γ粒子核素支架影响血管平滑肌细胞增殖与凋亡的实验研究

目的： 观察γ粒子钯-103［103Pd］核素支架对损伤血管中膜平滑肌细胞增殖与凋亡的影响。探讨核素支架防治支架内再狭窄的作用机制。方法： 选用雄性新西兰兔50只，随机分为普通支架组及核素支架组，设正常对照。分别于术后3、7、14、28及56 d取材，进行病理形态学、免疫组化（增殖细胞核抗原PCNA）、细胞凋亡(TUNEL)及原位杂交(bcl-2 mRNA及bax mRNA)的观察。结果： ①光镜下发现，核素支架组管腔狭窄程度明显低于普通支架组，术后第56 d最显著（P<0.01）；②免疫组化显示，术后3-28 d核素支架组PCNA表达均低于普通支架组，7 d为表达高峰，16.35%±0.79% vs 24.36%±0.55%(P<0.01)。③ TUNEL法检测发现术后3-28 d，核素支架组的平滑肌细胞凋亡较普通支架组更明显，术后第7 d达峰值，14.72%±0.53% vs 12.42%±1.13%（P<0.01）。④ PCNA阳性率与TUNEL法测得的细胞凋亡阳性率比率PCNA/apoptosis（P〖KG*6〗∶〖KG-*2〗A）显示，核素支架组 P〖KG*6〗∶〖KG-*2〗A 的值在术后3-28 d均显著小于普通支架组(P<0.05)。⑤原位杂交测定凋亡相关基因bcl-2 mRNA及bax mRNA表达并计算其比值显示，普通支架组术后第3、14、28 d均大于对照组(P<0.01)，核素支架组与对照组无显著差异(P>0.05)。术后第3-28 d，核素支架组的比值均小于普通支架组(P<0.05)。结论： γ粒子核素支架通过抑制平滑肌细胞的增殖，促进凋亡，使增殖与凋亡的比率降低，从而减轻再狭窄的程度。     

Different sensitivity of human endothelial cells, smooth muscle cells and fibroblasts to topography in the nano–micro range

Cell adhesion, orientation and migration are influenced by surface topographies in the micrometer and nanometer range. In this work, we demonstrate the stimulation by topographical signals of human fibroblast cells (FCs), endothelial cells (ECs) and smooth muscle cells (SMCs). We systematically quantified the contact guidance alignment and directed migration of FCs, ECs and SMCs adhering to grooved substrates with lateral dimensions of 2–10 μm and depths of 50–200 nm. We found a common quantitative response characteristic of all three cell types: contact guidance significantly increased when the cells were cultured on substrates with smaller lateral dimensions or deeper grooves. Despite their general behavior, the three cell types exhibited a cell-type specific sensitivity to the groove patterns. The minimum groove depth to induce an orientation response and change cell shape was 50 nm for FCs and about two times deeper for ECs and SMCs. The degree of alignment and directed migration of the FCs along the grooves was significantly stronger than for the ECs and SMCs. We demonstrate that ECs and SMCs can be stimulated by topographical signals but are less sensitive than FCs.     

剪切力条件下血管内皮细胞与平滑肌细胞的相互作用

血管内皮细胞(endothelial cells,ECs)与平滑肌细胞(smooth muscle cells,SMCs)是血管壁的主要细胞,它们之间的相互作用在维持血管正常的生理功能以及心血管疾病的发生发展过程中至关重要。为真实模拟ECs与SMCs体内条件下的位置关系与生长状态,人们建立了多种共培养系统。介绍当前几种常用的能够加载流动剪切力的共培养系统,并分别比较其优势与不足;简要总结剪切力条件下ECs与SMCs相互作用对ECs与SMCs表型与分布、SMCs生长与迁移、ECs表面相关黏附分子表达的影响。研究表明,一氧化氮(NO)、细胞因子、microRNA等可以作为信号分子介导ECs与SMCs之间的相互作用。     

