Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells

Summary Regulatory effects of extracellular magnesium ions ([Mg²⁺]_o) on intracellular free ionized magnesium ([Mg²⁺]_i) were exmained in cultured vascular smooth muscle cells (VSMCs) fromrat aorta by digital imaging microscopy using the Mg²⁺ fluorescent probe, Mag-fura-2. With normal Mg²⁺(1.2 mM)-containing incubation media, [Mg²⁺]_i in VSMCs was 0.63±0.09 mM. The ratio of [Mg²⁺]_i/[Mg²⁺]_o was 0.52±0.07. Elevation of [Mg²⁺]o up to 4.8 mM induced consistent increments in [Mg²⁺]_i (to a mean values of 1.63±0.08 mM) in 5 min and lowered the ratio of [Mg²⁺]_i/[Mg²⁺]_o to 0.34±0.02. Our data suggest that [Mg²⁺]_o can regulate [Mg²⁺]_i, which may be related to its effects on intracellular Ca²⁺ ([Ca²⁺]_i) and tone of VSMCs.     

慢性缺氧对大鼠肺动脉平滑肌细胞胞内钙的影响及其机制研究

目的:研究慢性缺氧对大鼠肺动脉平滑肌细胞(PASMCs)胞内钙浓度(［Ca²⁺］_i)的影响及L-型钙通道和胞内钙库的作用,为缺氧性肺动脉高压(HPH)发病机制的进一步研究提供理论依据。 方法:复制大鼠缺氧性肺动脉高压动物模型，利用Fura－2/AM钙离子成像方法测定PASMCs在不同钙离子浓度细胞外液及L-型钙通道阻滞剂nifedipine和IP3R钙通道抑制剂肝素干预前后 ［Ca²⁺］_i变化。 结果:(1)缺氧＋含钙外液组PASMCs ［Ca²⁺］_i 显著高于对照＋含钙外液组（P<0.05）。缺氧＋含钙外液组PASMCs ［Ca²⁺］_i显著高于缺氧＋无钙外液组（P<0.05）。(2)缺氧nifedipine组PASMCs［Ca²⁺］_i在加药前后无显著差异（P>0.05）。（3）缺氧未干预组与缺氧肝素组PASMCs ［Ca²⁺］_i无明显差异（P>0.05）。 结论:慢性缺氧可使PASMCs的［Ca²⁺］_i增加。慢性缺氧引起［Ca²⁺］_i增加可能与细胞外钙内流有关，L-型钙通道和IP3R钙通道在调节［Ca²⁺］_i的过程中可能不独立发挥作用。     

急性缺氧对大鼠远端肺静脉平滑肌细胞内钙浓度的影响

目的:研究急性缺氧对大鼠远端肺静脉平滑肌细胞(PVSMC)细胞内钙浓度([Ca2+]i)的影响及L型电压依赖性钙通道(VDCC)阻断剂硝苯地平的作用,为缺氧性肺动脉高压发病机制的进一步研究提供理论依据.方法:胶原酶消化法培养大鼠远端PVSMC,利用荧光显微镜和细胞内钙浓度检测系统观测急性缺氧(4％O2)、高钾(60 mmol/L KCl)溶液对PVSMC的[Ca2+]i影响及硝苯地平的干预作用.结果:对照组PVSMC的[Ca2+]i随时间变化维持基线水平;缺氧组PVSMC急性缺氧后,[Ca2+]i迅速升高并维持平台水平,△[Ca2+]i达82.83 nmol/L士23.03 nmol/L;硝苯地平干预组PVSMC予急性缺氧和5μmol/L硝苯地平干预后,[Ca2+]i升高幅度较小;高钾溶液孵育PVSMC后,[Ca2+]i迅速增高,5 μmol/L硝苯地平能完全阻断PVSMC的[Ca2+]i对高钾溶液的反应.结论:急性缺氧可使大鼠远端PVSMC的[Ca2+]i升高,其机制可能与激活PVSMC的VDCC和另外的非VDCC依赖的钙通道导致细胞外Ca2+内流有关.     

Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMP-elevating agents in cultured canine tracheal smooth muscle cells

The effects of increases in cellular adenosine 3′5′-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (−logEC₅₀) produced at concentrations of 7.0 ± 0.5 and 4.9 ± 0.4  respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca²⁺ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF₄ ⁻-induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF₄ ⁻- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca²⁺]_i increase or inhibit both responses independently. Received: 14 March 1996/Accepted: 10 April 1996     

Peroxynitrite induces apoptosis in canine cerebral vascular muscle cells: possible relation to neurodegenerative diseases and strokes

Considerable evidence is accumulating to suggest that in vivo formation of free radicals in the brain, such as peroxynitrite (ONOO⁻), and programmed cell death (i.e. apoptosis) play important roles in neurodegeneration and stroke. However, it is not known whether ONOO⁻ can induce apoptosis in cerebral vascular smooth muscle cells (CVSMCs). The present study was designed to determine whether or not canine CVSMCs undergo apoptosis following treatment with ONOO⁻. Direct exposure of canine CVSMCs to ONOO⁻ induced apoptosis in a concentration-dependent manner, as confirmed by means of fluorescence staining, TdT-mediated dUTP nick-end labeling and comet assays. Peroxynitrite treatment resulted in an elevation of [Ca²⁺]_i in the CVSMCs. Peroxynitrite-induced apoptosis may thus be brought about by activation of Ca²⁺-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis need to be further investigated, the present findings could be used to suggest that ONOO⁻ formation in the brain may play important roles in neurodegenerative processes and strokes via detrimental actions on cerebral microvessels and blood flow.     

尼可地尔降低血管平滑肌细胞内ATP诱导的游离钙浓度

目的：探讨尼可地尔对血管平滑肌细胞内游离钙（［Ｃａ２＋］ｉ）的影响及机理。方法：培养的兔主动脉平滑肌细胞加入Ｆｕｒａ－２ＡＭ２５μｍｏｌ／Ｌ,在３７℃下孵育５０ｍｉｎ,［Ｃａ２＋］ｉ用荧光分光光度计检测。结果：ＡＴＰ（０１ｍｍｏｌ／Ｌ）诱导的［Ｃａ２＋］ｉ峰相和持续相增加可被尼可地尔抑制,且呈剂量依赖性,尼可地尔（１０μｍｏｌ／Ｌ）的抑制作用可被优降糖（１０μｍｏｌ／Ｌ）完全阻断（峰相：５３０±３１ｖｓ５４４±４１ｎｍｏｌ／Ｌ,持续相：３７０±１９ｖｓ３８１±１１ｎｍｏｌ／Ｌ,Ｐ＞００５）;在无钙溶液中,先给尼可地尔能显著抑制ＡＴＰ诱导的［Ｃａ２＋］ｉ峰相增加。结论：尼可地尔抑制ＡＴＰ诱导的［Ｃａ２＋］ｉ增加,可能与减少细胞外钙内流及细胞内钙释放有关。     

急性低氧大鼠肺动脉平滑肌内质网钙信号的变化及意义

目的：观察急性低氧大鼠肺动脉平滑肌内质网钙信号的变化及意义。方法:细胞水平：钙荧光探针(Fura-2/AM)负载大鼠肺动脉平滑肌细胞（PASMCs），荧光分光光度法，细胞外液无Ca2+及含Ca2+，在常氧（37 ℃、5% CO2、21 %O2、74 %N2 ）和急性低氧(37 ℃、5% CO2、2% O2、93 %N2)时，检测理阿诺碱（RD）和环匹阿尼酸（CPA）等对细胞浆内游离钙离子浓度（［Ca2+］i）的影响；离体血管环水平：相同条件，离体血管灌流方法检测肺动脉环张力变化。结果:(1)急性低氧时［Ca2+］i升高：常氧组［Ca2+］i为（96.99±7.16） nmol/L，低氧组为(257.06±32.48) nmol/L (P<0.01)。(2)与低氧组比较，预先用RD或普鲁卡因(procain)抑制内质网理阿诺碱受体敏感钙库，随后再给予低氧刺激时［Ca2+］i不升高，为（100.91±11.21） nmol/L (P<0.01)；而用CPA或thapsigargin（TG）抑制内质网摄取Ca2+，再给低氧刺激时［Ca2+］i呈升高状态(P>0.05)，而在细胞外液含钙及低氧下CPA及TG引起［Ca2+］i进一步升高(P<0.05)。(3)低氧引起肺动脉环收缩：常氧对基础张力无影响，低氧引起肺动脉环收缩，最大收缩张力达（49.28±8.64） g/g,P<0.01。(4)与低氧组比较，预先用RD或procain抑制内质网理阿诺碱受体敏感钙库，再给予低氧刺激，肺动脉环不收缩，最大收缩张力（3.75±1.14） g/g, P<0.01；而用CPA或TG后，再给予低氧刺激，肺动脉环呈收缩状态(P>0.05)，而在细胞外液含钙及低氧下CPA及TG引起肺动脉环进一步收缩(P<0.05)。结论:急性低氧可以引起内质网释放Ca2+，至少来自理阿诺碱受体敏感钙库的Ca2+释放参与了低氧肺血管收缩的发病机制；这可能是PASMCs自身具有的，既不依赖细胞外Ca2+内流，也不依赖血管内皮。     

