted from 6th rat subcultured pulmonary artery smooth muscle cells(PASMC) exposed to continual chronic hypoxia or normoxia and the effects of chronic hypoxia on the changes of Kv1.3,Kv2.1,Kv3.1 mRNA in cultured PASMC induced by acute hypoxia were studied by semiquantitative RT-PCR in vitro. RESULTS:①Kv1.3,Kv2.1,Kv3.1 genes were found to be expressed in PASMC of rats exposed either to hypoxia or normxia.②The expression of Kv2.1 and Kv3.1 in 6th subcultured of PASMC in normaxia group could be upregulated by exposure to acute hypoxia,the levels of Kv2.1 and Kv3.1 mRNA were significantly increased from 0.646±0.092, 0.782±0.104 to 1.059±0.134, 0.985±0.116,respectively (P<0.01,n=5). ③PASMC cultured continuously in chronic hypoxia for 6 subcultures and then exposed to normoxia for 12 h,thereafter the expression of Kv2.1 and Kv3.1 were downregulated by acute hypoxia for 6 hours.The level of Kv2.1 mRNA was significantly decreased from 1.008±0.117 to 0.649±0.097 (P<0.01,n=5). CONCLUSION:Kv2.1,Kv3.1 genes might be oxygen sensitive genes.Chronic hypoxia might change the response of these Kv genes of PASMC to acute hypoxia and down-regulate its expression,which might probably decrease the role of Kv in HPV.     

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.     

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.     

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 SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells under hypoxia conditions

AIM: To explore the effect of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions. METHODS: PASMCs was cotransfected with pEFSOCS3 and pSV2neo by lipofectamine, and positive cell clones were obtained after being screened with G418. Expressions of SOCS3 protein in PASMCs before and after transfection were detected by immunocytochemistry, respectively. Before and after transfection, PASMCs were exposed to normoxic and hypoxia conditions at various time points, respectively, and the expressions of c-myc mRNA were assessed by semi-quantitive RT-PCR. ［3H］-TdR incorporation method was used to detect the cell proliferation. RESULTS: The expression of SOCS3 protein was confirmed by immunocytochemistry in PASMCs transfected with SOCS3 gene. c-myc mRNA level in the SOCS3 gene-transfected cells exposed to hypoxia were remarkablely lower than that in the control cells, respectively (P<0.01). Compared with the control groups at the same time points, ［3H］-TdR incorporation in SOCS3 gene-transfected cells was significantly low. CONCLUSION: SOCS3 protein may inhibit the proliferation of PASMCs by downregulating the c-myc gene expression under hypoxia conditions.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Roles of the Kv1.2, Kv1.5, Kv2.1 potassium channels in hypoxia pulmonary vasoconstriction

AIM: To determine the role of Kv1.2, Kv1.5, Kv2.1 in the hypoxia pulmonary vasoconstriction (HPV). METHODS: Male Wistar rats were divided into two groups: normoxic group and hypoxic group. The single smooth muscle cell was obtained from pulmonary artery of Wistar rats with acute enzymatic digestion method. The conventional whole-cell patch clamp technique was used to record the resting membrane potential (Em) and the potassium currents of voltage-gated potassium channel (IKv) in rat pulmonary arterial smooth muscle cells (PASMC). Intracellular application of Kv1.2/Kv1.5/Kv2.1 antibodies (1∶125) was conducted through the whole-cell patch clamp system. RESULTS: ① Em of PASMC was depolarized after 24 h hypoxia compared with that of control cells . IKv of PASMC was decreased after 24 h hypoxia, . ② The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies depolarized Em and inhibited IKv in PASMC from normoxic rat, whereas the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on them. ③ The mixture of Kv1.2/Kv1.5/Kv2.1 antibodies and the mixture of Kir2.1/Kir2.3/Kir4.1 antibodies had no effects on IKv and Em from rats hypoxic for 24 h. CONCLUSION: Kv1.2, Kv1.5, Kv2.1 might be oxygen sensitive potassium channels which mediated HPV.     

Effect of chronic cigarette smoking on BKCa and Kv1.5 expression in rat pulmonary arterial smooth muscle cells

AIM:To investigate the role of potassium channel expression alteration in chronic cigarette smoking-induced increase in pulmonary vascular responsiveness, the effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel (BK_Ca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) expression in rat pulmonary smooth muscle cells were investigated in vivo. METHODS:HE staining, immuno-histochemistry and in situ hybridization techniques were used. RESULTS: (1) Chronic cigarette smoking downregulates the protein and mRNA expression of BK_Ca in pulmonary arterial smooth muscles. (2) Chronic cigarette smoking downregulated the protein and mRNA expression of Kv1.5 in pulmonary arterial smooth muscles. (3) In big artery, BK_Ca decreased more makedly than Kv1.5, but in small artery, both of them decreased equally. CONCLUSION:Chronic cigarette smoking downregulates the levels of BK_Ca and Kv1.5 in rat pulmonary arterial smooth muscle cells in vivo, which maybe contribute to the mechanism of cigarette smoking-induced increase in pulmonary vascular responsiveness.     

