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1.
AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca2+]i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca2+ indicator Fura-2/AM was used to observe [Ca2+]i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca2+]i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca2+]i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K+-channel antagonist 4-aminopyridine (4AP), but not the Ca2+-activated K+-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K+-channel antagonist glibenclamide (Glib) increased [Ca2+]i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca2+]i , but Glib had no effect on [Ca2+]i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: KV plays an important role in the regulation of [Ca2+]i and proliferation of PASMCs. KCa serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. KATP has no effect on [Ca2+]i and proliferation of PASMCs in normoxic and hypoxic conditions.  相似文献   

2.
AIM:To investigate the role of K+ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K+ channels, 4-AP(voltage dependent K+ channel blocker), TEA(Ca2+ activated K+ channel blocker), GLIB(ATP sensitive K+ channel blocker) were used in isolated perfused rat lungs to detect the role of K+ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, KCa, KATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.  相似文献   

3.
AIM: To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension. METHODS: The intracellular calcium concentration ([Ca2+]i) was detected by laser-scanning confocal microscopy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique. RESULTS: Increased levels of [Ca2+]o or Gd3+ (an agonist of CaSR) induced the increase in [Ca2+]i and pulmonary artery constriction in a concentration-dependent manner. Additionally, the effects of Ca2+ and Gd3+ were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP3 receptor). However, U73343 (U73122 inactive analogue) did not take effect. CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by increasing [Ca2+]i through G-protein-PLC-IP3 pathway.  相似文献   

4.
AIM and METHODS: To determine the role of different K+-channels in attenuation of vasoreactivity of intrapulminary artery rings induced by chronic hypoxia. RESULTS: ①Acute hypoxia-induced pulmonary vasoconstriction (HPV) could be significantly attenuated by chronic hypobaric hypoxia for 15 days and for 30 days. ②HPV could be significantly potentiated by ATP-sensitive K+-channel (KATP) blocker or Ca2+-activated K+-channel (KCa) blocker, and the potentiated scope in chronic hypoxic group was much higher than that observed in control group. ③Delayed rectifier K+-channel (KDR) blocker showed no effect on HPV in both control group and chronic hypoxic group. CONCLUSION: Both KATP and KCa play an important modulating role in HPV and its potentiation may be a critical mechanism for the attenuated vasoreactivity to acute hypoxia following chronic hypobaric hypoxia.  相似文献   

5.
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   

6.
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.  相似文献   

7.
AIM: To investigate the role of intracellular free Ca2+ concentration (［Ca2+］i) in the regulation of calcium-activated chloride (ClCa) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca2+ indicator Fura-2/AM was used to observe ［Ca2+］i of rat PASMCs in normal and chronic hypoxic condition. The influences of ClCa channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The ClCa channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca2+］i was increased. In normoxic condition, ［Ca2+］i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca2+］i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca2+］i had no significant change and no effect on ClCa channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca2+］i which opened ClCa channels. The NFA and IAA-94 blocked them and decreased ［Ca2+］i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca2+］i which opened ClCa channels and had a positive-feedback to ［Ca2+］i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, ClCa channel may play a role in the regulation of PASMCs proliferation.  相似文献   

8.
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   

9.
Stromal interaction molecule 1 (STIM1) is a transmembrane protein of the endoplasmic reticulum (ER), a Ca2+ transducer in ER that activates the store-operated calcium channel. Through Orai1 protein, STIM1 adjusts the intracellular and extracellular calcium concentration. This way is called a store-operated Ca2+ entry. STIM1 plays a key role in phenotypic transformation of vascular smooth muscle cells, proliferation of endothelial cells, myocardial hypertrophy and myocardial fibrosis to regulate lots of cardiovascular diseases, such as atherosclerosis, congestive heart failure and systemic hypertension. STIM1 is closely related to cardiovascular diseases through calcium signal. The research progress of STIM1 in cardiovascular diseases is mainly discussed in this article.  相似文献   