天麻钩藤饮对SHR血清Ca2+浓度及血管平滑肌细胞钙通道的影响

目的：探讨天麻钩藤饮对自发性高血压大鼠(SHR)的血管平滑肌细胞钙通道电生理特征的影响，以期进一步阐明天麻钩藤饮对血压干预的作用机制。方法:选用12周龄自发性高血压雄性大鼠45只，随机分为5组，即天麻钩藤饮组（A组）、天麻钩藤饮去石决明组（B组）、硝苯地平组（C组）、石决明组（D组）、生理盐水对照组（E组）。治疗4周后，测定血清游离钙浓度；用全细胞模式膜片钳技术记录分析血管平滑肌细胞L型电压依赖性钙通道的特性。结果:用药前后天麻钩藤饮组和硝苯地平组血清游离钙浓度改变没有统计学意义(P>0.05)，天麻钩藤饮去石决明组、石决明组、生理盐水组血清游离钙浓度低于用药前 (P<0.05) ，以生理盐水组最明显(P<0.01)。天麻钩藤饮组、硝苯地平组有明显减少血管平滑肌细胞ICa-L内流的作用，石决明组作用较弱，天麻钩藤饮去石决明组、生理盐水组没有减少血管平滑肌细胞ICa-L内流的作用。结论:天麻钩藤饮可以提高血清游离钙离子浓度。天麻钩藤饮有明显的阻滞血管平滑肌细胞L型钙离子通道的作用，这可能是其降压作用机制之一。     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Proliferative effects of eosinophil lysates on cultured human airway smooth muscle cells

BACKGROUND: The hypertrophy/hyperplasia of airway smooth muscle (ASM) cells is one of the characteristic features of bronchial asthma. This structural change leads to the thickening of airway walls resulting in the amplification of airway narrowing. However, the pathogenesis of this structural change has not yet been determined. Eosinophils, which play a pathogenic role in asthma, have been demonstrated to have proliferative effects on fibroblasts and vascular smooth muscle cells. OBJECTIVE: We attempted to investigate the potential of eosinophils to induce the proliferation of ASM cells. METHODS: We examined the effect of lysates of eosinophils purified from peripheral blood of healthy donors on cultured human ASM cell proliferation. RESULTS: Eosinophil lysates significantly induced ASM cell proliferation in time- and dose-dependent manners, reaching a maximum on day 6 at 50% of eosinophil lysates (6.0 +/- 0.7 x 104 [mean +/- SD] /well, n = 5 vs. 4.5 +/- 1.1 x 104/well, n = 5; P < 0.05). This proliferative activity was heat-sensitive and recovered in the soluble fraction of the eosinophil lysates. Furthermore, the molecular weight of the mitogenic activity in the soluble fraction was identified as lower than 10 kDa. The inhibitory activity to ASM cell proliferation was also found in the insoluble fraction of the lysates. CONCLUSION: These results indicate that circulating eosinophils store mitogenic activity for ASM cells, suggesting that eosinophils might contribute to the development of the hyperplasia of ASM cells in asthmatics through the release of the stored mitogenic activity upon stimulation at the site of inflammation.     

