Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.     

组织基质金属蛋白酶抑制剂-2的表达纯化及活性鉴定

组织基质金属蛋白酶抑制剂-2(TIMP-2)抑制肿瘤迁移及侵袭。文中以人TIMP-2为研究对象,探索人TIMP-2蛋白的原核表达特征,并进行纯化及活性鉴定。以人肺癌A549细胞的总RNA反转录得到的cDNA为模板,克隆人TIMP-2基因,构建pET28a重组表达载体;经酶切检测和测序分析的重组表达载体pET28a-TIMP-2转入大肠杆菌Escherichia coli BL21(DE3)中,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并对表达条件进行优化。经镍亲和柱纯化后,用Westernblotting法鉴定融合蛋白His-TIMP-2,并用明胶酶谱法检测融合蛋白的活性。研究发现融合蛋白His-TIMP-2在E.coliBL21(DE3)中以包涵体的形式存在;在一定范围内,IPTG浓度对His-TIMP-2的表达量没有显著影响;而在该表达系统中,诱导温度和时间是关键参数,His-TIMP-2的表达量随诱导温度升高而增加;纯化并复性后的融合蛋白His-TIMP-2能有效抑制人肺癌A549细胞表达的基质金属蛋白酶的活性。具有活性的融合蛋白的获得为后续深入研究人TIMP-2的功...     

Identification,purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 (an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under non-reducing condition, non-denaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca+2, and is inhibited by EDTA, EGTA and 1:1 0-phenanthroline; (ii) it was inhibited by a, macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.     

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H₂O₂ (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na⁺ dependent Ca²⁺ uptake. Electron micrograph revealed that H₂O₂ (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 g/ml) reversed the effect produced by H₂O₂ (1 mM) on Na⁺ dependent Ca²⁺ uptake in the microsomes. However, H₂O₂ (1 mM) caused changes in MMP-2 activity and Na⁺ dependent Ca²⁺ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 g/ml of TIMP-2 which otherwise reversed MMP-2 (1 g/ml) mediated increase in ¹⁴C-gelatin degradation and inhibition of Na⁺ dependent Ca²⁺ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 g/ml) and H₂O₂ (0.5 mM) inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 g/ml) with H₂O₂ (1 mM) abolished the inhibitory effect of the inhibitor on ¹⁴C-gelatinolytic activity elicited by 1 g/ml of MMP-2. Thus, one of the mechanisms by which H₂O₂ activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na⁺ dependent Ca²⁺ uptake in the microsomes. (Mol Cell Biochem 270: 79–87, 2005)     

Fractalkine对大鼠肺动脉平滑肌细胞增殖的影响

目的：观察fractalkine（FKN）对体外培养的大鼠肺动脉平滑肌细胞（PASMCs）增殖的影响。方法：体外培养大鼠PASMCs,加入不同浓度（10-^10、10-^9和10-^8 mol／L）的FKN处理12h、24h和48h,采用四唑盐（MTT）法检测细胞增殖,流式细胞术（FCM）检测细胞周期。结果：MTT试验显示FKN显著促进大鼠PASMCs增殖,此作用呈浓度依赖性。FCM分析显示FKN使S期细胞比例和增殖指数P1值增加。FKN处理PASMCs 12h后,其S期细胞比例和H值即出现增加,24h达高峰。结论：FKN呈浓度依赖方式促进大鼠PASMCs增殖。     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Pathogenic A beta induces the expression and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid beta protein (A beta). Previously we have shown that pathogenic forms of A beta induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of A beta precursor, and apoptotic cell death. Here we show that pathogenic A beta stimulates the expression and activation of matrix metalloproteinase-2 (MMP-2). Furthermore, we demonstrate that the increase in MMP-2 activation is largely caused by increased expression of membrane type-1 (MT1)-MMP expression, the primary MMP-2 activator. Finally, treatment with MMP-2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic A beta. Our findings suggest that increased expression and activation of MMP-2 may contribute to HCSM cell death in response to pathogenic A beta. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.     

大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理检测

目的：研究大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理特性。方法：膜片钳全细胞和膜内向外记录模式检测大鼠肺动脉平滑肌细胞上钙激活氯通道全细胞电流和单通道电流。结果：大鼠肺动脉平滑肌细胞记录到稳定的钙激活氯通道电流（ICl（Ca））;ICl（Ca）表现出典型的外向整流特性和电压时间依赖性激活。结论：大鼠肺动脉平滑肌细胞膜上存在电压、时间依赖性氯通道电流,钙激活氯通道通过促进肺动脉平滑肌细胞去极化而成为调节肺动脉特性的关键调节因子。     

Heparin antiproliferative activity on bovine pulmonary artery smooth muscle cells requires both N-acetylation and N-sulfonation

The antiproliferative activity of Heparin (HP) on bovine pulmonary artery smooth muscle cells (BPASMC) in vitro requires both N-acetylation and N-sulfonation. This was demonstrated by quantifying the relative N-acetylation of three commercial heparins of known antiproliferative activities, using their Fourier-transform infrared (FTIR) band areas at 1381-1378 and 1320-1317 cm(-1), which combined resulted in 1.0, 1.0 and 1.3 cm2 for Choay, Elkins-Sinn and Upjohn HP, respectively. These results show that Upjohn HP, which is at least 44% more antiproliferative than the other two, is 30% more N-acetylated. Upjohn HP was also N-desulfonated chemically, and its antiproliferative activity was determined. Its total sulfonate (--SO 3 -) content (O- and N-sulfonate) was quantified using the FTIR band area at 1260-1200 cm(-1) for the S=O stretching; a drop in sulfonate content from 21.87% (w/w) before N-desulfonation to 16.51% (w/w) after N-desulfonation, resulted in a 67% decrease in its inhibitory potency. In addition to the requirement that approximately 24% of the sulfonate content be bonded to N, the data show a direct correlation between the extent of Upjohn HP N-acetylation and its antiproliferative activity on BPASMC.     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.     

