川芎嗪甲酸对平滑肌细胞游离钙浓度的影响

研究川芎嗪代谢产物川芎嗪甲酸（CTPZ）对大鼠阴茎海绵体平滑肌细胞（PCSMC）胞质内游离钙离子浓度的影响。方法：用新型Ca^2＋荧光染色剂Fluo-3／AM负载大鼠PCSMC,细胞分为氯化钾（KCl）和去甲肾上腺素（NE）作用组,应用激光扫描共聚焦显微镜（LSCM）实时测定胞质内[Ca^2＋]的变化,分别观察不同浓度的CTPZ对高钾和NE诱导胞质内钙浓度升高的影响,并与母药川芎嗪作用相比。结果：静息状态下,CTPZ对大鼠PCSMC胞质内[Ca^2＋]无明显影响。1,10,100μmol·L^-1 CTPZ能显著抑制高钾诱发的细胞内的钙浓度升高的影响,抑制率分别为（39．84-4．3）％,（49．2±3．6）％,（58．2±3．9）％。也能抑制1μmol·L^-1 NE诱发钙库释放的细胞内[Ca^2＋]升高,抑制率分别为（20．8±3．9）％,（32．3±2．5）％,（43．7±3．2）％。结论：CTPZ对大鼠PCSMC电压依赖性钙通道和细胞内钙库释放的抑制作用,能降低PCSMC胞质内[Ca^2＋]水平,其作用效果比母药川芎嗪佳。     

Alteration of Ryanodine-receptors in Cultured Rat Aortic Smooth Muscle Cells

Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic Ca(2+) response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure Ca(2+) release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated Ca(2+) entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the Ca(2+)- induced Ca(2+)-release pathway by directly measuring Ca(2+) release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with Ca(2+) stimulated Ca(2+) release from the SR. Caffeine and ryanodine also induced Ca(2+) release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and Ca(2+) failed to trigger Ca(2+) release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate Ca(2+) release from the SR by cytosolic Ca(2+) elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.     

小檗胺对血管平滑肌细胞钙动力学影响

本文以Fluo-3/AM 荧光技术和激光扫描共聚焦显微镜检查法, 研究小檗胺 (Ber) 对培养家兔胸主动脉血管平滑肌细胞(VSMC)内游离钙 ([Ca 2 ]i ) 的影响结果表明：在胞外钙 ([Ca 2 ]o ) 为?.0 mmol·L -1 条件下，Ber 抑制60 mmol·L -1 KCL1 mmol·L -1 哇巴因Ouabain 30 mmol·L -1 去甲肾上腺素 ( NE ) 1 mmol·L -1 5-羟色胺5-HT 和30 mmol·L -1 三磷酸腺苷 (ATP)引起的 [Ca 2 ]i 升高，而且荧光强度达峰时间亦延长。在无胞外钙条件下，Ber 对咖啡因 (Caffeine)引起的[Ca 2 ]i 升高没有作用。结论：Ber 抑制电压依赖性钙通道 (VDCC) 和受体调控性钙通道 (ROCC) 激活引起的外钙内流，对Caffeine 引起的内钙释放没有影响。Ber 可抑制Ouabain 诱导的细胞内钙升高。     

川芎嗪衍生物对人阴茎海绵体平滑肌细胞游离钙浓度的影响

目的研究川芎嗪（TMP）衍生物（A3 A6）对人阴茎海绵体平滑肌细胞（PCSMC）胞质内游离钙离子（Ca2+）浓度的影响。方法采用新型Ca2＋荧光染色剂Fluo 3/AM负载人PCSMC,应用激光扫描共聚焦显微镜（LSCM）实时测定胞质内［Ca2＋］i变化。以母体川芎嗪和经典钙拮抗药维拉帕米 （Ver）为阳性对照,分别观察A3 A6对去甲肾上腺素（NE）诱导胞质内钙浓度升高的影响。结果在浓度0.2 mmol•L 1时,A3 A6对NE诱发的人PCSMC内［Ca2＋］i升高有明显抑制作用,抑制率分别为49.03%,54.83%,51.48%和50.31%,优于TMP（18.96%）,远大于Ver（16.51%）（P＜0.05）。结论A3 A6对人PCSMC电压依赖性钙通道均有抑制作用,能降低PCSMC胞质内Ca2+水平,其作用效果均比TMP强。     