Smad通路参与ERK通路诱导血管平滑肌细胞增殖的过程

目的： 探讨Smad通路是否参与细胞外信号调节激酶(ERK)通路诱导血管平滑肌细胞（VSMCs）增殖的过程及其可能机制。方法：将人脐动脉平滑肌细胞（hUASMCs）分为对照组、血小板源性生长因子（PDGF）组、ERK阻断剂组和PDGF+ERK阻断剂组。用MTT法测hUASMCs的增殖活性(A值)，用免疫组化法测hUASMCs内细胞核增殖抗原（PCNA）、磷酸化ERK和磷酸化Smad蛋白的表达，用RT-PCR法测hUASMCs内Smad2/3 mRNA的表达。结果：PDGF组hUASMCs的增殖活性(A值)及hUASMCs内的PCNA、磷酸化ERK和磷酸化Smad2/3蛋白的表达都明显高于其它各组(P<0.01)；各组hUASMCs内Smad2/3 mRNA的表达没有差异。结论： Smad通路可在蛋白水平参与ERK通路诱导VSMCs的增殖过程。     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

利用激光扫描共聚焦显微镜实时监测体外诱导分化的人骨髓间充质干细胞内游离Ca2+浓度动态变化

目的 利用激光扫描共聚焦显微镜(LSCM)实时监测由人骨髓间充质干细胞(hMSC)诱导分化的心肌样细胞内游离Ca2+浓度的动态变化.方法 体外分离培养hMSC,5-氮胞苷诱导向心肌样细胞分化.分别以体外分离培养的原代搏动的出生2 d SD大鼠乳鼠心肌细胞(CM)和同代次的hMSC为阳性对照和阴性对照.Fluo-3/AM荧光探针分别标记hMSC、由hMSC诱导的心肌样细胞及CM,利用LSCM实时监测单细胞内Ca2+动态变化的优势,观察加入心脏正性肌力药物后的细胞反应及细胞内Ca2+水平的动态变化.结果 静息状态下(给药前),搏动的CM荧光强度有变化,而hMSC及心肌样细胞Ca2+的荧光强度保持在稳定的波动水平内,两者差异无统计学意义.给药后,hMSC的Ca2+离子流没有改变,与静息状态下趋于一致;而心肌样细胞则表现出一个明显的Ca2+离子流,且下降后细胞内Ca2+水平明显高于加药前静息钙水平,其Ca2+离子流的表现趋势与搏动的CM基本一致.比较给药后hMSC与心肌样细胞的Ca2+荧光强度落差值,差异具有统计学意义(64.04 ±22.68比180.75 ±34.04,P<0.01).结论 hMSC经5-氮胞苷诱导后,在电学特性方面具有向CM分化的趋势,心肌样细胞与CM具有某些相同的电生理特性.     

补体膜攻击复合物刺激血管内皮细胞胞浆游离钙离子浓度的变化

目的 在单个细胞水平，观测亚溶破补体膜攻击复合物（MAC）诱导的血管内皮细胞[Ca^2 ]i的动态变化。方法 将原代培养的人脐静脉内皮细胞，以Fluo-4/AM(1-6μmol/L）和0.02%PluronicF-127进行细胞钙荧光指示剂负载后，于0℃组装亚溶破剂量的MAC，在37℃条件下，以LSCM实时监测MAC沉积引起的胞浆钙荧光强度的变化。结果 MAC沉积后，多数细胞的钙荧光强度迅速增强，以后随时间的推移，升高的钙荧光逐渐下降，直至恢复至静息水平，定量分析选定的胞浆区域钙荧光强度的变化发现，不同细胞在[Ca^2 ]i的变化高度，持续时间和胞内钙振荡 的特征等方面存在异质性。结论 MAC沉积到内皮细胞表面后，可诱导胞浆游离钙离子的浓度增高，且不同细胞[Ca^2 ]i的变化存在明显的异质性。     

The inhibitory action of caffeine on calcium currents in isolated intestinal smooth muscle cells

The patch-clamp method has been used to investigate the action of caffeine on the calcium current (I _Ca) in single isolated smooth muscle cells of the guineapig ileum. Caffeine (10 mM) substantially inhibited I _Ca. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K⁺ in the pipette solution, application of caffeine evoked a transient Ca-dependent K⁺ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased I _Ca Ryanodine had only little effect on I _Ca, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of I _Ca. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the I _Ca inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of I _Ca inhibition was probably due to a direct blocking action of caffeine on Ca channels.     

