苹果多酚通过下调钙敏感受体改善大鼠低氧性肺动脉高压的作用

目的：探究苹果多酚通过下调钙敏感受体（CaSR）表达改善大鼠低氧性肺动脉高压的作用。方法：36只雄性SD大鼠随机分为对照组、低氧诱导组和苹果多酚干预组（n=12）。低氧诱导组和苹果多酚干预组大鼠每天在低氧舱（氧浓度为10%）中间断低氧暴露8 h,对照组在正常环境下饲养,为期四周。建模同时,苹果多酚干预组每日用苹果多酚按200 mg/kg的剂量灌胃,对照组、低氧诱导组用等量生理盐水灌胃,每日1次,共持续四周。建模完成后,检测各组大鼠的肺动脉平均压（mPAP）、肺血管阻力（PVR）、右心室肥厚指数（RVHI）、肺动脉中膜厚度（MT）、WA%、WT%及肺动脉组织中CaSR表达量。结果：低氧暴露组大鼠mPAP、PVR、RVHI、MT、WA%、WT%及肺动脉组织中CaSR表达量均较对照组明显升高（P<0.01）;苹果多酚干预组可改善上述效应。结论：苹果多酚能有效预防大鼠低氧性肺动脉高压,其机制与下调CaSR的表达有关。     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

慢性低氧高二氧化碳肺动脉高压小鼠肺组织Rho激酶的表达变化

慢性低氧高二氧化碳性肺动脉高压严重威胁着国民身体健康,但其发病机制尚未完全阐明。该研究通过检测正常对照组和慢性低氧高二氧化碳组小鼠右心室肥厚指数(right ven-tricular hypertrophy index,RVHI)、管壁厚度占血管外径的百分比(vessel wall thickness/total vasculardiameter,WT%)和管壁面积占血管总面积的百分比(vessel wall area/total vascular area,WA%),RT-PCR检测肺组织中Rho激酶(ROCK1,ROCK2)基因的表达,Western blot检测肺组织中ROCK1、p-MYPT1(phospho-myosin phosphatase target subunit 1)蛋白的表达,免疫组织化学法观察ROCK1的定位表达,探讨了Rho激酶在慢性低氧高二氧化碳性肺动脉高压形成中的作用。结果发现,慢性低氧高二氧化碳组小鼠RVHI、WT%、WA%值均显著升高(P<0.01),ROCK1、ROCK2基因表达明显增加(ROCK1 P<0.01,ROCK2 P<0.05),ROCK1、p-MYPT1蛋白表达显著增加(P<0.01),ROCK1蛋白表达于肺动脉、肺泡和支气管。以上结果提示,慢性低氧高二氧化碳条件下,小鼠肺组织中Rho激酶表达升高,可能参与了肺动脉高压的形成。     

肺高压大鼠肺动脉平滑肌细胞薄荷醇引起的胞浆游离Ca~(2+)浓度增加幅度减低

本文通过观察正常(control,CON)、慢性低氧(chronic hypoxia,CH)和野百合碱(monocrotaline,MCT)预处理肺高压大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)中,瞬时感受器电位M8(transient receptor potential melastatin8,TRPM8)通道特异激动剂薄荷醇(menthol,MT)引起的胞浆游离钙浓度([Ca2+]i)变化,探讨TRPM8通道在肺高压发病过程中的作用。大鼠处于CH环境或一次性腹腔注射MCT制备肺高压大鼠模型;原代培养CON和肺高压大鼠PASMCs,观察MT激动TRPM8通道引起的PASMCs[Ca2+]i变化,以及TRPM8通道特异阻断剂N-(4-叔-丁基苯基)-4-(3-氯吡啶-2-基)哌嗪-1-甲酰胺[N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide,BCTC]对MT引起[Ca2+]i变化作用的抑制效应;免疫组化检测TRPM8的细胞定位。结果显示,在2 mmol/L Ca2+和无Ca2+台氏液中,MT都可以引起CON组PASMCs的[Ca2+]i增高,且这两种增高作用均可以被BCTC阻断;在2 mmol/L Ca2+和无Ca2+台氏液中,与CON组相比,CH组和MCT组MT引起PASMCs的[Ca2+]i增高幅度均显著减小。免疫组化细胞定位实验显示,PASMCs除胞核外其余部分均有TRPM8棕黄色阳性表达。以上结果提示,PASMCs上的TRPM8通道既存在于胞膜,也存在于肌浆网;CH和MCT预处理可以分别减少PASMCs上TRPM8通道介导的Ca2+内流与Ca2+释放。     