不同剂量前列腺素E_2对离体培养主动脉平滑肌细胞的不同影响

以不同剂量前列腺素 E_2 作用于离体培养兔主动脉平滑肌细胞,观察其对细胞形态、生长性能和代谢的影响。结果显示不同剂量前列腺素 E_2 具有双相作用,即小剂量可促进 DNA 合成和细胞增生,轻度促进脂质过氧化反应,超微损伤较明显,而大剂量明显抑制 DNA 合成,轻度降低过氧化脂质的产生,超微变化轻微。上述所见与以往体内实验结果相一致。     

Interactions Between Cultured Bovine Arterial Endothelial and Smooth Muscle Cells: Studies on Uptake and Degradation of Low Density Lipoproteins by Smooth Muscle Cells

Abstract: This study was designed to investigate the effects of substances released from non-injured and injured bovine arterial endothelial cells on ¹²⁵I-low density lipoprotein uptake and degradation by smooth muscle cells in culture. It was demonstrated that endothelial cell-released non-dialysable (molecular weight cut off 12-14000) substances significantly stimulated ¹²⁵I-low density lipoprotein uptake and degradation by smooth muscle cells. Endothelial cell-released dialysable substances and endothelin-1 did not cause this stimulation. The increase in ¹²⁵I-low density lipoprotein uptake and degradation by smooth muscle cells could be dissociated from cell proliferation. However, in endothelial cell-smooth muscle cell co-culture ¹²⁵I-low density lipoprotein uptake and degradation by smooth muscle cells were not stimulated. Injury to endothelial cells by lipid-soluble smoke particles or ultraviolet light, which reduced total cellular protein by 15-25%, enhanced the endothelial cell release of the substances stimulating ¹²⁵I-low density lipoprotein uptake. The results are discussed in relation to atherogenesis.     

糖基化终产物对大鼠主动脉平滑肌细胞MMP-2表达的影响

目的：探讨糖基化终产物对大鼠主动脉平滑肌细胞基质金属蛋白酶-2（MMP-2）表达的影响。方法：采用组织块贴壁法培养大鼠主动脉平滑肌细胞。将50mmol／L葡萄糖孵育的糖基化终产物修饰牛血清白蛋白稀释成不同浓度分别干预24h和用同一浓度的糖基化终产物修饰牛血清白蛋白（浓度为200mg／L）分别干预0、4、8、16、24、48、72h，逆转录聚合酶链反应检测细胞中的MMP-2 mRNA表达水平，明胶酶图分析方法观察培养上清液中MMP-2活性。结果：随糖基化终产物修饰牛血清白蛋白干预浓度升高，MMP-2 mRNA表达增加（P〈0．01），以糖基化终产物修饰牛血清白蛋白（200mg／L）对平滑肌细胞干预4h时MMP-2 mRNA表达已升高，24h时最高，以后仍维持较高水平，与未干预组相比差异有统计学意义（P〈0．01），MMP-2降解明胶活性随着糖基化终产物修饰牛血清白蛋白干预浓度升高而增强（P〈0．05）。结论：糖基化终产物可促进大鼠主动脉平滑肌细胞MMP-2的表达。     

白介素-18对人脐动脉平滑肌细胞组织因子表达的影响

目的观察白介素-18(IL-18)对诱导体外培养的人脐动脉平滑肌细胞组织因子(TF)表达的作用.方法在培养的人脐动脉平滑肌细胞中加入IL-18(浓度为100 μg/L),用酶联免疫吸附分析法(ELISA)及细胞发色底物法分别测定培养基中TF抗原及细胞冻融液的TF活性.结果培养基中TF抗原含量在4、6、8 h组均高于24 h组和对照组(P＜0.05),且4h组最高(P＜0.05),4 h与6 h 2组间差别无统计学意义(P＞0.05),8 h组均低于4 h组和6 h组(P＜0.05),24 h组与对照组差别无统计学意义(P＞0.05).细胞冻融液中总TF活性在6、8、24 h组均高于对照组(P＜0.05),6h组和8 h组均高于24 h组(P＜0.05),6h与8h组间差别无统计学意义(P＞0.05).结论IL-18可以很快诱导体外培养的人脐动脉平滑肌细胞表达TF,使动脉血管平滑肌细胞呈现一过性促凝状态.     