Isolation and characterization of human smooth muscle cells from umbilical cord vein and their reconstitution in a vascular co-culture model with underlying endothelial cells

Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against smooth muscle actin, negative for antibodies against factor VIII and displayed typical hill and valley morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×10⁴ cells/cm² for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 M), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.Abbreviations BCA bicinchoninic acid - BSA bovine serum albumin - DMEM DMEM medium supplemented with 4.5 g/l glucose, 100 IU/ml penicillin, 100 g/ml streptomycin and 1.25 g/ml fungizone - DMEM-1 DMEM supplemented with 20% FBS, 300 IU/ml penicillin, 300 g/ml streptomycin and 3.75 mg/ml fungizone - DMEM-2 DMEM supplemented with 20% FBS, 100 IU/ml of penicillin, 100 g/ml of streptomycin and 1.25 g/ml of fungizone - DMSO dimethyl sulfoxide - FBS fetal bovine serum - HPF human pulmonary fibroblasts - HS human serum - HUVE human umbilical vein endothelial - HUVSM human umbilical vein smooth muscle - M199 M199 medium supplemented with 20% HS, 100 IU/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml fungizone and 2 mM L-glutamine - P passage number - PBS phosphate buffered saline - TCA trichloroacetic acid - vwF von Willebrand factor (factor VIII)     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

 The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells. Received: 23 May 1996 / Received after revision: 3 September 1996 / Accepted 16 September 1996     

内皮细胞Jagged1下调促进PDGF诱导的大鼠平滑肌细胞增殖迁移

目的： 探讨内皮细胞Jagged1表达对PDGF诱导的大鼠血管平滑肌细胞增殖迁移的调节作用。方法: 分离培养大鼠主动脉内皮和平滑肌细胞,将内皮接种于下室、平滑肌接种于上室建立细胞共培养体系,根据内皮是否行Jagged1小RNA干扰分为对照组、空载体组和Jagged1小RNA干扰组。用Western blotting检测内皮细胞Jagged1的干扰效率。于下室加入PDGF（10 μg/L）干预24 h后分别用［3H］-TdR 掺入和平滑肌迁移计数检测平滑肌细胞增殖迁移能力,用Western blotting检测平滑肌细胞α-SM-actin蛋白表达。结果: 与对照组相比空载体组内皮细胞Jagged1蛋白表达无明显差异,Jagged1小RNA干扰组内皮细胞Jagged1蛋白吸光度相对值明显降低（0.26±0.02 vs 0.67±0.02, P<0.05）;PDGF+空载体组平滑肌［3H］-TdR 掺入量和迁移数与PDGF组相比无明显差异,PDGF+Jagged1小RNA干扰组平滑肌［3H］-TdR 掺入量和迁移数高于PDGF组{［3H］-TdR 掺入(23 074±2 702)counts·min-1·well-1 vs (16 442±1 803)counts·min-1·well-1,n=5,P<0.05;迁移(27±4)cells/field vs (15±3) cells/field, n=5,P<0.05};PDGF+空载体组平滑肌α-SM-actin蛋白表达与PDGF组相比无明显差异,PDGF+Jagged1小RNA干扰组平滑肌α-SM-actin吸光度相对值低于PDGF组（0.25±0.06 vs 0.49±0.04, n=3,P<0.05）。结论: 内皮细胞Jagged1下调促进PDGF诱导的平滑肌细胞增殖迁移,提示血管内皮细胞Jagged1表达在维持平滑肌收缩表型、抑制平滑肌过度增殖迁移中起一定调控作用。     