不同剂量野百合碱诱导大鼠肺动脉高压模型的实验研究

目的:肺动脉高压患者的预后非常差并且目前对肺动脉高压的治疗缺乏有效的方法.因此,对肺动脉高压的研究十分迫切,而建立稳定、操作简便、稳定的肺动脉高压模型是研究肺动脉高压的基础.本实验的目的就是采用不同剂量野百合碱(monocrotaline,MCT)诱导建立SD大鼠肺动脉高压模型,探讨肺动脉高压模型的制作方法及其稳定性.方法:清洁级SD雄性大鼠75只,随机分为C组、M1、M2、M3和M4组,每组各15只.其中M1～M4组大鼠分别一次性腹腔注射MCT 30、40、50和60 mg/kg,诱导制作肺动脉高压动物模型;C组为对照组,给予相同剂量溶剂腹腔注射.腹腔注射4周后比较各组大鼠的生存率、检测平均肺动脉压力(mPAP),肺动脉收缩压(PASP),右心肥大指数(RVHI),并取肺组织行苏木素-伊红染色,观察肺的病理改变,采用肺小动脉形态计量学指标综合判断肺动脉高压模型的稳定性.结果:C组无死亡,M1～M4组大鼠的生存率分别为87％,87％,67％,40％.M1～M4各组SD大鼠平均肺动脉压、肺动脉收缩压、右心肥大指数依次增大(P＜0.05),肺小动脉形态计量学指标检测显示肺血管中膜厚度百分比依次增大(P＜0.05),而M3与M4组各测量结果无明显差异.结论:腹腔注射50 mg/kg、60 mg/kg剂量野百合碱均可引起大鼠肺动脉压力升高和肺血管重构,均可诱导稳定的肺动脉高压模型,而50 mg/kg剂量有更高的生存率.所以50mg/kg剂量野百合碱腹腔注射是成功诱导大鼠肺动脉高压模型的合适剂量.     

结缔组织生长因子在慢性阻塞性肺疾病血管重建中表达研究

目的:探讨结缔组织生长因子(CTGF)在慢性阻塞性肺疾病(COPD)血管重建中的表达及意义。方法:将30例有吸烟史的男性鳞癌需要手术的患者按其肺功能结果分成二组,对照组:(肺功能正常组);COPD稳定期组:(肺功能异常组),每组15例,标本来自于癌旁的肺组织,肺血管重塑的形态学观察行HE和MASSON三色染色,行免疫组化来观察CTGF蛋白、PCNA蛋白在肺血管平滑肌中的表达。结果:(1)COPD组肺动脉管壁面积/管总面积(WA%)、管壁的胶原厚度、肺动脉平滑肌中CTGF蛋白及PCNA蛋白的表达与对照组相比差异有统计学意义。(2)CTGF与管壁面积/管总面积(WA%)、管壁的胶原厚度及血管平滑肌中PCNA表达呈正相关(,r值分别为0.81、0.68、0.86,P<0.05)。吸烟指数与管壁面积/管总面积及PCNA的表达呈正相关(r=0.73,0.99,P<0.01)。结论:单纯吸烟者即有血管重建,吸烟伴COPD者血管重建更加严重,CTGF在COPD患者肺血管中的表达较对照组高,可能参与了COPD血管重建过程。     