Effects of Iptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective:To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1(ET-1) in vitro. Methods:A cell culture model, [3H]-thymidine([3H]-TdR) incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration([Ca2+]i) of rabbit PASMC induced by ET-1 in vitro. Results:The value of [3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10-7mol/L,10-6 mol/L,10-5 mol/L lowered [3H]-TdR incorporation by (19.8±4.6)%, (41.2±9.5)%, (54.7±10.1)%, respectively, compared with the value of the cells treated with ET-1(P＜0.01); The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70±10.12 to 143.84±28.23, significantly higher than that in control group(P＜0.01); whereas with Iptakalim,the fluorescence intensity(FI) was only increased from 74.30±10.20 to 86.03±9.82, significantly lower than that in ET-1 group(P＜0.01). Conclusion:Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.     

Effects of endurance training on intracellular calcium concentration in T lymphocytes

The purpose of the present study was to determine whether 12 months of endurance training reduced [Ca²⁺]i in T helper (CD4⁺) lymphocytes in trained (TR) men compared to untrained (UT). Fourteen trained (Ironman triathletes) and nine untrained (sedentary) men volunteered for the study. The TR group averaged 12 km of swimming, 300 km of cycling and 60 km of running per week during the year. Resting blood samples were taken from TR (VO_2peak 64 ± 2 ml kg⁻¹ min⁻¹) and UT (VO_2peak 42 ± 2 ml kg⁻¹ min⁻¹) subjects every 4 weeks for 52 weeks (October 1, 1999–October 1, 2000). Leukocyte concentration was measured using a full blood count. Unstimulated CD4⁺ lymphocytes were separated and analysed for changes in free ([Ca²⁺]i) and total ([Ca²⁺]t) calcium using flow cytometry. There were no significant differences in leukocyte concentration between UT and TR groups. There were significant differences between TR and UT in [Ca²⁺]i (October B and November), and [Ca²⁺]t (January and March). There were also significant sequential monthly changes in both [Ca²⁺]i and [Ca²⁺]t for TR and UT groups during the study. Significant increases in [Ca²⁺]i and [Ca²⁺]t during summer (January and March) for both TR and UT groups suggest an increase in intracellular signalling during hot weather. [Ca²⁺]i and [Ca²⁺]t were significantly lower in TR lymphocytes during November and March, suggesting that endurance training during warmer months may decrease [Ca²⁺]i through altered intracellular signalling, possibly to maintain lymphocyte function during heat stress.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.     

Characterization of voltage‐gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery

The aim of the present study was to characterize voltage‐gated Ca²⁺ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L^–1 external Ca²⁺. Calcium currents were identified on the basis of their voltage dependencies and sensitivity to nifedipine, Ni²⁺ and cinnarizine. In the majority of the cells studied, T‐ and L‐type currents were observed, while the remaining cells showed predominantly L‐type currents. In the latter group of cells, holding potential change from –50 to either –70 or –90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L‐type Ca²⁺ channels showed half‐maximal inactivation at –38 mV. A Ca²⁺‐dependent inactivation was also evident. Nifedipine (3 μmol L^–1) blocked L‐type but not T‐type Ca²⁺ currents. Ni²⁺ (50 μmol L^–1) as well as cinnarizine (1 μmol L^–1) suppressed the nifedipine‐resistant, T‐type component of the currents. At higher concentrations, both Ni²⁺ (0.3–1 mmol L^–1) and cinnarizine (10 μmol L^–1) blocked the net inward current. Replacement of Ca²⁺ with 10 mmol L^–1 Ba²⁺ significantly increased the amplitude of L‐type Ca²⁺ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L‐ and T‐type and the other only L‐type Ca²⁺ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited T‐type currents at low concentrations (1 μmol L^–1) but also blocked L‐type Ca²⁺ currents at higher concentrations (10 μmol L^–1).