双氢杨梅树皮素对兔胸主动脉条平滑肌收缩反应的影响

用兔胸主动脉条研究双氢杨梅树皮素对去甲肾上腺素（NE）、KCl和CaCl2所致兔胸主动脉条收缩反应量效曲线的影响。双氢杨梅树皮素能显著地抑制NP、KCl和CaCl2所致兔胸主动脉杂的收缩,量效曲线右移,最大反应降低,对高K 所致兔胸主动脉条收缩的抑制作用明显大于NE所致兔胸主动脉条收缩的抑制作用。双氢杨梅树皮素对NE引起的依赖内Ca2 释放的收缩有明显抑制作用,而对NE依赖细胞外Ca2 性收缩区在较高浓度时才显示抑制作用,提示双氢杨梅树皮素可能对电压依赖性钙通道（PDC）有选择性阻滞作用。     

Interactions between Cultured Bovine Arterial Endothelial and Smooth Muscle Cells: Effects of Injury on the Release of Growth Stimulating and Growth Inhibiting Substances

Abstract: Dimethylsulfoxide-soluble particles (DSP) from cigarette smoke and ultraviolet light caused a low degree (cell death < 30%) and high degree (cell death 60-90%) injury to bovine arterial endothelial cells and smooth muscle cells in culture. Conditioned medium from low degree injured endothelial cells and smooth muscle cells generally inhibited DNA synthesis in new smooth muscle cells or endothelial cells while high degree injury increased DNA synthesis in new cells. Specifically, the growth stimulating activity from endothelial cells was decreased after low degree injury but increased after high degree. UV light released more growth stimulating substances from smooth muscle cells after both low and high degree injury. The release of growth inhibiting substances was dependent on both cell kind and degree of injury. In co-culture low and high degree DSP injury to endothelial cells inhibited smooth muscle cell proliferation, which was in contrast to the effect of conditioned medium from high degree injured endothelial cells. Conditioned medium from endothelial cells treated with LDL and glucose inhibited DNA synthesis in smooth muscle cells. It is concluded that injury to endothelial cells or smooth muscle cells will modify the release of growth inhibiting and growth stimulating activity and that this release will depend on cell kind as well as degree and kind of the injurious stimulus.     

Effects of Calcium Channel Blockers on Urinary Tract Smooth Muscle

Abstract: Influx of calcium from the extracellular medium seems to be important for spontaneous as well as agonist-induced contractile activity in urinary tract smooth muscle. To a various extent this calcium influx occurs through pathways which can be blocked by calcium channel blockers. These drugs effectively suppress spontaneous ureteral activity in vitro. Whether they affect ureteral motility in vivo or whether they can counteract ureteral spasm associated with ureteral stones have not been established. Calcium channel blockers partially block electrically as well as agonist-induced detrusor contractions. Some of these drugs abolish even atropine-resistant contractile responses induced by electrical stimulation in detrusor muscle. Drugs with combined antimuscarinic and calcium channel blocking effect therefore have an attractive effect profile. Experiences with calcium channel blockers in the treatment of patients with ‘unstable bladder’ are limited, but results obtained with terodiline seem promising. Even if calcium channel blockers reduce agonist-induced contraction in isolated urethral muscle, their clinical effect on urethral function seems to be small. The effects of calcium blockers on urinary tract smooth muscle may be clinically useful and deserve further study.     