ING4、PHDs以及HIF-1α在低氧性肺动脉高压大鼠中的表达变化

目的研究大鼠低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)模型中ING4表达变化的规律及其与PHDs、HIF-1α间的相互关系。方法实验复制HPH大鼠模型,在缺氧不同时间点,测定大鼠平均肺动脉压(mPAP)及右心室肥大指数(RVHI),HE染色观察肺小动脉重塑(HPVR)。原位杂交、RT-PCR、检测肺内ING4、PHD1、PHD2、PHD3及HIF-1α的mRNA表达水平及部位,免疫组化、Western blot检测以上蛋白质表达水平及部位。结果 (1)缺氧7d后大鼠的mPAP开始上升,与对照组比较有显著性差异(P0.05),缺氧14d后出现肺血管重塑和RVHI增加,与对照组比较有显著性差异(P0.05)。HIF-1α蛋白在缺氧3d组表达均开始升高(P0.05,与对照组比),缺氧7d达高峰,14d和21d稍下降。HIF-1αmRNA在缺氧14d后表达略增高(与对照组比较P0.05)。(2)对照组PHD1蛋白质呈阳性表达,缺氧14d下降,缺氧21d保持较低水平。PHD1mRNA表达差异无显著性。对照组PHD2蛋白质和mRNA呈阳性表达,缺氧3d增高,缺氧14d达高峰,缺氧21d保持较高水平。对照组PHD3蛋白质呈弱阳性表达,缺氧3d明显增高,缺氧7d保持高水平,缺氧14d和21d下降。对照组肺小血管PHD3mRNA缺氧3d后迅速增高,缺氧14d达高峰,缺氧21d保持高水平。(3)对照组ING4蛋白质呈弱阳性表达,缺氧3d和7d后表达增高,缺氧14d达高峰,缺氧21d稍有下降。对照组ING4mRNA呈阳性表达,缺氧7d组比表达显著增高,直线相关分析表明,在缺氧组大鼠中ING4蛋白质与HIF-1α蛋白质及mPAP、RVHI、WA%负相关。结论肺动脉高压大鼠模型中ING4、PHDs和HIF-1α的表达变化,可能在HPH的发生和发展中发挥作用。     

慢性阻塞性肺疾病小气道NOX4、TGF-β的表达与气道重塑的关系

目的:探讨烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、转化生长因子β(TGF-β)的表达与慢性阻塞性肺疾病(COPD)小气道重塑的关系。方法:收集55例因肺部肿瘤行肺叶切除的患者,根据临床资料及术前肺功能分为COPD组(36例)及对照组(19例),术中留取病变远端正常的肺组织标本。采用HE染色观察COPD组及对照组小气道形态学变化;采用免疫组织化学法、免疫荧光双染法及免疫印迹检测小气道组织NOX4、TGF-β、α-SMA及胶原蛋白Ⅳ的表达情况,并分析其与肺功能气流受限严重程度的相关性。结果:(1)COPD组小气道平滑肌厚度占气道外径的百分比(WT%)、小气道平滑肌面积占支气管断面面积百分比(WA%)明显高于对照组(P0.05);WT%和WA%在不同肺功能分级的COPD组间比较,差别均有统计学意义(P0.05)。(2)COPD组上皮细胞增生明显,部分上皮细胞表达间质细胞表型α-SMA及胶原蛋白Ⅳ。COPD组上皮细胞α-SMA及Ⅳ型胶原蛋白免疫染色强度明显强于对照组(P0.05)。(3)NOX4在COPD组小气道平滑肌及上皮细胞中的表达明显高于对照组(P0.05),NOX4的表达在不同肺功能分级的COPD组间比较,差别均有统计学意义(P0.05)。(4)TGF-β在COPD组小气道平滑肌细胞及气道上皮细胞的表达均高于对照组(P0.05)。(5)COPD小气道平滑肌WT%、WA%与肺功能FEV1%pred、FEV1/FVC%呈负相关(P0.05);小气道平滑肌细胞NOX4的表达与肺功能FEV1%pred、FEV1/FVC%呈负相关(P0.05)。结论:COPD存在以小气道平滑肌增生及细胞外基质沉积为主要表现的气道重塑,且气道重塑的程度与气流受限严重程度呈正相关,COPD小气道上皮细胞和小气道平滑肌细胞表达NOX4、α-SMA和TGF-β明显增高,NOX4、α-SMA、TGF-β的表达与COPD气流受限严重程度呈负相关,提示TGF-β、NOX4信号机制可能参与COPD气道重塑的发生发展。     

