低氧对培养的不同内径的肺动脉平滑肌细胞增殖的影响

目的和方法：分离培养三种不同内径的肺动脉平滑肌细胞（PASMCs）,用^3H－TdR掺入速率和细胞计数作为细胞增殖的指标,观察低氧对其增殖作用的影响。结果：低氧对三种不同内径的PASMCs（内径分别为＞1000μm、500－800μm、300-400μm）增殖促进作用显著不同,其^3H－TdR掺入速率和细胞计数分别增加23.5％和11.1％、60.0％和33.8％、141.4％和52.0％,选择对低氧最敏感的PASMCs（内径为300－400μm）,进一步探讨低氧促PASMCs增殖作用的细胞机制：钙拮抗剂verapail、蛋白激酶C抑制剂staurosporine(Stau)和细胞Na-H交换抑制剂amiloride可显著降低低氧情况下PASMCs^3H－TdR掺入速率和细胞计数。结论：低氧对三种不同内径的PASMCs增殖促进作用显著不同; Ca^2 、蛋白激酶C和Na^2 －H^ 交换的激活,可能是低氧促PASMCs增殖的重要胞内信息转导机制。     

Isolation,culture and identification of pulmonary arterial smooth muscle cells from rat distal pulmonary arteries

The culture of pulmonary arterial smooth muscle cells (PASMCs) is one of the most powerful tools for exploring the mechanisms of pulmonary hypertension (PH). Both pulmonary vasoconstriction and remodeling occur predominantly in distal pulmonary arteries (PA). In this study, we provide our detailed and standardized protocol for easy isolation and culture of PASMCs from rat distal PA to supply every investigator with a simple, economical and useful method in studying PH. The protocol can be divided into four stages: isolation of distal PA, isolation of cells, growth in culture and passage of cells. Rat distal PASMCs were characterized by morphological activity and by immunostaining for smooth muscle α-actin and smooth muscle myosin heavy chain, but not for CD90/Thy-1 or von Willebrand factor. Furthermore, functional assessments were performed, confirming the presence of voltage-dependent Ca²⁺ channels and physiological characteristic of response to hypoxia. In conclusion, we have developed a detailed and simple protocol for obtaining rat distal PASMCs. These PASMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to further explore the pathophysiological mechanisms of PH.     

离子通道在肺动脉的分布及在低氧肺血管收缩反应中的作用

低氧性肺血管收缩反应（HPV）是指在急性低氧时,肺泡氧分压降到某一临界值,肺血管发生的快速、可逆的收缩反应,以纠正肺泡通气／灌流的不匹配。HPV的发生与肺动脉平滑肌细胞上K^＋、Ca^2＋、Cl^-通道的状态密切相关,而这些通道在不同部位的肺动脉上分布存在差异,因此不同部位的肺动脉在低氧中所表现的收缩反应程度也不同,本综述将对上述通道在肺动脉上的分布特点及其在HPV中的作用做一总结。     

Ion exchange activity in pulmonary artery smooth muscle cells: the response to hypoxia

The purposes of this study were to determine 1) the presence of the major ion transport activities that regulate cytoplasmic pH (pH(c)) in cat pulmonary artery smooth muscle cells, i.e., Na+/H+ and the Na+-dependent and -independent Cl-/HCO3- exchange, 2) whether pH(c) changes in cells from small (SPAs) and large (LPAs) pulmonary arteries during hypoxia, and 3) whether changes in pH(c) are due to changes in the balance of exchange activities. Exchange activities as defined by physiological maneuvers rather than molecular identity were ascertained with fluorescence microscopy to document changes in the ratio of the pH(c) indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Steady-state pH(c) was higher in LPA than in SPA normoxic smooth muscle cells. SPAs and LPAs possessed all three transport activities; in HCO3- containing normoxic solutions, Cl-/HCO3- exchange rather than Na+/H+ exchange set the level of pH(c); in HCO3- containing hypoxic solutions, pH(c) increased in SPA and decreased in LPA cells; altering the baseline pH(c) of a cell type to that of the other did not change the direction of the pH(c) response during hypoxia. The absence of Na+ prevented hypoxia-induced alkalinization in SPA cells; in both cell types, inhibiting the Cl-/HCO3- exchange activities reversed the normal direction of pH(c) changes during hypoxia.     

