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2.
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.  相似文献   

3.
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.  相似文献   

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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.  相似文献   

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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 PKCα, 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 PKCα, 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 PKCα 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 PKCα signaling functions.  相似文献   

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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.  相似文献   

9.
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.  相似文献   

10.
AIM:To investigate the expression of ET-1 mRNA in porcine pulmonary artery endothelial cells cultured in normoxic and chronic hypoxic conditions, and their different responses to acute hypoxia were also evaluated.METHODS:Insituhybridization and image -analysis system were used. RESULTS:Acute hypoxia enhanced the expression of ET-1 mRNA in both normoxic and chronic hypoxic group. The increment was more significant in the latter group.CONCLUSION:Chronic hypoxia increased the expression of ET-1 mRNA in response to acute hypoxia in porcine pulmonary artery endothelial cells.  相似文献   

11.
AIM: To detect the role of cyclic nucleotides in the alleviation of hypoxic pulmonary vasoconstriction (HPV) in chronic hypoxic animals. METHODS: The intracellular cAMP and cGMP of the cultured porcine pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were assayed by RIA. The length of single PASMC during acute hypoxia was measured by imaging analysis system. RESULTS: The basal levels of cAMP and cGMP in PASMC and cGMP in PAEC of Chronic hypoxic groups decreased remarkably compared with normoxic groups (P＜0.01). Under acute hypoxia, the contents of cAMP and cGMP in PASMC of chronic hypoxic groups increased significantly (P＜0.01). Meanwhile, the percentage of PASMC with weak constrictive response in chronic hypoxic group was higher than that of control group. CONCLUSION:It's suggested that the changes of cAMP and cGMP in chronic hypoxic PASMC and PAEC might contribute to the increase in the basic tension of pulmonary artery and the alleviation of HPV in chronic hypoxic animals.  相似文献   

12.
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.  相似文献   

13.
AIM: To investigate the effect of sulodexide (SDX) on the apoptosis of human dermal microvascular endothelial cells (HDMECs) exposed to hypoxia and its underlying mechanism. METHODS: The HDMECs were cultured and divided into normoxia control group cultured under normoxic condition; hypoxia control group cultured in a humid incubator maintained at 37℃ with 5% CO2 and 1% O2 for 24 h; treatment groups treated with SDX at 0.25, 0.5 and 1 LSU/mL for 24 h under hypoxic condition. The cell viability was measured by CCK-8 assay. The apoptotic rate of the HDMECs was analyzed by flow cytometry. The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53, caspase-3, Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis. Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3, while the expression of anti-apoptotic factor Bcl-2 was up-regulated. CONCLUSION: SDX significantly increases the viability and decreases the apoptosis of HDMECs exposed to hypoxia. Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.  相似文献   

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15.
AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 μmol/L) and G6983 (1 μmol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2.5 fold and 2.0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.  相似文献   

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17.
AIM：To establish a fast, accurate and economical technique for culturing mouse pulmonary arteriolar smooth muscle cells (PASMCs), and to explore the effects of hypoxia on the proliferation and apoptosis of the PASMCs. METHODS：In sterile condition, the pulmonary artery was isolated from the male BALB/c mice by digesting with collagenase I, and the cells were cultured in fetal bovine serum-coated flask. Centrifugal procedure was not used during the cell passage. The cell morphology was observed under an inverted phase-contrast microscope. α-Smooth muscle actin was identified by immunocytochemistry and immunofluorescence. The effects of hypoxia on the proliferation and apoptosis of the PASMCs were detected by CCK-8 assay and TUNEL assay. RESULTS：PASMCs were identified by the methods of immunocytochemistry, immunofluorescence staining and observation of morphology. Unlike the rat PASMCs with typical subcultured peak-vally pattern, the mouse PASMCs showed a lot different without a peak-vally pattern. The cells could be subcultured after 5 d to 7 d and there was 3 to 5 generations depending on the activity of the cells. CCK-8 assay demonstrated that the A values of PASMCs exposed to hypoxia increased after 24 h (P＜0.05) as compared with normoxia. TUNEL result showed that the apoptotic index of the PASMCs in hypoxia decreased after 24 h (P<0.05). CONCLUSION：This technique for obtaining cultured mouse PASMCs is simple, fast, accurate and economical. The digestion time is easy to control. Hypoxia promotes the proliferation and inhibits the apoptosis of PASMCs.  相似文献   

18.
AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O2) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.  相似文献   

19.
AIM: To investigate the effect of histamine and hypoxia on the expression of eNOS mRNA and protein in cultured porcine pulmonary artery and aorta endothelial cells. METHODS: Semi-quantitative RT-PCR and immuno-cytochemistry were used. RESULTS: (1) Histamine increased eNOS mRNA expression in a dose-and time dependent manner. For pulmonary endothelial cells, the effect reached peak when exposed to 10-5 mol/L histamine in 24 h. eNOS mRNA level was increased to 178.2%±7.7% (P<0.01) compared with control. eNOS protein was also enhanced to 173%±47% (P<0.01) compared with control. For aorta endothelial cells, the effect reach peak when exposed to 10-6 mol/L histamine in 24 h. The eNOS mRNA level was increased to 177.4%±14.3% (P<0.01) compared with control. The eNOS protein was also enhanced to 165%±54% (P<0.01). (2) The eNOS mRNA was enhanced in pulmonary endothelial cells after exposed to hypoxia for 12 h and reached peak in 24 h, increasing to 151.0%±9.1% (P<0.01). The protein expression was also enhanced to 216%±44% (P<0.01) compared with control. But there was no significant change in eNOS mRNA and protein expression in aorta endothelial cells during hypoxia. CONCLUSION: The experiments show that histamine increases the endothelial eNOS expression in both pulmonary and aorta endothelial cells, whereas hypoxia only increases eNOS expression in pulmonary endothelial cells. This may account partly for the different responses of pulmonary circulation and systemic circulation to hypoxia.  相似文献   

20.
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.  相似文献   

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