肺动脉高压模型大鼠肺动脉平滑肌细胞内钙的改变

目的分析肺动脉高压时肺动脉平滑肌细胞Ryanod-ine受体[Ca2+]i释放功能的改变。方法腹腔注射野百合碱建立大鼠肺动脉高压模型,原代培养肺动脉平滑肌细胞,Fura-2/AM负载培养细胞,荧光测钙技术测量Ryanodine受体激动剂对[Ca2+]i变化的影响。结果10nmol.L-1Ry-anodine使对照组[Ca2+]i平均增加(93.31±12.41)nmol.L-1,使PAH组[Ca2+]i平均增加(141.71±13.59)nmol.L-1。两组样本[Ca2+]i增加的数值差异有显著性(P<0.01);10mmol.L-1Caffine使对照组[Ca2+]i平均增加(149.02±13.02)nmol.L-1,使PAH大鼠PASMC的[Ca2+]i平均增加(191.2±21.26)nmol.L-1,两组样本[Ca2+]i数值的变化差异有显著性(P<0.01)。结论肺动脉高压大鼠原代培养的肺动脉平滑肌细胞对Ryanodine受体激动剂的敏感性增强,提示肺动脉高压时Ryanodine受体释放[Ca2+]i的功能发生了异常改变。     

羊角拗苷对豚鼠心室肌细胞内游离钙离子浓度的影响

目的观察羊角拗苷(Div)对豚鼠心室肌细胞内游离钙离子([Ca2+]i)浓度的影响,以探讨Div正性肌力作用的机制。方法Fura-2/AM荧光探针标记豚鼠心室肌细胞,应用荧光离子成像系统观察Div800nmol.L-1对心室肌细胞[Ca2+]i的影响。结果在正常台氏液中,Div使心室肌细胞[Ca2+]i显著升高(243±36)%,在无钙液中对[Ca2+]i无明显影响。在无Na+、无K+台氏液中,Div使[Ca2+]i升高(96±20)%。给予L型钙通道阻滞剂CdCl2100μmol.L-1预处理后,Div仍使[Ca2+]i升高(63±10)%。给予T型钙通道阻滞剂NiCl240μmol.L-1预处理后,Div引起的[Ca2+]i升高基本被阻断。结论体外应用Div800nmol.L-1时使豚鼠心室肌细胞[Ca2+]i升高,该升高作用依赖于细胞外Ca2+的存在,可能主要由T型钙通道介导,L型钙通道和Na+-Ca2+交换蛋白亦参与其中。其正性肌力作用与心室肌细胞[Ca2+]i升高有关。     

P物质对大鼠垂体前叶细胞游离Ca2+影响的研究

目的:探讨SP是否影响培养的大鼠AP细胞[Ca2+]i,在第二信使信息转导途径上进一步阐述SP产生生物学效应的机制。方法:原代培养成年雌性S-D大鼠的AP细胞48h,加入SP孵育不同时间,采用Fura-2/AM检测[Ca2+]i。结果:当SP浓度为100nmol/L,孵育时间由10min增加到30min时[Ca2+]i增加,但孵育时间为40min、50min时,[Ca2+]i呈现减少趋势,即最大反应的孵育时间为30min。当孵育时间为20min、30min、40min、50min时,[Ca2+]i分别为211±16nmol/L、369±51nmol/L、358±24nmol/L、239±36 nmol/L,与10min(117±17nmol/L)相比较,呈显著性差异(P<0.05)。结论:通过Fura-2/AM可精确反映[Ca2+]i,SP兴奋AP细胞SPR后可通过Ca2+来完成其部分的生物学效应,说明了SP对生殖轴(下丘脑-垂体前叶-卵巢/睾丸)有调控作用。     