阿魏酸钠抑制LDL氧化修饰及其致兔主动脉平滑肌细胞的增殖

本文观察阿魏酸钠(SF)对细胞诱导的人低密度脂蛋白(LDL)的氧化修饰及兔主动脉平滑肌细胞(SMC)增殖的作用。在LDL存在的培养的SMC系统中,SF可明显抑制Cu2 诱导的MDA含量增高,剂量在40~120 mgmL-1内呈剂量依赖性。同时,SF 40~120 mgmL-1 可降低3H-TdR 细胞掺入量及MTT法显示细胞数降低,提示SF作为抗氧化剂可抑制LDL 的氧化修饰及SMC 的增殖。     

心脉龙注射液对大鼠缺氧-复氧心肌细胞内游离钙离子及脂质过氧化物的影响

目的:观察心脉龙注射液(XML)对大鼠缺氧及缺氧-复氧心肌细胞[Ca2+]及丙二醛(MDA)、超氧化物歧化酶(SOD)的影响,以进一步阐明其作用机制。 方法:采用Fura-2/AM及相应试剂盒测定该药对大鼠缺氧及缺氧-复氧心肌细胞|Ca2+|i,MDA和SOD的影响。结果:7.5×102μg/mL XML能升高缺氧及缺氧-复氧心肌细胞|Ca2+|i(P<0.01,P<0.05),此作用与去乙酰毛花苷相当3.75 × 102,1.5×103μg/mL XML则略降低心肌细胞|Ca2+|i(P>0.05),同时应用维拉帕米均不能抑制去乙酰毛花苷及XML各剂量组对细胞内|Ca2+|i的影响。 3.1 ×102,6.3×102,1.25×103μg/mL XML能升高缺氧及缺氧-复氧心肌细胞悬液的SOD含量(P<0.01,P<0.05),虽能降低MDA,但无统计学意义 结论:对缺氧及缺氧-复氧状态的衰竭心脏,在强心时要严格掌握XML的剂量,其强心作用与心肌细胞|Ca2+|i的升高有关,该药同时还具有一定的抗氧化作用。     

首乌复方冲剂对ox-LDL引起的血管平滑肌细胞增殖的影响

采用氧化型低密度脂蛋白(ox-LDL)造成小牛主动脉平滑肌细胞(VSMC)增殖,观察首乌复方冲剂对ox-LDL诱导VSMC增殖的影响。将培养的VSMC分为对照组、ox-LDL组和用药组(ox-LDL 不同剂量首乌复方冲剂),应用硫代巴比妥酸比色法测定VSMC培养液中丙二醛(MDA)的水平。用MTT法测定VSMC数量和应用流式细胞仪测定VSMC周期。结果显示首乌复方冲剂能明显降低细胞培养液中MDA的水平,MTT检测显示首乌复方冲剂抑制ox-LDL诱导的VSMC增殖,流式细胞仪检测表明,用药组(S G2M)期降低。首乌复方冲剂能抑制ox-LDL刺激的VSMC的增殖,并具有抗脂质过氧化作用。     

Enhancement in Anti-proliferative Effects of Paclitaxel in Aortic Smooth Muscle Cells upon Co-administration with Ceramide using Biodegradable Polymeric Nanoparticles

PURPOSE: Using a combination of paclitaxel (PTX), and the apoptotic signaling molecule, C6-ceramide (CER), the enhancement in anti-proliferative effect of human aortic smooth muscle cells (SMC) was examined by administering in polymeric nanoparticles. METHODS: PTX- and CER-loaded poly(ethylene oxide)-modified poly(epsilon caprolactone) (PEO-PCL) nanoparticles were formulated by solvent displacement and characterized. The uptake and intracellular localization of the nanoparticle in SMC was examined using Z-stack fluorescent confocal microscopy. Anti-proliferative and pro-apoptotic effects of SMC were determined upon administration of PTX and CER, either as single agent or in combination, in aqueous solution and in PEO-PCL nanoparticle formulations. RESULTS: High encapsulation efficiencies (i.e., >95%) of PTX and CER at 10% (w/w) loading were attained in the PEO-PCL nanoparticles of around 270 nm in diameter. Fluorescence confocal analysis showed that nanoparticle delivery did facilitate cellular uptake and internalization. Additionally, combination of PTX and CER delivery in PEO-PCL nanoparticles was significantly more effective in decreasing the proliferation of SMC, probably by enhancing the apoptotic response. CONCLUSIONS: The results of this study show that combination of PTX and CER when administered in PEO-PCL nanoparticles can significantly augment the anti-proliferative effect in SMC. This strategy may potentially be useful in the treatment of coronary restenosis.     