成肌纤维细胞在大鼠低氧性肺动脉高压发病中的作用

目的探讨低氧性肺动脉高压(HPH)无肌性肺动脉肌化的细胞来源.方法雄性Wistar大鼠40只,分为对照组和低氧(3、7、14、21d组),每组8只,低氧组复制HPH大鼠模型.测定各组平均肺动脉压(mPAP),右心室肥大指数(RVHI),肺小动脉病理及其形态计量学;免疫组化测定各组肺微血管α-平滑肌肌动蛋白(α-SMA)表达;透射电镜观察微血管的超微结构改变.结果 (1)低氧7d起大鼠mPAP、管壁面积/管总面积(WA%)、管腔面积/管总面积(LA%)分别为(18.41±0.37)mmHg、(52.2±0.8)%、(47.8±0.8)%与对照组(14.02±0.41)mmHg、(64.5±1.3)%、(35.5±1.3)%比较差异有显著性(P<0.05),低氧14天起稳定于高水平;低氧14d RVHI为(25.0±1.8)%与对照组(23.6±0.5)%比较差异也有显著性(P<0.05).(2)从低氧7d开始无肌型动脉、部分肌型动脉、肌型动脉的构成比分别是39%、46%、15%与对照组60%、35%、5%比较差异有显著性(P＜0.005).(3)免疫组化发现,腺泡内肺动脉管壁α-SMA的表达随着低氧时间的延长,α-SMA表达量逐渐增多.(4)透射电镜观察21d组增厚的重塑血管壁,位于内外弹性膜之间的细胞为成肌纤维细胞表型,外层具有与它紧密联系的成纤维细胞.结论在低氧性肺动脉高压时,成纤维细胞转化为成肌纤维细胞是腺泡内肺动脉重塑的重要原因之一.     

Ca~(2+)转运通路对金雀异黄酮舒张大鼠脑血管作用的影响

目的:研究Ca2+转运通路对金雀异黄酮舒张大鼠脑血管作用的影响。方法:75只大鼠被随机分为3组,分别经由二甲亚砜、金雀异黄酮和酪氨酸磷酸化抑制剂A47处理基底动脉及Willis环血管。每组大鼠进一步划分成5个亚组,每个亚组用不同浓度的细胞外Ca2+处理,分为:0、0.6、1.2、1.8和3.6 m M Ca2+组。5-羟色胺诱导血管收缩。测定大鼠基底动脉管壁厚度与官腔周长的比值;荧光成像分析法测定血管平滑肌细胞细胞内Ca2+浓度;免疫印迹分析检测肌球蛋白轻链激酶(MLCK),蛋白质磷酸酶催化亚基1(PP1),肌凝蛋白磷酸酶目标亚基1(MYPT1)的表达来测定血管平滑肌细胞Ca2+敏感性。结果:金雀异黄酮和酪氨酸磷酸化抑制剂A47显著降低大鼠基底动脉管壁厚度与官腔周长的比值(P0.01),Ca2+内流(P0.01,P0.05)及MLCK的表达(P0.01);增加PP1和MYPT1的表达(P0.01)。细胞外Ca2+与金雀异黄酮及酪氨酸磷酸化抑制剂A47有协同效应。硝苯地平和毒胡萝卜素可废除该效应。结论:低细胞外Ca2+水平增强了金雀异黄酮和酪氨酸磷酸化抑制剂A47的血管舒张作用。L型电压门控Ca2+通道(L-VGCC)和肌浆网Ca2+库(SR)参与交互效应。     

脂肪干细胞移植对野百合碱诱发的肺动脉高压大鼠肺动脉压的量效和时效作用

目的探索脂肪干细胞（ADSC）移植治疗野百合碱（MCT）诱导的肺动脉高压（PAH）大鼠的适宜细胞数和干预时间。 方法（1）MCT的建模时效和量效：雄性SD大鼠48只分为正常对照组,20 mg/kg、30 mg/kg、40 mg/kg MCT组分别予腹腔注射生理盐水、MCT 20 mg/kg、30 mg/kg、40 mg/kg,4和8周后,右心室插管法检测平均肺动脉压（mPAP）,称重法计算右心室肥厚指数（RVHI）。（2）ADSC的治疗量效作用：雄性SD大鼠分别予腹腔注射MCT（30只）和生理盐水（30只）,1周后通过颈静脉注射分别移植0.5×10⁶、1.0×10⁶、3.0×10⁶、5.0×10⁶ADSC,其他组予等量生理盐水。移植3周后检测mPAP和RVHI。（3）ADSC的治疗时效作用：雄性SD大鼠30只,分别注射40 mg/kg MCT（24只）和生理盐水（6只）。MCT腹腔注射1 d,1、2周后分别移植1.0×10⁶个ADSC。MCT注射4周后检测mPAP和RVHI。多组间比较采用单因素或双因素方差分析,两两比较采用LSD检验。 结果（1）腹腔注射4周后,30 mg/ kg或40 mg/kg MCT组mPAP和RVHI均升高[mPAP值（24.89±3.31）mmHg,（27.19±2.11）mmHg比（15.80±0.42）mmHg,差异有统计学意义（P均< 0.05）;RVHI值0.42±0.06,0.47±0.04比0.25±0.02,差异有统计学意义（P均< 0.05）]。8周后,20 mg/kg或30 mg/ kg MCT组mPAP和RVHI均恢复正常,而40 mg/kg MCT组大鼠全部死亡。（2）40 mg/ kg MCT诱导的PAH大鼠mPAP和RVHI均升高。移植1.0×10⁶个ADSC可降低PAH大鼠的mPAP[（17.24±0.66）mmHg比（27.19±1.73）mmHg,P < 0.05]。移植0.5×10⁶、3.0×10⁶、5.0× 10⁶个ADSC不能降低PAH大鼠的mPAP和RVHI。（3）MCT腹腔注射1周和2周后,移植1.0×10⁶个ADSC可降低PAH大鼠的mPAP。 结论40 mg/kg MCT造模4周可建立稳定的PAH大鼠模型;造模1或2周后移植1.0×10⁶个ADSC能有效降低PAH大鼠的mPAP。     