低浓度的哇巴因引起豚鼠心室肌细胞内钙增高的可能途径

目的观察哇巴因(ouabain,OUA)对豚鼠心室肌细胞内游离钙浓度([Ca2+]i)的影响。方法酶解分离豚鼠心室肌细胞,负载Fluo3-AM,激光共聚焦显微镜术测定单个心室肌细胞[Ca2+]i的荧光密度,结果用相对荧光强度(FI-FI0)/FI0(%)表示,其中,FI0:给药前的荧光密度值,FI:给药后的荧光密度值。结果在正常台氏液及无钙台氏液中,OUA(1×10-9~1×10-6mol·L-1)浓度依赖性地升高细胞内钙浓度,在正常台氏液分别为16.7±6.8(P<0.01)、26.0±4.7(P<0.01)、183.0±101.0(P<0.01)、295.0±172.0(P<0.01),而在无钙台氏液中OUA升高[Ca2+]i不如在正常台氏液中,分别为9.19±4.73(P<0.05)、20.75±5.2(P<0.05)、85.79±64.7(P<0.05)、231.0±26.0(P<0.05)。肌浆网钙通道抑制剂理阿诺碱Ryanodine(1×10-5mol·L-1)可部分抑制正常台氏液时OUA的效应5.3±2.1(P<0.01)。在无钠无钾台氏液中,OUA(1×10-9~1×10-6mol·L-1)对心室肌细胞[Ca2+]i的影响分别为16.5±6.5、25.0±5.0、162.0±45.0、280.0±96.0与正常台氏液比较无差别(P>0.05)。蛋白酪氨酸激酶抑制剂三羟异黄酮(genistein,GST;1、10、50、100μmol·L-1)可浓度依赖性地抑制正常台氏液中的OUA效应,分别为17.5±3.1、14.2±8.9、0.8±7.6(P<0.05)、-1.9±6.7(P<0.01)。L-型钙通道激动剂BayK8644,肌浆网钙通道开放剂Ryanodine(1×10-7mol.L-1)在正常台氏液中均可提高[Ca2+]i为13.3±3.2(P<0.05)、6.4±5.6(P<0.05)。三羟异黄酮可取消其效应为-13.0±21.0(P<0.01)、-1.6±5.9(P<0.01)。结论低浓度的OUA升高豚鼠心室肌细胞内游离钙浓度,此作用与其开放钙通道及促进内钙释放有关,且信号转导通过此二途径起作用。     

吸烟、硝酸甘油对动脉粥样硬化家兔血管平滑肌细胞钙浓度的影响

目的研究吸烟、硝酸甘油对动脉粥样硬化血管平滑肌细胞胞内游离钙浓度([Ca2+]i)的影响,探讨吸烟对动脉粥样硬化形成及硝酸甘油的生物效应的作用。方法复制家兔动脉粥样硬化模型,分离血管平滑肌细胞。Fluo-3/AM负载细胞,应用流式细胞仪检测血管平滑肌细胞[Ca2+]i,激光扫描共聚焦显微系统测定单个血管平滑肌细胞内Ca2+的时空变化。结果各组动物主动脉壁均见到程度不同的动脉粥样硬化斑块形成。动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i明显升高(48.45±5.31,与生理盐水对照组38.09±2.57比较,P<0.01);吸烟能增加动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i(56.48±2.99,与单纯动脉粥样硬化组48.45±5.31比较,P<0.01);硝酸甘油则能明显降低动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i(41.91±3.16,与单纯动脉粥样硬化组48.45±5.31比较,P<0.05);吸烟能明显抑制硝酸甘油降低血管平滑肌细胞[Ca2+]i的作用(47.99±5.10,与硝酸甘油组41.91±3.16比较,P<0.05)。结论吸烟能明显增加动脉粥样硬化血管平滑肌细胞[Ca2+]i,硝酸甘油则能明显降低动脉粥样硬化血管平滑肌细胞[Ca2+]i,吸烟能抑制硝酸甘油的生物学效应。     

