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。     

马齿苋总黄酮对缺血缺氧刺激下血管平滑肌细胞蛋白激酶C活性的影响

目的观测马齿苋总黄酮(PTF)对缺血缺氧刺激下血管平滑肌细胞钙超载及蛋白激酶C(PKC)的影响。方法用Fura-2/AM作Ca2+指示剂,检测PTF对正常培养及缺血缺氧作用下家兔主动脉血管平滑肌细胞Ca2+浓度及PKC的活性的改变。结果 PTF对正常培养家兔主动脉血管平滑肌细胞[Ca2+]i浓度无明显影响;PTF(8,16,32,64 mg·L-1)剂量依赖性抑制缺血缺氧缺氧致血管平滑肌细胞[Ca2+]i升高;PTF对正常培养家兔主动脉血管平滑肌细胞胞浆、胞膜PKC活性均升高;PTF处理后胞浆PKC活性下降,胞膜PKC活性上升。结论PTF可能抑制缺氧致血管平滑肌细胞钙超载,调节缺氧条件下血管平滑肌细胞PKC活性。     

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。     

小檗胺对ROCC介导的血管平滑肌细胞内游离钙的影响

目的　研究小檗胺 (BA)对受体调控性Ca2 +通道介导的家兔胸主动脉血管平滑肌细胞内游离钙 ([Ca2 +]i)的影响。方法　家兔主动脉血管平滑肌以Fluo 3/AM负载 ,通过激光扫描共聚焦显微镜 (LSCM )测定 [Ca2 +]i。结果　在细胞外Ca2 +存在的条件下 ,BA 30 μmol·L-1不影响静息[Ca2 +]i;但对去甲肾上腺素 (NE) 30mmol·L-1、5 羟色胺 (5 HT) 1μmol·L-1诱导的 [Ca2 +]i 升高有明显的抑制作用。在无胞外钙时 ,对咖啡因 40mmol·L-1诱导的 [Ca2 +]i 升高没有作用。结论　BA对ROCC激活后的外钙内流有明显的抑制作用 ,对内钙释放没有影响。其作用与维拉帕米相似     

尼氟灭酸对急性低氧人肺动脉收缩的影响

目的研究钙激活氯离子(ClCa)通道阻滞剂尼氟灭酸(NFA)对急性低氧人肺动脉收缩的影响。方法在常氧(37℃、5%CO2、21%O2、74%N2)和急性低氧(37℃、5%CO2、2%O2、93%N2)条件下,采用荧光分光光度计法、离体血管灌流法,观察ClCa阻滞剂NFA和indaryloxyacetic acid(IAA-94)对急性低氧人肺动脉平滑肌细胞质内游离钙离子浓度([Ca2+]i)及肺动脉张力的影响。结果①急性低氧引起[Ca2+]i升高,由常氧时的(103.26±4.72)nmol.L-1升高至(253.89±9.82)nmol.L-1(P<0.01);加入NFA、IAA-94使[Ca2+]i明显下降,最低至(107.18±14.99)nmol.L-1(P<0.01);②急性低氧引起肺动脉环收缩,由常氧时的(4.54±0.52)g.g-1至最大收缩张力(49.51±1.30)g.g-1(P<0.01);加入NFA和IAA-94后,它们均能抑制由低氧引起的血管收缩,使收缩张力最低降至(4.50±0.40)g.g-1(P<0.01)。结论 NFA能有效抑制急性低氧引起的肺动脉收缩,这一效果可能是通过抑制ClCa通道活性来实现的。     