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。     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。     

Role of hydroxyl radical in the oxidant H2O2-mediated Ca2+ release from pulmonary smooth muscle mitochondria

We sought to investigate the mechanism(s) by which the oxidant H₂O₂ stimulates Ca²⁺ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H₂O₂ dramatically stimulated hydroxyl radical generation as measured by methane (CH₄) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH₄ production caused by H₂O₂. In the absence of EGTA, H₂O₂ caused stimulation of Ca²⁺ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca²⁺ loaded mitochondria resulted an immediate loss of Ca²⁺ and that has been found to be augmented by H₂O₂. The release of Ca₂₊ by H₂O₂ did not appear to occur with concommitant increase in sucrose entry into, K⁺ release from, and swelling of mitochondria when the Ca²⁺ cycling was prevented by EGTA. These observations suggested that H₂O₂-mediated Ca²⁺ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific pore formation.Abbreviations H₂O₂ hydrogen peroxide - OH· hydroxyl radical - t-buOOH tert-butyl hydroperoxide - CH₄ methane - GLC gas liquid chromatography - DMTU dimethylthiourea - EGTA ethylene glycol bis(-aminoethyl ether) - N Ntetraacetic acid - DMSO dimethyl sulfoxide - CH₄ methane - HBPS Hank's buffered physiological saline - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MOPS 3-(N-morpholino)propane sulfonic acid - Tris tris (hydroxymethyl aminomethane)     

Matrix metalloproteinase-9,-3 and tissue inhibitor of matrix metalloproteinase-1 in colorectal cancer: relationship to clinicopathological variables

The balance between matrix metalloproteinases (MMPs) and their physiological tissue inhibitors of matrix metalloproteinases (TIMPs) is crucial in tumour invasion and progression. The aim of this study was to investigate the levels of MMP-9, MMP-3 and TIMP-1 in colorectal cancer (CRC) and to evaluate these proteinases and their inhibitor with respect to clinicopathological variables. Activities of pro- and active MMP-9 were measured in paired tumour and distant normal tissue specimens from 43 patients with CRC using gelatin zymography. ELISA was employed for the determination of MMP-9, MMP-3 and TIMP-1 protein expressions. The activity levels of pro- and active MMP-9 and protein expression levels of MMP-9, MMP-3 and TIMP-1 were higher in tumour tissues than in the corresponding normal tissues; the differences being significant for all (p < 0.05), except TIMP-1. Similarly, active MMP-9/proMMP-9 and the ratio of protein expression level of MMP-9-TIMP-1 were found to be significantly higher in tumour tissues ( p < 0.01). Among all the clinicopathological variables investigated, significant correlations were found between MMP-9 and presence of perineural invasion, MMP-3 and lymph node status, TIMP-1 and tumour differentiation, MMP-9/TIMP-1 ratio and histological types ( p < 0.05). In conclusion, MMP-3 was not as notably increased as MMP-9 in tumour tissues. However, different roles may be attributed to MMP-9 and MMP-3 in CRC development and progression. Additionally, assessment of TIMP-1 in relation to MMPs appeared to be crucial in CRC studies to provide a basis for the re-evaluation of the clinical usefulness of TIMP-1 in colorectal cancer.     

Role of membrane-associated CA2+ dependent matrix metalloprotease-2 in the oxidant activation of Ca2+ATPase by tertiary butylhydroperoxide

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca²⁺⁺ATPase activity. The smooth muscle plasma membrane possesses a Ca²⁺⁺ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca²⁺⁺ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca²⁺⁺ dependent protease that degrades ¹⁴C-gelatin. The oxidant triggered protease activity and the Ca²⁺⁺ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca²⁺⁺ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca²⁺⁺ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca²⁺⁺ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.     

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.     

体外转染NHE-1核酶对大鼠肺动脉平滑肌细胞增殖的影响

目的：探讨锤头状核酶对大嫌肺动脉平滑肌细胞Na^ /H^ 变换器（NHE－1）表达和活性、pHi的影响,及其与细胞增殖的关系。方法：构建含锤头状核酶序列的pLXSN反转录病毒重组载体,将其转染体外培养的大鼠肺动脉平滑肌细胞,G418筛选后获取含有重组载体的细胞克隆,检测细胞NHE－1 mRNA表达、^22Na摄取量、pHi和^3HTdR掺入量。结果：与结果pLXSN空载体的细胞和正常对照组细胞比较,转染重组载体的肺动脉平滑肌细胞中NHE－1 mRNA表达、^22Na摄取量减少,同时伴有pHi降低和^3H-TdR掺入量减少,正常对照组和空载体组间无显著差异。结论：NHE－1特异性锤头状核酶可对NHE－1 mRNA进行特异性切割,减少其表达,从而诱导细胞酸化,抑制肺动脉平滑肌细胞增殖。