供体年龄影响融合生长内皮祖细胞对平滑肌细胞表型转换及增殖迁移的作用

目的:探讨老龄和年轻个体来源融合生长状态内皮祖细胞(EPCs)对血管平滑肌细胞(SMCs)表型转换以及增殖和迁移的调节作用。方法:脱臼处死1~2月龄、19~26月龄SD大鼠,应用含15%FBS的DMEM/F12培养基(含内皮细胞生长添加剂100 mg/L、肝素100 mg/L、青霉素、链霉素各1×105U/L)培养EPCs,取1~2月龄大鼠腹主动脉,组织块法培养血管SMCs,应用Di I-Ac-LDL与FITC-UEA-1荧光双染以及α-SM-actin免疫荧光分别对EPCs和SMCs进行鉴定。建立细胞共培养体系,上室为融合生长状态的EPCs,下室为SMCs,实验分4组:(1)第3代SMCs(P3)组;(2)第4代SMCs(P4)组;(3)第4代SMCs与年轻大鼠来源EPCs共培养(P4YE)组;(4)第4代SMCs与老龄大鼠来源EPCs共培养(P4AE)组。Western blotting检测α-SM-actin和osteopontin蛋白的表达;[3H]-TdR掺入法检测SMCs增殖;细胞划痕实验检测SMCs的迁移能力。结果:与P3组相比,P4组的SMCsα-SM-actin表达显著下调,而osteopontin表达显著增强;P4YE组SMCs的α-SM-actin及osteopontin表达与P3组比较未见有显著差别;与P4组相比,年轻和老龄大鼠来源的EPCs均显著促进第4代SMCs的α-SM-actin和下调osteopontin的表达,抑制第4代SMCs的增殖和迁移;与老龄大鼠来源的EPCs相比,年轻大鼠来源的EPCs更能够显著延迟SMCs表型由收缩型向合成型转换,抑制SMCs增殖和迁移。结论:共培养融合生长状态的EPCs使血管SMCs表型转换延迟、抑制SMCs增殖和迁移,年轻大鼠来源的EPCs较老龄大鼠来源的EPC更显著延迟血管SMCs表型由收缩型向合成型转换,并具有更强的抑制血管SMCs增殖和迁移的能力。     

Longitudinally orientated smooth muscle cells in rabbit arteries

Intima formation in vessels, spontaneous or experimentally induced, is generally characterized by the presence of longitudinally orientated smooth muscle cells (LSMC). During an experiment of neo-intima induction in carotid arteries in rabbits, by application of a nonconstrictive silastic cuff, a study was performed to investigate the presence of LSMC in the systemic and pulmonary circulations, in both elastic and muscular arteries. Three patterns could be distinguished: intimai cushions in muscular arteries, single or small groups of LSMC in the intima in elastic and larger muscular arteries, and intra-medially located layers or columns of LSMC in the aorta, the pulmonary artery, at the bifurcation of the aorta and around orifices of branches. In order to understand this peculiar orientation a biomechanical approach was used: this showed that near the lumen the circumferential stress is 4.5 times higher than the longitudinal. Because the cell surface of the smooth muscle cells exposed to this stress per unit vessel length is much less in the longitudinal than in the circular direction we conclude that the LSMC align in the direction which allows them to cope most effectively with the mechanical stresses.     

Effect of chronic nicotine delivery on the proliferation rate of endothelial and smooth muscle cells in experimentally induced vascular wall plaques

To study the effect of nicotine, cholesterol feeding, and their combination on endothelial and smooth muscle cells in vascular wall plaques an experimental method was established which allows the immunohistochemical detection and quantification of the fractions of endothelial and smooth muscle cells in DNA synthesis under the effect of these stimuli. For this purpose standardized fibromuscular plaques were produced by electrostimulation in the common carotid arteries of rabbits. The animals received either nicotine via implanted osmotic minipumps or a cholesterol diet or both. Plaque size was determined at the end of the experiments after 7 or 14 days as well as the fraction of endothelial and smooth muscle cells in DNA synthesis during exposure to bromodeoxyuridine (BrdU). The BrdU labeling index of endothelial cells clearly increased under chronic nicotine administration for either 7 days or 14 days compared to controls. The combination of nicotine and cholesterol diet led to a more significant increase. In contrast, the BrdU labeling index of smooth muscle cells was not increased under nicotine delivery. The combination of nicotine and cholesterol, however, led to a significant increase of the BrdU labeling index of smooth muscle cells in the plaques compared to cholesterol feeding. Measurement of the plaque size revealed no difference between controls and nicotine-treated animals after 14 days of nicotine delivery, whereas the combination of cholesterol and nicotine produced increased plaque formation compared to a group of animals which received a cholesterol diet alone.Abbreviations BrdU bromodeoxyuridine - EC endothelial cell - HDL high-density lipoprotein - LDL low-density lipoprotein - SMC smooth muscle cell     