Effects of Kv1.5 on proliferation and apoptosis of rat PASMCs under hypoxia+hypercapnia condition and relationship with MAPK pathway

AIM：To investigate the effects of voltage-dependent K＋ channel 1.5 (Kv1.5) on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia+hypercapnia condition and the relationship with mitogen-activated protein kinase(MAPK) signal pathway. METHODS：The PASMCs isolated from the male SD rat were cultured under hypoxia+hypercapnia condition, and randomly divided into normal group (N group), hypoxia+hypercapnia group (HH group), hypoxia+hypercapnia+DMSO incubation group (HD group), hypoxia+hypercapnia+U0126 (an extracellular signal-regulated kinase 1/2 inhibitor) incubation group (HU group), hypoxia+hypercapnia+SB203580 (a p38 mitogen-activated protein kinase inhibitor) incubation group (HS group), and hypoxia+hypercapnia+anisomycin (an agonist of MAPK) incubation group (HA group). Cell Counting Kit-8 was used to detect the cell viability. The protein expression of Kv1.5, PCNA and Bax was detected by Western blotting. RESULTS：Compared with N group, the cell viability and PCNA protein expression in HH group and HD group were significantly raised (P<001), but Kv1.5 and Bax proteins were significantly decreased (P<0.01). No difference between HH group and HD group was observed (P>005). Compared with HD group, the cell viability and PCNA protein expression in HU group, HS group and HA group were decreased (P<0.05 or P<0.01), but Kv1.5 protein and Bax protein were raised (P<0.01), with the most significant changes in HA group. ^{CONCLUSION：}The regulation of Kv1.5 to the proliferation and apoptosis of PASMCs under hypoxia+hypercapnia condition might have a relationship with the activation of MAPK signal pathway.     

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.     

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.     

Effect of ginsenoside Rb1 on proliferation and serotonin transporter and 5-HT1B R expression in hypoxia-induced rat pulmonary artery smooth muscle cells via Rho/Rho-kinase pathway

AIM: To observe the effect of ginsenoside Rb1 on the proliferation and the expression of serotonin transporter (SERT), 5-hydroxytryptamine 1B receptor (5HT_1BR) in rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition and the relationship with Rho/Rho-kinase signal pathway.METHODS: PASMCs were isolated from the adult male SD rats and primarily cultured. The subcultured cells from the 4th generation to the 6th generation were harvested and divided into normal group, and hypoxia group, different concentrations of Rb1 incubation groups treated with 50, 100 and 200 mg/L ginsenoside Rb1 under hypoxia (HR50, HR100 and HR200 groups, respectively). The viability of the PASMCs was measured by CCK-8 assay. BrdU positive cells were determined using flow cytometry. The expression of serotonin transporter and 5HT_1BR at mRNA and protein levels was detected by RT-PCR and Western blot, respectively. The PASMCs were randomly divided into normal group, hypoxia group, HR200 group and hypoxia+Y-27632 incubation group (HY group). The mRNA expression of Rho-kinase and phosphorylated myosin phosphatase target subunit 1 (p-MYPT1) protein level were investigated by RT-PCR and Western blot, respectively.RESULTS: Compared with normal group, the proliferation of PASMCs in hypoxia group was significantly increased (P<0.01). The cell viability and the expression of SERT and 5HT_1BR at mRNA and protein levels in all different concentrations of Rb1 groups were obviously decreased compared with hypoxia group (P<0.05). The mRNA expression of Rho-kinase and protein level of p-MYPT1 were markedly decreased in HR200 group, and no significant difference compared with HY group was observed (P<0.01).CONCLUSION: Treatment with ginsenoside Rb1 might prevent hypoxia-induced proliferation of PASMCs and over-expression of SERT and 5HT_1BR through inhibiting the Rho/Rho-kinase pathway.     