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。     

P物质对大鼠垂体前叶细胞游离Ca~(2+)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。     

滨蒿内酯对原代和传代培养豚鼠气道平滑肌细胞内钙的影响

目的探讨滨蒿内酯(scoparone,Scop)对原代及短期传代培养的豚鼠气道平滑肌细胞(airway smooth musclecells,ASMCs)内钙的影响,同时,比较原代与传代ASMCs在形态、生长曲线及内钙释放受Scop及caffeine影响的异同。方法细胞计数法绘制原代及传代培养ASMCs的生长曲线,应用Fluo-3/AM为细胞内Ca2+示踪剂,通过倒置荧光纤维镜观察和记录原代及短期传代培养的ASMCs的细胞形态及其细胞内钙离子浓度([Ca2+]i)的改变。结果原代和传代培养ASMCs的倍增时间分别为(31.89±1.24)h和(22.91±6.82)h,传代培养ASMCs的倍增时间明显缩短(P<0.05),传代培养的ASMCs相对原代培养ASMCs体积增大。在细胞外液无钙条件下,不同浓度的Scop(10-6、10-5、10-4mol.L-1)可降低静息状态下培养的ASMCs的[Ca2+]i,并与给药浓度有关,原代与传代培养的ASMCs比较,对不同浓度的Scop的降钙反应无明显异同(P>0.05);不同浓度咖啡因(caffeine,10-4、10-3、10-2mol.L-1)在10-4mol.L-1Scop存在下,可升高ASMCs的[Ca2+]i,传代培养的ASMCs[Ca2+]i较原代对caffeine的反应下降(P<0.01)。结论Scop可降低培养的ASMCs的[Ca2+]i,并且不受细胞传代影响。短期传代培养的ASMCs相对于原代细胞,形态及内钙释放通道特性发生了改变。     

T型钙通道在心肌肥厚大鼠心肌细胞钙内流中的作用

目的研究T型钙通道在心肌细胞钙离子内流中的作用及其对心脏兴奋收缩耦联的可能影响。方法测定选择性T型钙通道阻滞剂米贝拉地尔对培养的SD乳大鼠心室肌细胞和二肾一夹心肌肥厚大鼠心室肌细胞[Ca2+]i的影响。结果血管紧张素Ⅱ(AngⅡ)刺激使乳大鼠心室肌舒张期细胞[Ca2+]i增高,收缩期细胞[Ca2+]i降低,[Ca2+]i上升和下降的时间延长。米贝拉地尔1.25～5μmol·L-1浓度依赖性降低AngⅡ引起的细胞[Ca2+]i变化。在心肌肥厚模型大鼠,咖啡因刺激后,[Ca2+]i增幅和最高[Ca2+]i明显降低。而米贝拉地尔25mg·kg-1·d-1(灌胃给药7～9周)组加入咖啡因刺激后细胞内[Ca2+]i增幅和最高[Ca2+]i明显增高。结论T型钙通道异常开放可以引起心肌细胞内钙超载。阻断T型钙通道,可能通过改善肌浆网摄取及释放钙的功能而抑制心肌细胞钙超载。     

哇巴因对血管内皮细胞ECV304细胞凋亡及胞内游离钙离子和活性氧浓度的影响

目的研究哇巴因(毒毛花苷G)对人脐静脉血管内皮细胞ECV304凋亡的诱导作用,并探讨其可能的作用机制。方法哇巴因0.01,0.05,0.1,0.5,1和10μmol·L-1与ECV304细胞作用24,48和72h,MTT法检测细胞存活率,Hoechst33342/碘化丙锭双荧光染色法和流式细胞仪检测细胞凋亡百分率,激光共聚焦显微镜观察细胞内游离Ca2+浓度([Ca2+]i)和活性氧(ROS)浓度,逆转录PCR和Western印迹法检测胱天蛋白酶3mRNA和蛋白表达。结果哇巴因在0.01～10μmol·L-1浓度范围内与ECV304细胞分别作用24,48和72h,对细胞存活的抑制率明显增加,且呈浓度和时间依赖性,24,48和72h浓度-效应相关系数分别为0.984,0.994和0.997(P<0.05);哇巴因作用24,48和72h的IC50值分别为0.624,0.184和0.041μmol·L-1,时间-效应相关系数为0.974(P<0.05)。哇巴因0.1μmol·L-1与ECV304细胞作用24h,细胞凋亡百分率由正常对照组的(4.2±0.5)%升高到(26.0±3.2)%,作用48h,细胞凋亡率由(4.7±0.5)%升高到(36.5±5.3)%,差异有统计学意义(n=3,P<0.01);同时细胞出现染色质凝集。哇巴因0.01,0.1和0.5μmol·L-1分别与ECV304细胞作用12,24和36h,[Ca2+]i和ROS浓度呈浓度和时间依赖性增加,在哇巴因0.5μmol·L-1时[Ca2+]i和ROS浓度的时间-效应相关系数分别为0.912和0.924,作用36h时[Ca2+]i和ROS浓度的浓度-效应相关系数分别为0.889和0.907(P<0.05)。逆转录PCR和Western印迹法分析显示,哇巴因0.1和0.5μmol·L-1作用ECV304细胞24h后,胱天蛋白酶3mRNA表达增加,差异有统计学意义(P<0.05);哇巴因0.01,0.1和0.5μmol·L-1作用ECV304细胞24h,胱天蛋白酶3蛋白表达明显增加,差异有统计学意义(P<0.05)。结论哇巴因可诱导人脐静脉血管内皮细胞ECV304凋亡,其机制可能与增加[Ca2+]i和ROS浓度及胱天蛋白酶3表达有关。     