Effects of Calcium-Channel Blockers on Cytosolic Free Calcium and Amylase Secretion in Rat Pancreatic Acini

Abstract: We investigated the effects of verapamil and diltiazem on cytosolic free calcium and amylase secretion in rat pancreatic acini. Verapamil and diltiazem reduced a rise in cytosolic free calcium and amylase release stimulated by the maximal concentration (10^-5 M) of carbachol in a dose-dependent manner. High concentrations (500 μM) of verapamil and diltiazem inhibited both the initial and the sustained amylase secretion stimulated by 10^-5 M carbachol. However, at low concentration (1 μM), they showed no effect on amylase secretion by 10^-5 M carbachol. These calcium-channel blockers did not affect calcium mobilization and amylase secretion stimulated by either caerulein or neuromedin C. Binding of ³H-N-methylscopolamine to pancreatic acini was inhibited by verapamil and diltiazem in a dose-dependent manner. These findings suggested that verapamil and dilitiazem reduced carbachol-induced amylase secretion probably not due to their calcium-channel blocking activities but due to their non-competitive effects on the level of muscarinic receptors.     

胰岛素对平滑肌细胞产生一氧化氮的影响

目的：探讨胰岛素和血管紧张素Ⅱ（AngⅡ）对人脐动脉平滑肌细胞产生一氧化氮（NO）和超氧阴离子（O^-2）的影响.方法：不同浓度的AngⅡ和胰岛素加入体外培养的人脐动脉平滑肌细胞,分别测定细胞培养液中一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）活性,NO、环鸟苷磷酸（cGMP）和O^-2浓度.结果：胰岛素（10,100,1 000mU/L）使平滑肌细胞NOS活性升高,NO、cGMP产生增加,对SOD活性和O^-2无影响;1.0 nmol/LAngⅡ使NOS活性下降,NO、cGMP产生减少;0.1,0.5,1.0 nmol/L AngⅡ使SOD活性下降,O^-2水平升高;胰岛素和AngⅡ同时存在时NOS活性下降,NO和cGMP产生减少,SOD活性下降,O^-2水平升高;氯沙坦（losartan）可以改善AngⅡ引起的NOS、SOD活性下降,NO、cGMP产生减少,O^-2水平升高.结论：胰岛素使平滑肌细胞NO产生增加,高浓度AngⅡ使NO产生减少,O^-2水平升高,且可以被Losartan阻断,胰岛素和AngⅡ同时存在时平滑肌细胞NO产生减少,O^-2水平升高.     

The Effect of Sodium Transport and Calcium Channel Inhibitors on Phorbol Ester-induced Contraction of Bovine Airway Smooth Muscle

Summary: We studied the role of sodium transport and calcium channels in protein kinase C mediated signal-transduction pathways in bovine airway smooth muscle. 4-β phorbol 12,13 dibutyrate (PDB), an activator of protein kinase C, caused dose-related slowly developing contraction in bovine bronchial rings with a peak effect at 60-90 min. 4-α PDB, an inactive analogue, was without effect. Mean peak PDB-induced contraction (measured as a percentage of the maximum response to methacholine) was reduced from 122% to 20% when experiments were carried out in calcium-free fluids + EDTA (10^-3 M). Similar reductions were seen in the presence of nifedipine and verapamil, inhibitors of voltage-dependent calcium channels. Amiloride at high concentrations (10^-3 M) reduced the contractile response to PDB from 87% to 20%, but at a concentration which inhibits the sodium entry channel (10^-6 M), amiloride was without effect. 5-N,N-hexamethylene amiloride (10^-5 M), a specific inhibitor of Na⁺/H⁺ exchange, did not alter the contraction produced by PDB. Frusemide (10^-5 M), an inhibitor of Na⁺-K⁺-Cl^- cotransport, was without effect on PDB contractions. We conclude that phorbol ester-induced contraction of bovine airway smooth muscle is dependent on calcium entry via voltage-dependent calcium channels but is independent of Na⁺ entry, Na⁺/H⁺ exchange or Na⁺-K⁺-Cl^- cotransport.