金丝桃苷对新生大鼠神经细胞缺氧/再给氧损伤的保护作用及其机制

目的探讨金丝桃苷(hyperin,Hyp)对新生大鼠脑细胞缺氧/再给氧损伤的保护作用及其机制。方法将分离的新生大鼠脑细胞缺氧30min或缺氧30min后再给氧40min造成细胞缺氧或再给氧损伤模型,测定细胞培养上清液中乳酸脱氢酶(LDH)、丙二醛(MDA)和一氧化氮(NO)水平的变化,用Fura2-AM方法测定脑细胞内游离钙离子浓度([Ca2+]i)的变化。结果脑细胞缺氧时,细胞培养上清液中LDH从(62.0±13.0)U·L-1(Sham组)增高到(116.0±16.6)U·L-1(Control组,P<0.01),再给氧时,LDH和MDA分别从(45.6±9.2)U·L-1和(9.1±0.9)μmol·L-1(Sham组)增高到(106.0±17.4)U·L-1和(16.4±2.7)μmol·L-1(Control组,P<0.01)。Hyp在1.0～16.0μmol·L-1范围内,明显地抑制缺氧及再给氧损伤诱导的上述LDH和NO增高,并呈一定的浓度依赖性。1.0～16.0μmol·L-1的Hyp不仅可明显地抑制缺氧诱导的细胞培养上清液中NO和脑细胞内[Ca2+]i的增高(P<0.05或P<0.01),也可明显地抑制再给氧时NO和脑细胞内[Ca2+]i的升高(P<0.05或P<0.01),16.0μmol·L-1的Hyp可将再给氧时NO和[Ca2+]i分别从(34.4±6.3)μmol·L-1和(640±94)nmol·L-1(Control组)降至(25.0±5.1)μmol·L-1(P<0.05)和(331±56)nmol·L-1(P<0.01)。结论Hyp对神经细胞缺氧/再给氧损伤保护作用可能与抑制NO的释放、钙超载及自由基脂质过氧化有关。     

埃他卡林对兔肺动脉平滑肌内皮细胞钙浓度的影响

目的观察埃他卡林(IPT)对内皮素1(ET-1)诱导培养的兔肺动脉平滑肌细胞(PASMC)增殖的影响,并探讨其作用机制。方法应用细胞培养、氚-胸腺嘧啶核苷([3H]-TdR)参入实验、Fluo-3和激光扫描共聚焦显微镜技术评价IPT对ET-1诱导的兔PASMC增殖及PASMC[Ca2+]i调节的作用。结果ET-1(10-7mol.L-1)使PASMC[3H]-TdR参入量增加146.8%,与对照组比较,差异有显著性(P<0.01);在相同条件下,加入ET-1的同时,分别向培养基中加入IPT10-7、10-6、10-5mol.L-1,细胞[3H]-TdR参入量分别下降(19.8±4.6)%、(41.2±9.5)%、(54.7±10.1)%,与ET-1组比较差异有显著性(P<0.01);对照组PASMC[Ca2+]i荧光强度和荧光光密度值较低;ET-1组中[Ca2+]i荧光光密度值明显增高,从73.7±10.1增加到143.8±28.2,两者比较差异有显著性(P<0.01);而IPT组细胞内荧光光密度值明显降低,仅从74.30±10.2增加到86.03±9.82,与ET-1组比较差异有显著性(P<0.01)。结论IPT可明显抑制ET-1诱导的兔PASMC增殖、DNA合成;减少钙通道的开放时间,抑制细胞内Ca2+浓度增加。     

肿瘤坏死因子-α对肾入球动脉平滑肌细胞胞内钙离子浓度的影响

目的观察肿瘤坏死因子-α(TNF-α)对肾入球动脉平滑肌细胞(RASMCs)内钙离子浓度([Ca2+]i)的影响。方法应用筛网及酶消化法进行原代雄性SD大鼠RASMCs的分离与培养,将原代培养的RASMCs间接免疫荧光α-肌动蛋白(α-actin)抗体、平滑肌肌球蛋白重链(SM)单克隆抗体染色,进行鉴定。实验分组:A.无钙缓冲液组;B.无钙缓冲液+TNF-α组;C.无钙缓冲液+内皮素组;D.无钙缓冲液+2-氨基乙基二苯硼酸盐(2-APB)+内皮素组;E.无钙缓冲液+TNF-α+内皮素组;F.无钙缓冲液+TNF-α+2-APB+内皮素组。采用confocal显微镜测定单个活细胞内Ca2+荧光强度,计算细胞内[Ca2+]i。结果 A组和B组RASMCs内[Ca2+]i分别为(109.51±52.60)和(128.07±78.56)nmol/L,两组间无显著差异(P>0.05);C组和E组分别为(240.36±56.78)和(375.45±60.39)nmol/L,两组间有显著差异(P<0.01),与前A组和B组比较也均有显著差异(P<0.01);D组和F组分别为(101.66±48.03)和(116.35±53.48)nmol/L,与A组比较无显著差异(P>0.05)。结论 TNF-α可增强内皮素刺激引起的RASMCs内[Ca2+]i增加,且通过1,4,5-三磷酸肌醇受体(IP3R)发挥作用。     