Expression of volume-activated chloride channel in rat PASMCs treated with hypoxia and hypercapnia and its relationship with MAPK pathway

AIM：To investigate the expression of volume-activated chloride channel (CLC3) in rat pulmonary artery smooth muscle cells (PASMCs) treated with hypoxia and hypercapnia and its relationship with MAPK pathway. METHODS：The method of enzyme digestion was used to isolate the PASMCs in male SD rat for cell primary culture. The cells were identified by immunofluorescence cytochemical method with mouse anti-rat α-smooth muscle actin antibody. The rat model of hypoxia and hypercapnia was established. The protein expression of CLC3 was detected by Western blotting. The mRNA expression of CLC3 was determined by RT-PCR. RESULTS：Compared with control group, the mRNA and protein expression of CLC3 in PASMCs was significantly raised in hypoxia and hypercapnia group. Compared with hypoxic and hypercapnic group, the expression of CLC3 was significantly reduced in ERK inhibitor U0126+ hypoxia and hypercapnia group, and was up-regulated in p38 inhibitor SB203580+ hypoxia and hypercapnia group. p38 activator anisomycin significantly decreased the expression of CLC3 at mRNA and protein levels in hypoxia and hypercapnia group. CONCLUSION：The expression of CLC3 at mRNA and protein levels in PASMCs increases under hypoxia and hypercapnia conditions. The ERK1/2 pathway mediates CLC3 expression in PASMCs induced by hypoxia and hypercapnia. Activation of p38 MAPK pathway down-regulates the expression of CLC3 at mRNA and protein levels induced by hypoxia and hypercapnia.     

Downregulated transient outward potassium channel protein Kv4.2 and Kv4.3 expression in PVN contributes to sympathoexcitation in rats with chronic heart failure

AIM: To investigate the transient outward potassium channel protein expression in paraventricular nucleus(PVN) and its contribution to renal sympathetic nerve activity(RSNA) in rats with chronic heart failure(CHF).METHODS: A rat model of CHF was prepared by acute myocardial infarction that was induced by ligation of the left anterior descending coronary artery. Four weeks after heart failure, echocardiogram was applied to identify the CHF model and plasma norepinephrine(NE), serum NH₂-terminal pro-brain natriuretic peptide(NT-proBNP) were detected by ELISA. The expression of ransient outward potassium channel proteins Kv4.2 and Kv4.3 at mRNA and protein levels was determined by real-time PCR and Western blot. The mean arterial pressure(MAP), heart rate(HR) and RSNA were measured in anesthetized rats with PVN microinjection of potassium channel blockers 4-AP. RESULTS: In CHF group, the rat cardiac function and Kv4.2 and Kv4.3 expression in PVN were obviously lower while plasma NE and serum NT-proBNP were obviously higher than those in sham group. Microinjection of 4-AP into PVN induced an increase in MAP, HR and RSNA in both sham and CHF rats, while the CHF rats exhibited smaller responses to 4-AP than sham-operated rats.CONCLUSION: Downregulation of Kv4.2 and Kv4.3 expression in the PVN may be a potential mechanism for sympathoexciation in the rats with chronic heart failure.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions

AIM: To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia.METHODS: PASMCs were transfected with Gax gene by Ad-Gax.Under normal oxygentention (21% O₂) or hypoxia (2.5% O₂) for 12 h condition,expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry.The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR.［³H］-TdR incorporation was used to measure the PASMCs proliferation.RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry.Under normal oxygentention or hypoxia,the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group,respectively (P<0.05).［³H］-TdR incorporation in the transfection group was lower than that in non-transfection group (P<0.05,P<0.01).CONCLUSION: Gax overexpression might inhibit the PASMCs proliferation induced by hypoxia through downregulating the expressions of c-fos and c-jun.     

Inhibitory effect of notoginsenoside monomer R1 on activation of p38 MAPK signaling pathway induced by hypoxic hypercapnia in PASMCs

AIM: To explore the mechanism of notoginsenoside monomer R₁ (R₁) against hypoxic hypercapnia-induced pulmonary vasoconstriction (HHPV) by investigating the effect of R₁ on p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in pulmonary arterial smooth muscle cells (PASMCs) under the condition of hypoxia and hypercapnia. METHODS: Primary cultured PASMCs, which were isolated from Sprague-Dawley rats, were incubated in logarithmic growth phase from the 2nd to 5th generation with different concentrations (8, 40 and 100 mg/L) of R₁ under the condition of 6% CO₂ plus 1% O₂ for 24 h. The expression of p38 at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. RESULTS: The results of Western blotting and RT-PCR analysis indicated that the protein and mRNA expression levels of p-p38 MAPK were significantly higher in hypoxic hypercapnia group with DMSO control than those in normoxia control group (P<0.01). In R₁ treatment groups, the levels of p-p38 MAPK protein and p38 MAPK mRNA were markedly decreased (P<0.01) in a dose-dependent manner. CONCLUSION: p38 MAPK signaling pathway may mediate hypoxic hypercapnia pulmonary vasoconstriction in rats. Notoginsenoside monomer R₁ attenuates HHPV, which may be related to blockage of p38 MAPK signal pathway.