10.
AIM: Nitroxyl(HNO) increases myofilament Ca2+ responsiveness relative to increases in intracellular Ca2+ in cardiac muscle. In this study, we further investigated this effect of HNO on trabecular muscles from phospholamban knockout(PLB-KO) and wide-type(WT) mice using a novel HNO donor, 1-nitrosocyclohexyl acetate(NCA). METHODS: Trabecular muscles were dissected from the right ventricles of the rat hearts and mounted between a force transducer and a motor arm. The muscles were superfused with K-H solution(pH 7.4) at room temperature. Fura-2 was loaded into the trabecular muscles via electrophoresis. The length of the sarcomere was set to 2.2~2.3μm. During steady-state activations, the maximal Ca2+-activated force and Ca2+ required for 50% activation were measured. RESULTS: The intracellular Ca2+ transients and force of the PLB-KO muscles at baseline were higher than those of the WT muscles and exhibited a negative force-frequency relationship(FFR). NCA(2.5μmol/L) increased systolic force in both PLB-KO group and WT group at any given[Ca2+]o. However, there was more dramatic increase in the force development due to moderate increases in the intracellular Ca2+ transients in the WT muscles when external Ca2+ increased from 1.5 to 4.5 mmol/L under NCA. NCA did not affect the negative FFR in PLB-KO muscle. Steady-state force-Ca2+ relations obtained from skinned muscles were not different between the 2 groups, while NCA increased Ca2+ responsiveness in skinned muscles from both PLB-KO and WT mice.CONCLUSION: HNO increases force development in both PLB-KO and WT muscles as a result of increases in myofilament Ca2+ responsiveness. The increased intracellular Ca2+ transients are accompanied by greater force development in WT mice, suggesting that HNO improves Ca2+ activation and establishes HNO as a positive inotropic agent with novel mechanisms.  相似文献   

11.
AIM: To investigate the possible role of prostaglandin, NO and potassium channel in the adaptive blunting of hypoxic pulmonary vasoconstriction (HPV) in high altitude animal (pika). METHODS: The effect of L-NAME, indomethacin and 4-AP on the response of isolated lung strips of pika and Wistar rats instead of pulmonary artery to acute hypoxia were studied. RESULTS: (1) After inhibition of prostaglandins by indomethacin, the percentage increase in hypoxic constriction in lung tissue strip of pikas was greater than that in Wistar rats , P＜0.05; (2) After inhibition of NO synthesis by L-NAME, the percentage increase in hypoxic constriction in lung tissue strip of pikas was greater than that in Wistar rats , P＜0.05; (3) After administration of 4-AP, there was no significant difference in the percentage decrease in hypoxic constriction of lung tissue strip between pika and Wistar rat. CONCLUSIONS: (1) Prostaglandins and NO might modulate HPV in either rat of pika; the closure of Voltage-gated K+ channels might mediate HPV in either rat or pika. (2) Prostaglandins and NO might play an important role in the blunting of HPV in pika, Voltage-gated K+ channels may play a minor role in the blunting of hypoxic pulmonary vasoconstriction in pika.  相似文献   

12.
AIM: To study the relaxation effect of isoliensinine on high K+-induced isolated mouse airway smooth muscle (ASM) and the underlying mechanism. METHODS: The muscle tension transducer was used to detect the effects of isoliensinine on high K+-induced precontraction and Ca2+ influx in ASM. The technique of patch-clamp and calcium imaging system were respectively used to examine the effects of isoliensinine on LVDCC currents and[Ca2+]i of the ASM cells (ASMCs). RESULTS: Isoliensinine significantly relaxed precontracted ASM induced by high K+ in a concentration-dependent manner. The maximum relaxation ratio was(95.3±3.9)% by isoliensinine at 100 μmol/L. In addition, LVDCC currents were measured using the whole-cell patch-clamp technique, which were abolished by isoliensinine. High K+-induced 340/380 nm fluorescence ratio of Fura-2 was 0.63±0.10 in ASMCs, while it decreased to 0.36±0.05 after the addition of isoliensinine (P<0.01). When isoliensinine was added at the peak point of[Ca2+]i, the ratio rapidly decreased from 0.74±0.02 to 0.42±0.05 (P<0.01). Moreover, isoliensinine inhibited high K+-induced Ca2+ influx-mediated contraction of ASM. CONCLUSION: Isoliensinine inhibits LVDCC currents, terminates Ca2+ influx and reduces[Ca2+]i, eventually resulting in relaxation of the ASM, indicating isoliensinine might be a potential bronchodilator.  相似文献   