Cytoskeletal mechanics in airway smooth muscle cells

Mechanical properties and contractility of airway smooth muscle tissue are largely responsible for airway narrowing and airway hyperresponsiveness in asthma. To explain these pathological phenomena, investigators have studied the mechanical behaviour of airway smooth muscle cells and its relationship to the underlying cellular biophysical and biochemical mechanisms. During the past decade, a growing body of evidence has indicated that a deformable intracellular polymer network, known as the cytoskeleton, plays a major role in transmitting and distributing mechanical forces within the cell and in their conversion into biochemical responses. We review here evidence suggesting that the tensed and crosslinked cytoskeletal lattice, the contractile apparatus, and the cytoskeleton–extracellular matrix interactions are key determinants of mechanical properties and mechanosensing of airway smooth muscle cells, with the mechanical distending stress of the cytoskeleton playing the central role.     

In vitro effects of tibolone and its metabolites on human vascular coronary cells

OBJECTIVES: Tibolone is a tissue selective compound with estrogenic, androgenic and progestogenic properties in classical bioassays. It is used for alleviation of menopausal symptoms and for osteoporosis prophylaxis in postmenopausal women. Only few data are available regarding the effects of tibolone on the cardiovascular system. We investigated therefore the in vitro effects of tibolone and its metabolites on the vasculature under special controlled conditions, using human female coronary endothelial and smooth muscle cells. METHODS: The effect on the production of the following markers in endothelial cells from human female coronary arteries was evaluated: nitric oxide synthase, prostacyclin, endothelin, plasminogen-activator-inhibitor-1 (PAI-1), E-Selectin, Intercellular adhesion molecule (ICAM-1), monocyte attracting protein-1 (MCP-1) and the precursor of matrix metalloproteinase-1 (pro-MMP-1). Tibolone, its metabolites, estradiol (E2), E2/norethisterone (NET) and E2/medroxyprogesterone acetate (MPA) were tested at 0.1 microM and 1 microM. The markers were determined by enzyme immunoassays in the cell supernatant. Cell proliferation of smooth muscle cells from female coronary artery was measured by an adenosine triphosphate-assay. RESULTS: Tibolone, its 3-hydroxy metabolites, E2/NET, E2/MPA and estradiol alone had significant effects on the synthesis of all markers tested. The magnitude of the tibolone effects, however, was mostly smaller than that of E2/NET and E2/MPA. Concerning smooth muscle cells tibolone and its 3-hydroxy metabolites also elicited an inhibition of the proliferation compared to control values. The strongest effect here was found for E2/NET and E2 alone, whereas E2/MPA had no effect. CONCLUSION: The results of this in vitro study conducted with cells of the most important vascular bed with respect to the problem of cardiovascular risk suggest that tibolone can positively influence the vasculature. However, these tibolone effects may depend on intact vascular cells and may vary due to the different atherosclerotic stages of the vessels. Thus, experimental studies are useful to explore mechanisms, but clearly cannot replace clinical studies.     

糖皮质激素对前列腺素E2介导的主动脉平滑肌细胞cAMP生成的影响

目的观察地塞米松对前列腺素Ｅ２介导的离体培养兔主动脉平滑肌细胞的ｃＡＭＰ生成的影响。方法用蛋白竞争结合法测定细胞ｃＡＭＰ含量,并确定腺苷酸环化酶的活性。结果从１０ｐｍｏｌ／Ｌ到１０ｎｍｏｌ／Ｌ地塞米松对培养细胞的ｃＡＭＰ生成有明显抑制作用并呈现剂量依赖性,作用时间愈长,该抑制作用愈强。经地塞米松预处理８小时的培养细胞,其前列腺素Ｅ２介导的腺苷酸环化酶活性显著下降。结论糖皮质激素在腺苷酸环化酶水平抑制前列腺素Ｅ２介导的培养平滑肌细胞ｃＡＭＰ生成。另外还观察到地塞米松拮抗前列腺素Ｅ２的抑制培养细胞增生效应,但未发现地塞米松对培养平滑肌细胞增生的直接促进作用     