缺氧时肺动脉内皮细胞与肺动脉平滑肌细胞增殖的相互影响

血管内皮细胞和血管平滑细胞在结构和功能上关系密切,两者的相互在与血管舒缩笔血和壁结构。本文观察了培养的小牛肺动脉内皮细胞（ＰＡＥＣｓ）和肺动平滑肌细胞（ＰＡＳＭＣＳ）缺氧时在细胞增殖方面的相互影响。ＰＡＳＭＣＳ常氧条件培养基（ＣＭ）可使ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入降低约５８％,缺氧ＣＭ对ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入无明显的抑制作用;ＰＡＥＣＳ的常氧ＣＭ使ＰＡＳＭＳ的＾３Ｈ－ＴｄＲ掺入升高约６０     

常氧和急性低氧下大鼠传导性肺动脉平滑肌细胞的电生理特性

本文旨在研究大鼠传导性肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）的电生理特征及对急性低氧的反应。用酶解法急性分离出1-2级分支的PASMCs,通过全细胞膜片钳方法研究常氧及急性低氧状况下细胞钾电流的差异,并在常氧下先后使用iBTX和4-AP阻断大电导钙激活钾离子（large conductance Ca-activated K^＋,BKCa）通道及延迟整流性钾离子（delayed rectifier K^＋,KDR）通道后,观察细胞钾电流特征。根据细胞的大小、形态及电生理特征可将PASMCs分为Ⅰ、Ⅱ、Ⅲ类。iBTX对Ⅰ类细胞几乎无作用,而4-AP几乎完全阻断它的钾电流;Ⅱ类细胞的钾电流在加入iBTX后大部分被抑制,其余的对4．AP敏感;Ⅲ类细胞的钾电流对iBTX及4-AP均敏感。急性低氧对三类细胞的钾电流均有不同程度的抑制,并使Ⅰ类细胞的膜电位显著升高,而Ⅱ、Ⅲ类细胞膜电位升高的程度不如Ⅰ类显著。结果表明,传导性肺动脉有3种形态及电生理特性不同的PASMCs,在急性低氧时其钾电流不同程度地受到抑制,同时静息膜电位也有不同程度去极化,这些可能参与急性低氧时传导性肺动脉舒缩反应的调节。KDR及BKCa通道在3种细胞中的比例不同可能是急性低氧对3种PASMCs影响不同的离子基础。     

大鼠肺动脉平滑肌细胞缺氧后STAT3活性变化与细胞增殖关系的研究

目的：探讨信号转导及转录活化因子3（STAT3）对缺氧大鼠肺动脉平滑肌细胞（PASMCs）增殖的影响及作用机制。方法：组织块法原代培养PASMCs,用AG490预孵育后进行缺氧处理,半定量RT-PCR,Westernblot法分别检测缺氧2h、6h、12h、16h、24h组STAT3酪氨酸活性水平变化;半定量RT-PCR检测缺氧条件下上述时相点c-mycmRNA水平变化;^3H-TdR掺入法观察缺氧条件下细胞增殖变化。结果：Western blot定量分析显示缺氧培养6h组STAT3酪氨酸磷酸化水平升高,12h组达高峰,16h略有下降;缺氧培养2h组c-mycmRNA表达升高,4h达高峰,6h下降,12h恢复至正常水平;^3H-TdR掺入法结果显示缺氧6h组细胞^3H-TdR掺入量的增加,并随缺氧时间延长变化更为显著。AG490抑制缺氧诱导STAT3酪氨酸磷酸化及c-mycmRNA表达。结论：①STAT3活化和c-myc表达参与缺氧PASMcS增殖;②在缺氧PASMCs增殖过程中STAT3上调c-myc表达。     

Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia and mitochondria inhibitors on the capacitative Ca(2+) entry (CCE) in cultured smooth muscle cells from rabbit small pulmonary arteries. Cyclopiazonic acid (CPA) depleted Ca(2+) from sarcoplasmic reticulum (SR) in Ca(2+)-free medium and subsequent addition of Ca(2+) led to the nifedipine-insensitive, La(3+)-sensitive Ca(2+) influx. The presence of CCE was further verified by the measurement of unidirectional Mn(2+) influx. During the decay phase of the CCE-induced [Ca(2+)]c transients, hypoxia (P(O2) < 50 mmHg) and the mitochondria inhibitor FCCP reversibly increased [Ca(2+)]c, that is La(3+)-sensitive. Once SR is depleted by CPA, subsequent treatment of FCCP slowed the decay of CCE-induced [Ca(2+)]c transients but it did not attenuate Mn(2+) influx. Mitochondrial uptake of incoming Ca(2+) through CCE was demonstrated by additional increase in [Ca(2+)]c with Ca(2+) ionophore after terminating CCE. Together, it is suggested that the augmentation of CCE-induced [Ca(2+)]c transients by hypoxia and FCCP reflects a net gain of [Ca(2+)]c by the inhibition of mitochondrial Ca(2+) uptake.     

三七皂苷单体Rg1对低氧高二氧化碳肺动脉平滑肌细胞p38MAPK表达的影响

目的:研究三七皂苷单体Rg1对低氧高二氧化碳肺动脉平滑肌细胞(PASMCs)p38MAPK表达的影响。方法:分离、纯化SD大鼠PASMCs,实验用2至5代细胞,实验分六组:常氧组(N组),低氧高二氧化碳组(H组),DM-SO对照组(HD组),Rg1干预组(RgL、RgM、RgH组)。采用Western blot检测磷酸化p38MAPK蛋白表达,RT-PCR检测p38MAPK mRNA的表达。结果:Westernblot、RT-PCR结果显示,HD组p-p38MAPK蛋白和p38MAPK mRNA表达明显高于N组(P<0.01),RgL、RgM、RgH组不同程度抑制了p-p38MAPK蛋白和p38MAPK mRNA和的表达(P<0.01),并呈剂量依赖关系。结论:三七皂苷单体Rg1对低氧高二氧化碳条件下PASMCs有保护作用,其机制可能与抑制p38MAPK的表达有关。     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells

Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K(+) channels play an important role in the regulation of vascular tone. In the fetus, Ca(2+)-activated K(+) channels (K(Ca)) are predominant, whereas from birth voltage-gated K(+) channels (K(V)) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K(+) channels in adulthood. We evaluated the effects of perinatal hypoxia on K(V) and K(Ca) channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only K(V) channels contributed to this current, whereas in mice born in hypoxia both K(V) and K(Ca) channels were implicated. K(V) channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased K(Ca) and K(V) channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance K(Ca) alpha-subunit and K(V)1.5 alpha-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,l-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by K(Ca) channels and only slightly by K(V) channels, whereas following perinatal hypoxia both K(Ca) and K(V) channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K(+) channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity.     

红细胞生成素3‘端增强子在肺动脉平滑肌细胞代氧性增殖调控中的作用

用噻唑蓝比色法（ＭＴＴ法）、Ｈ＾３－胸腺嘧啶核苷（Ｈ＾３－ＴｄＲ）掺入法和流式细胞术,观察红细胞生成素（ＥＰＯ）３’端增强子片段对培养的猪肺动脉平滑肌细胞（ＰＡＳＭＣｓ）的内皮依赖性和非内皮依赖性低氧性增殖的影响。结果为：（１）低氧２４ｈ后ＰＡＳＭＣｓ明显增殖,转入野生型ＥＰＯ３’端增强子片段可被抑制,而转入突变型片段无此作用;（２）肺动脉内皮细胞（ＰＡＥＣｓ）低氧２４ｈ,其条件培养液有明显的促Ｐ     

P2X7 receptor-mediated phenotype switching of pulmonary artery smooth muscle cells in hypoxia

Molecular Biology Reports - P2X7R activation contributes to the pathogenesis of pulmonary hypertension. However, the molecular mechanism through which P2X7R participates in pulmonary vascular...     

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.     

Alterations of endothelium and smooth muscle function in monocrotaline-induced pulmonary hypertensive arteries

We examined how monocrotaline (MCT), which impairs the endothelium and causes pulmonary hypertension, altered the endothelial regulation of pulmonary artery functions. Rats were given a single injection of MCT (60 mg/kg sc). Pulmonary arteries were depolarized to -48.3 +/- 2.6 and -39.8 +/- 2.2 mV at 2 and 3 wk after treatment with MCT, respectively (control arteries -59.9 +/- 1.9 mV). The basal tone in the resting state was only slightly elevated at 3 wk in endothelium-intact arteries. Removal of the endothelium caused further depolarization in MCT-affected arteries at 2 wk, but not at 3 wk, and greatly elevated the basal tone at 2 and 3 wk. N(omega)-nitro-L-arginine (200 microM), a nitric oxide synthase inhibitor, also caused depolarization in endothelium-intact arteries in both groups and elevated the basal tone of MCT-affected arteries. The relaxant responses of pulmonary arteries to ACh and A-23187 were depressed at 2 and 3 wk after MCT treatment. Thus chronic impairment of the endothelium altered the property of the pulmonary artery leading to depolarization. During the early stage of depolarization, a rise in the basal tone was offset by nitric oxide released from the injured endothelium.     

Regulation of activin A expression in mast cells and asthma: its effect on the proliferation of human airway smooth muscle cells

Activin A, a homodimeric protein (betaAbetaA) and a member of the TGF-beta superfamily, is involved in the inflammatory repair process. Using cDNA microarray analysis, we discovered strong induction of the activin betaA gene in human mast cells (MC) on stimulation with PMA and calcium ionophore (A23187). Activin betaA mRNA was also highly induced in primary cultured murine bone marrow MC (BMMC) after stimulation by IgE receptor cross-linking. Secretion of activin A was evident in human mast cell-1 line cells 3 h after stimulation and progressively increased over time. Activin A was present in the cytoplasm of activated but not unstimulated murine bone marrow MC as demonstrated by immunofluorescence studies, suggesting that secretion of activin A by MC was due to de novo synthesis rather than secretion of preformed protein. Activin A also colocalized with human lung MC from patients with asthma by double-immunofluorescence staining. Furthermore, secretion of activin A was significantly increased in the airway of wild-type mice after OVA sensitization followed by intranasal challenge. Secretion of activin A, however, was greatly reduced in MC-deficient WBB6F(1)-W/W(v) mice as compared with wild-type mice, indicating that MC are an important contributor of activin A in the airways of a murine asthma model. Additionally, activin A promoted the proliferation of human airway smooth muscle cells. Taken together, these data suggest that MC-derived activin A may play an important role in the process of airway remodeling by promoting the proliferation of airway smooth muscle.