POTENTIATION BY ALDOSTERONE OF VASOPRESSIN-INDUCED CALCIUM KINETICS IN VASCULAR SMOOTH MUSCLE CELLS

1. The present study was undertaken to examine the effect of aldosterone on arginine vasopressin (AVP)-induced Ca2+ kinetics in cultured rat vascular smooth muscle cells (VSMC). The pre-incubation of cells with 1 x 10(-6) mol/L aldosterone for 24 h did not affect the basal cytosolic free Ca2+ [( Ca2+]i) but enhanced the AVP-induced mobilization of [Ca2+]i (1 x 10(-8) mol/L AVP; 287 vs 401, 1 x 10(-6) mol/L AVP; 430 vs 714 nmol/L). 2. The pre-incubation of cells with 1 x 10(-7) mol/L aldosterone for 24 h did not show this enhancing effect on the AVP-induced mobilization of [Ca2+]i. Without the preincubation, aldosterone did not change the basal [Ca2+]i or the AVP-induced mobilization of [Ca2+]i. This enhancement was still observed in the Ca2+-free solution containing 0.1 mmol/L EGTA (1 x 10(-8) mol/L AVP; 169 vs 341 nmol/L). 3. The enhancement by aldosterone of the AVP-mobilized [Ca2+]i was completely blocked by the simultaneous administration of 1 x 10(-4) mol/L spironolactone (1 x 10(-8) mol/L AVP; 258 vs 265 nmol/L). The treatment with aldosterone also stimulated the AVP-produced [45Ca2+] efflux during a 3 min period (1 x 10(-8) mol/L AVP; 32 vs 49, 1 x 10(-6) mol/L AVP; 50 vs 58% released from the resting intracellular [45Ca2+]-contents). 4. The present results indicate that aldosterone enhances the vascular action of AVP mediated through the stimulation of Ca2+ kinetics which may be dependent on the changes in the cellular signal transduction systems.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.     

Resveratrol decreases calcium sensitivity of vascular smooth muscle and enhances cytosolic calcium increase in endothelium

Resveratrol causes endothelium dependent and independent relaxation of vascular smooth muscle. This study investigated the mechanisms behind the effect of resveratrol on vascular tone. Resveratrol (0.1 mM) inhibited KCl-stimulated contractions in endothelium-denuded rat aorta and this inhibition was not reversed by tetraethylammonium (TEA) (5 mM), glyburide (3 microM), ouabain (0.1 mM), thapsigargin (1 microM), or indomethacin (10 microM). KCl (90 mM) increased the intracellular free calcium concentration ([Ca2+]i) in the isolated smooth muscle cells from the rat aorta and resveratrol (0.1 mM) did not inhibit the KCl-stimulated [Ca2+]i increase. The CaCl2 (0.1-100 microM) stimulated contractions were inhibited by resveratrol (0.1 mM) in the Triton X-100 skinned smooth muscle of the aorta. In heart valve endothelium, resveratrol (0.1 mM) augmented the acetylcholine (10 microM) stimulated [Ca2+]i increase. Resveratrol-induced augmentation of the acetylcholine-stimulated [Ca2+]i elevation was reversed by glyburide (3 microM), but not by TEA (5 mM). The present study indicated that resveratrol affected vascular smooth muscle and endothelium in different ways. Resveratrol decreased the Ca2+ sensitivity but did not affect the KCl-stimulated [Ca2+]i increase in the vascular smooth muscle. In the endothelial cells, resveratrol enhanced the agonist-stimulated [Ca2+]i increase that might trigger nitric oxide synthesis from endothelial cells.     

Ca2+ localization and sensitivity in vascular smooth muscle

An increase in cytosolic Ca2+ level ([Ca2+]i) is a prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+]i and muscle tension give direct information on the Ca2+ regulation of smooth muscle. The photoprotein aequorin and the fluorescent Ca2+ indicator fura-2 are widely used for this purpose. Although there are some inconsistencies between the results obtained with these two indicators, comparison between [Ca2+]i and muscle tension in vascular smooth muscle indicates that stimulation of alpha-adrenoceptors increases, whereas stimulation of beta-adrenoceptors decreases, both the Ca2+ sensitivity of contractile elements and [Ca2+]i. Thus, as Hideaki Karaki explains, contractility of vascular smooth muscle may be regulated not only by [Ca2+]i but also by the Ca2+ sensitivity of the contractile elements.     