大黄素增强结肠近端平滑肌细胞钙离子依赖氯离子通道的表达

目的:研究大黄素对结肠近端平滑肌细胞钙离子依赖氯离子通道(ClCa channel)的作用。方法:采用RM6200四道仪记录黄体酮对平滑肌的等长收缩活动作用,相对定量的单细胞RT-PCR法检测平滑肌细胞ClCa通道mRNA的表达。结果:大黄素显著增强离体结肠平滑肌肌条和单个平滑肌细胞的收缩,并可使ClCa电流显著增强,作用与剂量呈正相关。DIDS和NFA可阻滞大黄素的作用。豚鼠近端结肠平滑肌细胞有ClCa1和ClCa2基因的mRNA表达,ClCa1基因的mRNA表达强度是ClCa2基因的mR-NA的5.3±0.71(n=5,P<0.01)倍。50μM大黄素孵育可以使ClCa1基因的mRNA表达增强(n=5,P<0.01)。结论:大黄素可通过兴奋氯离子通道电流引起结肠平滑肌收缩,大黄素对氯离子通道兴奋作用可能与增强结肠平滑肌细胞ClCa1基因表达有关。     

细胞内钙在脑心综合征心律失常中作用研究

目的通过研究脑心综合征心肌细胞内游离钙离子浓度([Ca2+]i)的变化及其来源,以探讨其与脑心综合征心律失常的关系。方法线栓法建立脑心综合征模型,监测心电图,酶法分离心肌细胞,激光共聚焦扫描显微镜观察心室肌[Ca2+]i的变化及其途径。结果模型组QT间期较正常组和假手术组明显延长(P<0.01);在正常台氏液中,KCl诱发模型组心肌[Ca2+]i升高幅度高于假手术组(P<0.01),用1μmol·L-1和10μmol·L-1维拉帕米预孵育模型组后,10μmol·L-1维拉帕米明显降低KCl诱发的心肌[Ca2+]i升高幅度(P<0.01);在无钙台氏液中,caffeine诱发模型组心肌[Ca2+]i升高幅度高于假手术组(P<0.01)。结论通过心肌细胞L-型钙通道和肌浆网雷诺定受体介导的内钙异常升高可能是脑心综合征心律失常的原因之一。     

The Cl(-) channel blocker niflumic acid releases Ca(2+) from an intracellular store in rat pulmonary artery smooth muscle cells

The effect of the Cl- channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl- channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl- channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.     

Dynamics of Ca2+-dependent Cl- channel modulation by niflumic acid in rabbit coronary arterial myocytes

Calcium-activated chloride channels (Cl(Ca)) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. Niflumic acid (NFA), a potent blocker of Cl(Ca) in vascular myocytes, was shown recently to cause inhibition and paradoxical stimulation of sustained calcium-activated chloride currents [I(Cl(Ca))] in rabbit pulmonary artery myocytes. The aims of the present study were to investigate whether NFA produced a similar dual effect in coronary artery smooth muscle cells and to determine the concentration-dependence and dynamics of such a phenomenon. Sustained I(Cl(Ca)) evoked by intracellular Ca(2+) clamped at 500 nM were dose-dependently inhibited by NFA (IC(50) = 159 microM) and transiently augmented in a concentration-independent manner (10 microM to 1 mM) approximately 2-fold after NFA removal. However, the time to peak and duration of NFA-enhanced I(Cl(Ca)) increased in a concentration-dependent fashion. Moreover, the rate of recovery was reduced by membrane depolarization, suggesting the involvement of a voltage-dependent step in the interaction of NFA, leading to stimulation of I(Cl(Ca)). Computer simulations derived from a kinetic model involving low (K(i) = 1.25 mM) and high (K(i) < 30 microM) affinity sites could reproduce the properties of the NFA-modulated I(Cl(Ca)) fairly well.     