13.
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.  相似文献   

14.
AMI:To clarify whether OX-LDL and simvastatin can induce the changes of PKC activity and cytosolic free Ca2+ in rat aortic smooth muscle cells (ASMC). METHODS:PKC activity and cytosolic free Ca2+ were measured by its ability to transfer phosphate from [32P]ATP to lysine-rich histone and flow cytometric analysis after loading with the Ca2+dye fluo-3/Am, respectively. RESULTS:OX-LDL increased PKC total activity in a dose-dependent manner and induced translocation of PKC from the cytosolic to membrane, while OX-LDL induced biphasic [Ca2+]i responses including the rapid initial transient phase and the sustained phase. When simvastatin was added, the translocation of PKC was markedly decreased and simvastatin did not impair the initial peak response to OX-LDL but significantly reduced the subsequent plateau phase. CONCLUSSION:OX-LDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca2+ in ASMC and these two events are closely linked.  相似文献   

15.
AIM: To explore the mechanism of ET-1, NO and PGI2 release from coronary artery endothelial cells(CAEC) induced by acute hypoxia. METHODS: Bovine coronary artery endothelial cells were cultured and [45 Ca2+] was used to investigate the difference of calcium uptake between normoxia group and hypoxia group(3% O2). The contents of ET-1, NO and PGI2 in media of normoxia group, hypoxia group and hypoxia + verapamil group were measured 24 h after hypoxia. RESULTS: [ 45 Ca2+] uptake by CAEC in hypoxia group was 1.9 times more than normoxia group(P< 0.01). Hypoxia + verapamil group released more PGI2, ET-1 and less NO than hypoxia group(P< 0.05). CONCLUSION: Changes of ET-1, NO and PGI2 releases during hypoxia may be caused by the inflow of Ca2+ into coronary artery endothelial cells.  相似文献   

16.
AIM: To investigate changes of Ca2＋ activated potassium channels (KCa) in autogenous vein grafts. METHODS: The contraction of venous ring was measured by means of perfusion in vitro. The intimal proliferation and proliferation of cultured smooth muscle cells (vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method, respectively. Furthermore, whole cell mode of patch clamp was used to record KCa of VSMCs isolated from autogenous vein grafts. RESULTS: 1 week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P＜0.05, n=8 vs control), and they were more enhanced 4 weeks after vein transplantation (P＜0.01, n=8 vs control). TEA (blocker of Ca2＋ activated potassium channels) increased MTT A490 value of VSMCs from femoral vein in a dose-dependent manner (P＜0.05, n=8). KCa current density was significantly attenuated in VSMCs from autogenous vein grafts 1-4 weeks after transplantation (P＜0.05, n=5). CONCLUSION: KCa was inhibited in autogenous vein graft, which accounted for vasospasm and intimal proliferation.  相似文献   

17.
AIM: To investigate the role of reactive oxygen species (ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose. METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis. The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h. The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose (35 mmol/L) for 24 h. RESULTS: After high glucose treatment, the cell proliferation was inhibited. The early apoptosis and total cell death increased to (24.16?3.53)% and (63.74?4.32)%,respectively. High glucose treatment significantly increased intracellular levels of ROS and Ca2+. The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM. Inhibition of store-operated Ca2+ channels by La3+ also decreased the intracellular level of Ca2+ and cell apoptosis induced by high glucose. CONCLUSION: High glucose increases intracellular ROS level and the release of Ca2+ through the store-operated Ca2+ channels, thus resulting in intracellular Ca2+ overload and leading to apoptosis of osteoblasts.  相似文献   