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。     

Effect of serum withdrawal on the contribution of L-type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.     

An increase in opening of BK(Ca) channels in smooth muscle cells in streptozotocin-induced diabetic mice

INTRODUCTION Diabetes mellitus (DM) is a kind of disease thatmetabolism decompensates with hyperglycemia and re-sults in multi-organ damage[1]. Thus, the risk of coro-nary disease, cerebrovascular disease, and other car-diovascular complications increase. These changes, atleastpartially, due todiabetes functionalchanges inbloodvessels including endothelial cell dysfunction. Simulta-neously, altered ion channelfunction in vascular smoothmuscle are also involved[2,3]. In vascular…     

Ca2+-images of smooth muscle cells and endothelial cells in one confocal plane in femoral artery segments of the rat.

Simultaneous recording of Ca2+-images in one confocal plane from vascular smooth muscle cells (SMCs) and endothelial cells (ECs) of an intact rat femoral artery segment was performed using indo-1 and a confocal microscope. During application of 10 microM acetylcholine (ACh), elevation and oscillation of intracellular Ca2+ concentration ([Ca2+]i) were observed in ECs but not in SMCs. Sequential conduction of Ca2+ oscillation from an EC to the neighboring ECs in one longitudinal direction was often observed in the presence of ACh. On the other hand, the activation of voltage-dependent Ca2+ channels by external 30 mM K+ resulted in the elevation of [Ca2+]i only in SMCs. When 10 microM ACh was added in the presence of 30 mM K+, it was observed in one confocal plane that [Ca2+]i in ECs and SMCs was almost simultaneously increased and decreased, respectively. The simultaneous recording method in this intact preparation will provide a line of valuable information about the interactions between SMCs and ECs, based on spatio-temporal analyses of absolute values of [Ca2+]i in individual cells.     

Magnesium modulates contractile responses of rat aorta to thiocyanate: A possible relationship to smoking-induced atherosclerosis.

Thiocyanate anions (SCN-) as the end products of tobacco smoke and found in the blood of cigarette smokers have been implicated in atherogenesis and heart diseases. Magnesium deficiency has also been implicated in the etiology of atherogenesis. The contractile responses of rat aorta to SCN- and the modulation of extracellular magnesium ions ([Mg2+]o) on the effect of SCN- were, therefore, studied in isolated rat aortic rings. SCN- exposure at a range of concentrations (from 10(-5) to 5 x 10(-2) M) induces contractile responses of isolated rat aortic rings with and without endothelium in a concentration-dependent manner. Significant differences in responsiveness to SCN- were found in rat aortic ring segments with and without endothelial cells. Preincubation of these vessels with low [Mg2+]o markedly shifted the contractile concentration-effect curves to the left, and the contractile effects of SCN- in rat aortic rings were potentiated. In contrast to lowering [Mg2+]o, increasing [Mg2+]o to 2.4 mM was found to dramatically attenuate the contractile responses to SCN-. In the absence of extracellular Ca2+ ([Ca2+]o), SCN--induced contractions were, however, almost abolished after exposure to Mg2+-free medium. In order to investigate the mechanisms of [Mg2+]o modulation of SCN--induced contractile response of rat aorta, changes in intracellular Ca2+ ([Ca2+]i) were measured in cultured primary smooth muscle cells isolated from rat aorta. The resting level of [Ca2+]i in the rat aortic smooth muscle cells was 80.6 +/- 6.6 nM. Exposure of these cells to SCN- (5 x 10(-5) to 5 x 10(-3) M) produced rises in [Ca2+]i in a concentration-dependent manner. Preincubation of these cells with low [Mg2+]o (0 or 0.3 mM, the lowest physiological range) for 24 h significantly potentiated increments in [Ca2+]i induced by SCN-. These rises in [Ca2+]i induced by SCN- were completely inhibited by pretreating the cells with 2.4 mM [Mg2+]o for 24 h. These results support a hypothesis whereby cigarette smoking or exposure to smoking can induce cardiovascular diseases, at least partly, probably by causing spasm and thickening of arterial blood vessels as a consequence of large rises in [Ca2+]i in vascular smooth muscle cells. The chronic presence of or exposure to both thiocyanate and low Mg2+ in the blood of smokers can result in rapid flux of Ca2+ into vascular smooth muscle cells, thus accelerating or initiating atherosclerotic processes in smokers.