Cl-通道阻断剂对A10细胞α1-AR介导的Ca2+内流的影响

目的 :在胚胎大鼠主动脉平滑肌细胞 (A10 ) ,探讨Cl- 通道与Ca2 + 内流的关系及酪氨酸磷酸化对Ca2 + 内流的作用。方法 :采用Fura 2荧光探针双波长测定胞浆游离Ca2 + 浓度 ([Ca2 + ]i)。结果 :Ca2 +通道阻断剂nifedipine和SK&F96 36 5可阻止肾上腺素 (Adr)触发的Ca2 + 内流 ;氯通道阻断剂niflumicacid(NFA)和furosemide呈浓度依赖性抑制Ca2 + 内流。在Ca2 + 内流被SK&F96 36 5最大限度抑制后 ,NFA和furosemide可进一步抑制Ca2 + 内流 ;而Ca2 +内流被NFA和furosemide分别最大抑制 2 8%和 35 %后 ,SK&F96 36 5也可进一步抑制Ca2 + 内流达 5 3%和5 2 %。genistein呈浓度依赖性抑制Ca2 + 内流 ;vana date浓度依赖性促进Ca2 + 内流。结论 :在A10细胞 ,肾上腺素受体触发的Ca2 + 内流涉及电压依赖性Ca2 + 通道 (VDC)和受体操纵性Ca2 + 通道 (ROC) ;氯通道参与了VDC及ROC介导的Ca2 + 内流 ;蛋白酪氨酸磷酸化的水平影响Ca2 + 内流。     

Phosphorylation alters the pharmacology of Ca2+-activated Cl? channels in rabbit pulmonary arterial smooth muscle cells

Background and purpose:

Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca²⁺-activated Cl⁻ channel (Cl_Ca) blocker niflumic acid (NFA) produces a paradoxical dual effect on I_Cl(Ca), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I_Cl(Ca) were modulated by phosphorylation.

Experimental approach:

I_Cl(Ca) was elicited with 500 nM free internal Ca²⁺ in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 µM).

Key results:

Dephosphorylation enhanced the ability of 100 µM NFA to inhibit I_Cl(Ca). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <−50 mV, ∼70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I_Cl(Ca) in the range of 0.1–250 µM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 µM, which converted to block at concentrations >1 µM when cells were dialysed with 5 mM ATP.

Conclusions and implications:

Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl_Ca channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.     

Vardenafil, but not sildenafil or tadalafil, has calcium-channel blocking activity in rabbit isolated pulmonary artery and human washed platelets

BACKGROUND AND PURPOSE: Phosphodiesterase type-5 (PDE5) inhibitors constitute a novel and important therapeutic option for the treatment of pulmonary hypertension. The effects of the PDE5 inhibitors sildenafil, tadalafil and vardenafil on rabbit isolated pulmonary artery ring preparations and on intracellular Ca2+ concentration of thrombin-stimulated human platelets were investigated. EXPERIMENTAL APPROACH: Rabbit pulmonary artery rings were mounted in 10 mL organ bath containing Krebs solution. Tissues were connected to force-displacement transducers, and changes in isometric force were recorded. Ca2+ flux in human washed platelets was measured. KEY RESULTS: Sildenafil, tadalafil and vardenafil (0.0001-10 microM) concentration-dependently relaxed endothelium-intact and endothelium-denuded pulmonary artery rings. Endothelium denudation caused rightward shifts in the concentration-response curves to sildenafil, tadalafil and vardenafil (9-, 12- and 123-fold, respectively). Incubation with N(omega)-nitro-L-arginine methyl ester (100 microM) or ODQ (1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one) (10 microM) caused similar reductions of PDE5-induced vasorelaxations in intact rings. Sildenafil and tadalafil did not affect the phenylephrine-induced contractions, whereas vardenafil reduced the maximal responses, and shifted the phenylephrine-induced contraction curves to the right in endothelium-denuded rings (5- and 19-fold for 1 and 10 microM, respectively). Vardenafil (but neither sildenafil nor tadalafil) caused a marked rightward shift and a decrease of maximal contractile response to CaCl2. Vardenafil, but neither sildenafil nor tadalafil, significantly reduced the Ca2+ mobilization and Ca2+ influx in thrombin-stimulated washed platelets. CONCLUSIONS AND IMPLICATIONS: Our results indicate that vardenafil, in contrast to sildenafil or tadalafil, also blocked Ca2+ fluxes, thus enhancing its vasorelaxation of the pulmonary artery.     