The importance of smooth muscle cells in the development of foam cells in the gastric mucosa

Summary Foam cells in lipid islands of the stomach can develop from both histiocytes and smooth muscle cells. With increasing storage of lipid vacuoles in smooth muscle cells, loosening of the myofilament arrangement and decrease of the dense areas subjacent to the plasma membrane occurs. Endoplasmic reticulum and the cisternae of the Golgi-apparatus dilate, the cell organelles increase initially and the basement membrane of the smooth muscle cells is fragmentarily formed. Only in incompletely formed foam cells can the origin from smooth muscle cells be recognised, in their final state their histiogenesis is seldom apparent.The authors are grateful to H. Gerdes for leaving the specimens and to R. Naumann and H. Staubitz for technical and photographic assistance.     

Multiple types of voltage-dependent Ca channels in mammalian intestinal smooth muscle cells

(1) Whole-cell and single channel recording techniques have been applied to smooth muscle cells isolated from guinea-pig taenia coli to examine whether multiple types of Ca channels exist. (2) Whole-cell recordings under physiological Ca concentration (1.8 mM) revealed two current components with fast and slow inactivating kinetics. The fast inactivating component was present when cells were held at very negative potentials (–80 mV). It was insensitive to the dihydropyridine (DHP) derivative, nifedipine. In contrast, the slow inactivating component was present at less negative holding potentials. It was blocked by nifedipine. (3) The two current components were found to have closely similar voltage dependencies for activation. (4) These results suggest that the fast inactivating decay of the Ca current was mediated not only by the entry of Ca into the cell but also by a voltage-dependent process via a different type of Ca channel with fast inactivating kinetics. (5) Recordings from cell-attached membrane patches with 100 mM external Ba clearly showed the existence of multiple types of Ca channels with different conductances. (6) The large conductance channels (30 pS) activated at more positive potentials (0 mV) and their averaged current decayed much more slowly. The DHP Ca antagonist, nifedipine, inhibited the large conductance channels increasing the proportion of blank sweeps and reducing the averaged current. On the other had, the DHP Ca-agonist, BayK 8644, increased the averaged current by increasing the mean open-times of the large conductance channels. The presence of micromolar Cd in the patch pipettes produced a flickering block of the large conductance channels. (7) The small conductance channels (7 pS) activated at more negative potentials (–40 mV 30 mV) and the averaged current decayed rapidly within 100 ms. They showed no sensitivity to nifedipine and Cd ions. (8) In summary, we have identified at least two distinct types of Ca channels with different conductances, different pharmacological sensitivities, and different voltage- and time-dependent kinetics in smooth muscle cells isolated from guinea-pig taenia coli. The large and small conductance channels could be classified asl- andt-type Ca channels, respectively, which have been described in neuronal and heart cells. The contribution of those channels to the macroscopic component of whole-cell Ca current is also discussed.     

大鼠远端肺动脉平滑肌细胞分离与原代培养

目的探索简便、高效原代培养大鼠远端肺动脉平滑肌细胞的方法,为研究肺动脉高压发病机制提供实验材料。方法采用显微操作和分次酶消化法进行原代培养并与传统酶消化法比较。对培养的细胞进行形态学观察、平滑肌α-actin免疫荧光细胞化学法鉴定、激光扫描共聚焦显微镜计算纯度。结果两种酶消化法均可获得高纯度的远端肺动脉平滑肌细胞。镜下细胞呈典型的"峰-谷"状生长,胞质特异的α-actin阳性表达,细胞纯度达98%。分次酶消化法获得的细胞数及细胞存活率均大于传统酶消化法,并且提前了3d得到足够进行实验的细胞数量。结论本法简便、可靠、低成本,短期内可获得大量高纯度、功能良好的远端肺动脉平滑肌细胞。     