Insulin secretion and intracellular Ca rises in monolayer cultures of neonatal rat β-cells

Glucose-induced insuline release, glucose-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and voltage-dependent Ca²⁺ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca²⁺]_i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca²⁺ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca²⁺]₂ rise and, like deprivation of extracellular Ca²⁺, prevented the glucose-induced rise in [Ca²⁺]_i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca²⁺ channels was demonstrated directly by measuring Ca²⁺ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca²⁺]₁ after membrane depolarization by 45 mMm K⁺ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca²⁺ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Dissociation of Intracellular Ca Release and Ca Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca²⁺ pool and those discharged by the Ca²⁺-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i), followed by a sustained plateau phase that was dependent on extracellular Ca²⁺. In such cells, TG produced a concentration-dependent increase in [Ca²⁺]_i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca²⁺. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca²⁺]_i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca²⁺-ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca²⁺-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca²⁺ stores is sufficient for activation of Ca²⁺ influx. Some characteristics of the Ca²⁺-influx activated by depletion of internal Ca²⁺ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca²⁺ influx was inhibited by La³⁺, membrane depolarisation, and the novel Ca²⁺-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca²⁺ influx by TG also was blocked by La³⁺, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca²⁺ stores in TSMCs is sufficient for activation of Ca²⁺ influx, and (2) the agonist-activated Ca²⁺ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool are indistinguishable.     

川芎嗪对模拟失重大鼠颈动脉平滑肌Ca2+敏感性的影响

目的：探讨川芎嗪对模拟失重大鼠颈动脉平滑肌收缩蛋白Ca²⁺敏感性的影响及机制。方法：21只雌性SD大鼠分为对照组（CON）、模拟失重组（TS）、模拟失重+川芎嗪灌饲组（LTZ）（n=7）。4周后测定大鼠颈动脉收缩性、收缩蛋白Ca²⁺敏感性及肌球蛋白轻链激酶抑制剂ML-7对以上指标的影响。结果：TS组苯肾上腺素（PHE）收缩力比CON组高25.14%（P<0.01）,LTZ组较TS组低13.46%（P<0.01）,较CON组高8.30%;ML-7可使CON、TS、LTZ组PHE收缩力分别降低8.84%、16.24%（P<0.01）和3.40%;TS组KCl收缩力比CON组高40.46%（P<0.01）,LTZ组较TS组低18.80%,较CON组高14.05%;ML-7可使CON、TS、LTZ组KCl收缩力分别降低21.97%（P<0.05）、21.88%（P<0.01）和10.84%;TS组pD2[Ca²⁺]比CON组高10.03%（P<0.01）,LTZ组较TS组低7.01%（P<0.01）,较CON组高2.32%;ML-7可使CON、TS、LTZ组pD2[Ca²⁺]分别降低2.42%、7.43%（P<0.01）和2.51%。结论：模拟失重大鼠颈动脉收缩增强可能是由动脉平滑肌收缩蛋白Ca²⁺敏感性增强所导致的。川芎嗪可改善模拟失重大鼠颈动脉的收缩功能,其机制可能与抑制血管平滑肌肌球蛋白轻链激酶继而降低Ca²⁺敏感性有关。     

Ca(2+) as second messenger in PTTH-stimulated prothoracic glands of the silkworm,Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.