Pharmacologically distinct intracellular calcium pools regulate tonic and oscillatory responses in porcine thoracic duct

The present study investigated the mechanisms by which the thromboxane A2 mimetic U46619 can elicit phasic and tonic contractions in the pig thoracic duct, whereas other agonists like 5-hydroxytryptamine (5-HT) produce tonic contractions only. Tonic contractions in response to either agonist were abolished by the l-type voltage-operated calcium channel (VOCC) inhibitor nifedipine, the store-operated calcium channel inhibitor SKF 96365, the calcium-sensitive chloride channel (ClCa) inhibitor niflumic acid, and by removal of extracellular Cl-. Superimposed phasic responses to U46619 were abolished by only nifedipine. Inhibitors of K+ channels did not prevent phasic contractions to U46619. The IP3 receptor antagonist 2-APB attenuated tonic contractions only, whereas ryanodine and removal of extracellular Na+ selectively abolished phasic contractions to U46619. Therefore, selective initiation of phasic contractions by U46619 appears to depend on intracellular Ca2+ from a ryanodine-sensitive store that causes depolarization via Na+/Ca2+ exchange, whereas tonic contractions to U46619 and 5-HT are mediated primarily by release of IP3-mobilized intracellular Ca2+ that subsequently causes ClCa opening, membrane depolarization, and Ca2+ entry via l-type VOCC.     

氯通道阻断剂对α1—肾上腺素受体亚型引起的Ca^2＋内流的影响

目的探讨氯通道阻断剂在α１Ａ、α１Ｂ、α１Ｄ肾上腺素受体（ＡＲ）亚型触发的Ｃａ２＋内流中的作用。方法采用Ｆｕｒａ－２荧光探针双波长测定胞浆游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）。结果在３种亚型的细胞上,肾上腺素（Ａｄｒ）触发的Ｃａ２＋内流均不受ｎｉｆｅｄｉｐｉｎｅ影响; ＳＫ＆Ｆ９６３６５可部分抑制α１Ａ、α１Ｂ－ＡＲ介导的Ｃａ２＋内流,而对ａ１Ｄ－ＣＨＯ细胞无影响。在ＢＢ－ＣＨＯ细胞上,ｎｉｆｌｕｍｉｃ　ａｃｉｄ（ＮＦＡ）和ｆｕｒｏｓｅｍｉｄｅ呈浓度依赖性抑制Ｃａ２＋内流;当Ｃａ２＋内流被ＳＫ＆Ｆ９６３６５最大限度抑制后,ＮＦＡ和ｆｕｒｏｓｅｍｉｄｅ可进一步抑制Ｃｄ２＋内流。α１Ａ－ＡＲ介导的Ｃａ２＋内流可被ｆｕｒｏｓｅｍｉｄｅ抑制,抑制率达 １４％ １５％。 ＮＦＡ可抑制ａ１Ｄ－ＡＲ引起的Ｃａ２＋内流,抑制率为３９％±９％。结论氯通道参与α１Ａ、α１Ｂ及α１Ｄ－ＡＲ引起的经非电压依赖性Ｃａ２＋通道介导的Ｃａ２＋内流,其间存有异同。ＮＦＡ敏感Ｃａ２＋内流及ｆｕｒｏｓｅｍｉｄｅ敏感的Ｃａ２＋内流与ＳＫ＆Ｆ９６３６５敏感的Ｃａ２＋内流存在非同一性。     

Effects of Ba2+ on F&F 96365-sensitive sustained contraction of rat pulmonary artery

The effects of Ba2+ on receptor-mediated sustained contraction of rat pulmonary artery and guinea-pig oesophageal muscularis mucosae were studied in-vitro. In rat isolated pulmonary artery, sustained contraction induced by noradrenaline (1 microM) was resistant to nicardipine (1 microM), but this same sustained contraction was completely inhibited by SK&F 96365 (30 microM), a blocker of voltage-dependent L-type Ca2+ channels and receptor-activated Ca2+ influx. The SK&F 96365-sensitive sustained contraction induced by noradrenaline (1 microM) may be due primarily to Ca2+ influx through receptor-activated Ca)+ channels resistant to nicardipine. Cumulatively applied BaCl2 (0.1 - 10 mM) increased the noradrenaline (1 microM)-induced sustained contraction of the pulmonary arterial preparation in the absence of nicardipine, but in the presence of nicardipine (1 microM), BaCl2 (0.1 - 3 mM) did not affect this contraction. A higher concentration of BaCl22 (10 mM), however, weakly inhibited the noradrenaline (1 microM)-induced tone. In addition, BaCl2 (3-10 mM) increased the tone induced by KCl (60 mM), and the BaCl2-elevated KCl tone was markedly inhibited by nicardipine (1 microM) treatment. In the guinea-pig isolated oesophageal muscularis mucosae, sustained contraction induced by acetylcholine (3 microM) was resistant to nicardipine (1 microM) but was returned to its basal level by SK&F 96365 (30-60 microM). The SK&F 96365-sensitive, acetylcholine-induced sustained contraction of the oesophageal muscularis mucosae is also likely to link with receptor-activated Ca2+ channels resistant to nicardipine. In contrast to the rat pulmonary artery, cumulatively applied BaC12 (0.3 - 10 mM) inhibited the acetylcholine (3 microM)-induced sustained contraction of the oesophageal muscularis mucosae in a concentration-dependent manner in the presence of nicardipine (1 microM). In conclusion, Ba2+ presumably activates voltage-dependent Ca2+ channels by depolarizing plasma membrane and also passes through voltage-dependent Ca2+ channels to contract the pulmonary artery in the absence of nicardipine, and Ba2+ also has a minor effect on the nicardipine-resistant, SK&F 96365-sensitive sustained contraction induced by noradrenaline in rat isolated pulmonary artery.     