microRNA-133b在低切应力诱导血管内皮细胞影响血管平滑肌细胞增殖中的作用

目的研究血管内皮细胞(endothelial cells,ECs)直接感受低切应力刺激后分泌类胰岛素生长因子-1(insulinlike growth factor-1,IGF-1)影响血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖这一过程中microRNAs(miRs)的作用。方法用平行平板流动腔系统对ECs施加1.5 Pa正常切应力(normal shear stress,NSS)和0.5 Pa低切应力(low shear stress,Low SS),Real-time PCR检测VSMCs的miRs变化。用miRs预测网站预测miR-133b靶基因并验证。Western blotting检测核糖核酸结合蛋白1(polypyrimidine tract binding protein 1,Ptbp1)和N-myc下游调节基因1(N-myc downstream regulated 1,Ndrg1)的蛋白水平变化。Ed U流式检测miR-133b对VSMCs增殖的影响。结果 IGF-1静态刺激后,VSMCs的miR-133b和miR-378a表达上升。Low SS条件下,VSMCs的miR-133b表达显著上升,miR-378a表达无明显变化。下调VSMCs的miR-133b表达,Ptbp1、Ndrg1的mRNA水平均显著升高,上调VSMCs的miR-133b的表达,Ptbp1、Ndrg1的mRNA和蛋白水平显著降低,并且显著促进VSMCs增殖。结论在Low SS条件下ECs分泌IGF-1可能通过调控联合培养VSMCs的miR-133b和靶基因Ptbp1和Ndrg1促进VSMCs增殖。研究结果为心血管疾病治疗提供了一个新的潜在靶标。     

Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H₂O₂ (200 μM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA₂) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H₂O₂ resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H₂O₂ treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 μM) and quercetin (50 μM). In quiesced VSMC, incubation with H₂O₂-conditioned medium resulted in increased cPLA₂ activity compared to the controls (P<.05). This activity was partially inhibited by the ET_A-receptor antagonist, PD 142893 (10 μM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H₂O₂-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.     

Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens

The electrophysiological effect of adenosine triphosphate (ATP) on the enzymatically dispersed smooth muscle cells from rat vas deferens was investigated. ATP always induced depolarization accompanied with a reduction in membrane resistance. In a whole cell voltage clamp experiment, an inward current was recorded when the cell was exposed to ATP-containing solution. The ATP-induced current disappeared within 2min even in the continuous presence of ATP, which may indicate that the cells were desensitized to this compound. The ATP-induced current was also recorded in the cells superfused with 10^–5M nicardipine or in the Cs-loaded cells, eliminating the possible involvement of voltage-gated Ca and K current. During cell-attached patch clamp, an elementary current having a mean conductance of 20pS was observed when the intrapipette solution was changed to ATP-containing solution. The estimated zero current potentials of the ATP-activated macroscopic current and elementary current were about 0mV. These results suggest that ATP exerts its transmitter-like action by activating ion channels in smooth muscles.     

三种实用而简单的急性酶分离动脉平滑肌细胞的方法

目的:探讨有效而简便的获取单个动脉平滑肌细胞的方法。方法:应用三种急性酶分离细胞的方法分离人体肠系膜动脉平滑肌细胞,采用单通道膜片钳和穿孔膜片钳技术,记录该细胞上的大电导钙激活钾通道(Large-conductance Ca2+-activated potassium channels,BKCa)电流。结果:三种急性酶分离细胞方法均可有效的获取较高质量的单个人体肠系膜动脉平滑肌细胞,并成功应用于电生理膜片钳实验中,且记录的电流稳定,记录时间长。结论:这三种分离单个血管平滑肌细胞的急性酶分离方法简单而实用,为今后的进一步研究提供有效而简便的方法。