18.
AIM: The effect of urotensin II (U-II) on proliferation of cultured pulmonary arterial smooth muscle cells (PASMCs) of rabbits and its mechanism are investigated. METHODS: PASMCs were isolated using explant technique. RPASMCs were incubated in serum-free medium with different concentrations of nicardipine, calcimodulin antagonist W7, PKC inhibitor H7 or MAPK inhibitor (PD98059), with or without U-II. RPASMC proliferation was examined by MTT assay and by the increase in [3H]-thymidine incorporation into DNA. RESULTS: U-II (10-9 mol/L-10-7 mol/L) increased A value of PASMCs by MTT assay and [3H]-thymidine incorporation in PASMCs in a dose-dependent manner. U-II induced a maximal effect at a concentration of 10-7 mol/L. A value and [3H]-thymidine incorporation rose 42.9% and 68.5% (P<0.05), respectively. U-II had no effect at a concentration of 10-10 mol/L. Nicardipine, W7, H7, PD98059 (10-7 mol/L-10-5 mol/L) inhibited the effect of U-II in inducing increase of A value and -thymidine incorporation in a dose-dependent manner, with the maximal inhibitory rate of 42.3%, 19.6%, 23.2%, 10.5% (P<0.05) in A value and 46.6%, 9.8%, 21.7%, 14.7% (P<0.05) in [3H]-thymidine incorporation at concentration of 10-5 mol/L, respectively. CONCLUSION: Our results suggest that U-II may induce proliferation of PASMCs of rabbits by Ca2+, CaM, PKC and MAPK signal transduction pathway.  相似文献   

19.
AIM: To study the expression of Jagged2/Notch3 signaling molecules in pulmonary vascular wall of pulmonary hypertensive rats induced by monocrotaline. METHODS: SD rats were randomly divided into normal control group (C group,n=15), solvent control group (S group,n=15) and monocrotaline model groups (M group,n=15). The model of pulmonary hypertension was established by a single subcutaneous injection of monocrotaline (50 mg/kg). The rats in S group were given a single subcutaneous injection of the same dose of solvent. After 4 weeks, the pulmonary vascular remodeling was assessed by HE staining, and the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) were determined by right heart catheterization. The expression of Jagged2/Notch3/Hes5 molecules in the pulmonary vascular wall was detected by immunohistochemical method and real-time PCR. RESULTS: Compared with S group and C group, the percentage of medial wall thickness of smaller arteries in model group increased significantly (P<0.01). The levels of mPAP and RVSP in M group were significantly higher than those in S group and C groups (P<0.01). The results of real-time PCR showed that the expression of Jagged2, Notch3 and Hes5 was significantly increased in M group compared with S group and C group. The data from immunohistochemical detection indicated that Jagged2 mainly expressed in the intima of small lung artery, Notch3 and Hes5 mainly expressed in the medial smooth muscle cells. Compared with S group and C group, the expression of Jagged2 and Notch3 was significantly increased in the lung small arteries of M group. CONCLUSION: The activation of Jagged2/Notch3 signaling pathway might play an important role in the formation of pulmonary hypertension.  相似文献   

20.
AIM:These studies aimed at exploring the alteration of intracellular Ca2+ level in the course of macrophage-derived foam cell formation as well as its mechanism.METHODS:Foam-like cell was generated by peritoneal macrophage of C57BL/6J mouse, which is susceptible to atherosclerosis, incubated in 10 mg·L-1 oxidized low density lipoprotein for 96 hours. With the technique of Ca2+ fluorescent indicator and the assay of NADH-oxidizing coupling spectrum-alteration, the intracellular Ca2+ level and membranous Ca2+-ATPase activity of the above foam-like cell were determined.RESULTS:The foam-like macrophage Ca2+ level was 2.7 times higher than the control macrophage, and the former Ca2+-ATPase activity was 24% of the later.CONCLUSION:The results suggested that macrophage-derived foam cell formation was connected with slow Ca2+ entry or release, which possibly derived from long-lasting opening of membranous Ca2+ channels at the early stage and irreversible inactivating of membranous Ca2+ pump at the late stage.  相似文献   

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