超氧阴离子对兔肺动脉平滑肌细胞内钙的影响

AIM: To study the effect of superoxide anion on the Ca2+ homeostasis in smooth muscle cells isolated from the rabbit pulmonary artery. METHODS: Intracellular Ca2+ concentration ([Ca2+]i) was investigated using cell suspension of freshly isolated smooth muscle cells from rabbit pulmonary artery (PASMC). Fura-2 fluorescent ratio obtained at 340 nm and 380 nm wave lengths was measured as an indicator of [Ca2+]i. RESULTS: ATP 30 mumol.L-1 induced a transient increase in the ratio (Ca2+ transient). Thapsigargin, an inhibitor of sarcoplasmic Ca2+ ATPase, induced a phasic increase in the ratio due to Ca2+ leak from intracellular store sites, but not the sustained increase, thereby suggesting the absence of Ca2+ release-activated Ca2+ entry (CRAC) mechanism in PASMC. When PASMC were exposed to superoxide anion by the pretreatment with xanthine and xanthine oxidase (X/XO) for 30 min, sustained component of ATP-induced Ca2+ transient was elevated. The ratios at 5 and 10 min after ATP application (delta ratio5 min and delta ratio10 min) were increased from 0.091 +/- 0.022 to 0.149 +/- 0.048 (P < 0.05) and from 0.021 +/- 0.020 to 0.117 +/- 0.047 (P < 0.01), respectively. But, thapsigargin-induced [Ca2+]i transient was not affected by X/XO. CONCLUSION: Superoxide anion makes ATP-induced Ca2+ transient sluggish, and does not affect Ca2+ leak pathway in PASMC.     

Amiodarone-mediated increase in intracellular free Ca2+ associated with cellular injury to human pulmonary artery endothelial cells

The cardiac antidysrrhythmic drug amiodarone can give rise to potentially fatal pulmonary toxicity in large numbers of patients. The effect of amiodarone on Ca2+ homeostasis and cell injury has been studied using human pulmonary artery endothelial (HPAE) cells in vitro. Amiodarone produced a concentration-dependent increase in intracellular free Ca2+ concentration ( [Ca2+]i) to micromolar levels that are similar to those seen with physiological stimuli that increase [Ca2+]i. Unlike physiological stimuli, the rise in [Ca2+]i produced by amiodarone developed slowly and was maintained over at least 30 min. Omitting Ca2+ from the external medium reversibly prevented the amiodarone-induced rise in [Ca2+]i. Amiodarone treatment increased the apparent first order rate constants for 45Ca2+ influx and efflux in intact HPAE cells. 45Ca2+ accumulation into the endoplasmic reticulum of saponin-permeabilized HPAE cells was decreased by amiodarone treatment. The release of 45Ca2+ from the endoplasmic reticulum stores by the putative intracellular second messengers inositol-1,4,5-trisphosphate, arachidonic acid, and Ca2+ was blocked by amiodarone treatment. The changes in Ca2+ homeostasis coincide with an increase in [3H]deoxyglucose release as a measure of early cell injury by amiodarone. It is concluded that amiodarone can produce an increase in [Ca2+]i by an action on the plasma membrane that allows the influx of external Ca2+. This increase in [Ca2+]i, together with other changes in Ca2+ homeostasis, may be responsible for the early cell injury associated with amiodarone toxicity.