NO诱导血管平滑肌细胞凋亡中Ca^2＋的作用

探讨了NO诱导血管平滑肌细胞凋亡与细胞内游离Ca^2 之间的关系,通过粘附式细胞仪和Ca^2 荧光探针Fluo-3/AM,检测分析了NO在供体SNAP的作用下,血管平滑肌细胞中游离Ca^2 浓度的变化;又通过SNAP与维拉帕米、EGTA、肝素钠、普鲁卡因共同孵育的方法,测了Ca^2 浓度变化在细胞凋亡中的作用,得出SNAP能使细胞中游离Ca^2 浓度升高,而胞外Ca^2 内流在其中起主要作用;并且阻断胞外Ca^2 内流能够抑制SNAP所诱导的血管平滑肌细胞的凋亡,提示了胞内Ca^2 浓度升高可能是SNAP诱导血管平滑机细胞凋亡的一条途径。     

小鼠卵孤雌激活及激活过程中细胞内游离Ca^2＋的变化

小鼠ＭⅡ期卵母细胞经８％乙醇或电刺激后在体外可孤雌发育至囊胚。利用Ｃａ２＋荧光探针ｆｕｒａ－２测定人工激活小鼠卵过程中胞内游离Ｃａ２＋浓度的动态变化结果表明，乙醇和电刺激激活卵时均诱导胞质游离Ｃａ２＋浓度较大幅度升高；而未被激活的卵胞质Ｃａ２＋浓度维持稳定。表明胞质游离Ｃａ２＋浓度升高可能是卵激活的启动信号。在无Ｃａ２＋或用ＥＧＴＡ螯合细胞外Ｃａ２＋的条件下电刺激不能诱导卵内游离Ｃａ２＋升高，而乙醇却仍可引起卵内游离Ｃａ２＋较小幅度升高。这表明，电刺激必导的卵内游离Ｃａ２＋升高主要来源于细胞外Ｃａ２＋内流。乙醇则可诱发细胞内钙库Ｃａ２＋释放。     

黄精凝集素Ⅱ对大鼠主动脉Ca^2＋跨膜流动的影响

用４５Ｃａ跨膜流动技术测定黄精凝集素Ⅱ对大鼠主动脉Ｃａ２＋跨膜内流的影响．黄精凝集素Ⅱ在０．１～１０μｍｏｌＬ－１时,不影响静息状态下Ｃａ２＋的内流,但能抑制ＮＥ和ＫＣｌ引起的Ｃａ２＋内流,其抑制能力依赖于凝集素的浓度;在１０μｍｏｌＬ－１时,可完全阻滞ＮＥ引起的Ｃａ２＋增加,并能有效地阻滞ＫＣｌ引起的内流作用．表明黄精凝集素Ⅱ能阻滞细胞外的Ｃａ２＋通过受体操纵钙通道（ＲＯＣ）和电压依赖钙通道（ＰＤＣ）的内流     

Ebselen 抑制 ONOO~- 对神经元[Ca~(2+)]_i 的影响

Ｆｕｒａ－２显微荧光测钙技术研究发现,过氧亚硝基阴离子（ＯＮＯＯ－）作用于ＭＮ９Ｄ细胞,数ｓ内即可导致其胞内游离钙离子浓度（［Ｃａ２＋］ｉ）的急剧升高．胞外液换为无钙液或向胞外液中加入硝苯吡啶（Ｎｉｆｅｄｉｐ－ｉｎｅ）、二硫苏糖醇（ＤＴＴ）均可抑制ＯＮＯＯ－对［Ｃａ２＋］ｉ的影响,提示Ｌ－型钙通道的激活是ＯＮＯＯ－引起［Ｃａ２＋］ｉ升高的主要原因,ＯＮＯＯ－的这种作用可能与其氧化特性有关．Ｅｂｓｅｌｅｎ（２－苯基－１,２－苯并异硒唑－３（２Ｈ）酮）明显抑制ＯＮＯＯ－对［Ｃａ２＋］ｉ的影响,并且存在一定的剂量效应关系．     

^4^5Ca跨膜流动测定技术用于中药钙拮抗作用的研究

应用４５Ｃａ跨膜流动测定研究的钙拮抗剂ｖｅｒａｐａｍｉｌ，中药西红花（Ｃｒｏｃｕｓ ＳａｔｉｖｕｓＬ．），“地奥心血康”（ＤＡＸＸＫ），川红花及银杏叶等对大鼠主动脉４５Ｃａ跨膜内流的作用，以确定其对细胞膜Ｃａ＾２＾＋通道影响。实验结果表明：西红花，川红花，ＤＡＸＸＫ对平滑肌细胞膜上电压依赖Ｃａ＾２＾＋通道（ＰＤＣ）和受体操纵Ｃａ＾２＾＋通道（ＲＯＣ）都有阻滞作用，并以西红花的阻滞作用最强；而银杏叶     

重芳烃石油磺酸盐与M~(2+)和地层砂的沉积与吸附

利用分光光度法和容量法分别考察了重芳烃石油磺酸盐（简称ＨＡＰＳ）与Ｃａ２＋、Ｍｇ２＋的沉积规律及在地层砂上的吸附规律．实验表明：当固定Ｃａ２＋、Ｍｇ２＋混合液浓度改变ＨＡＰＳ浓度时，ＨＡＰＳ与Ｃａ２＋、Ｍｇ２＋的作用呈现出沉淀——溶解——再沉淀的普遍规律，且沉淀量随着ＨＡＰＳ和Ｃａ２＋、Ｍｇ２＋混合液浓度的增加而增加；ＨＡＰＳ在地层砂上的吸附等温线呈“Ｓ”型，表现出多层吸附的特征．     

￣（45）Ca跨膜流动测定技术用于中药钙拮抗作用的研究

应用￣（４５）Ｃａ跨膜流动测定技术研究钙拮抗剂ｖｅｒａｐａｍｉｌ、中药西红花（ＣｒｏｃｕｓＳａｔｉｖｕｓＬ．）、“地奥心血康”（ＤＡＸＸＫ）、川红花（ＣａｒｔｈａｍｕｓｔｉｎｃｔｏｒｉｕｓＬ．）及银杏叶（ＧｉｎｋｇｏｂｉｌｏｂａＬ．）等对大鼠主动脉￣（４５）Ｃａ跨膜内流的作用，以确定其对细胞膜Ｃａ￣（２＋）通道影响。实验结果表明：西红花、川红花、ＤＡＸＸＫ对平滑肌细胞膜上电压依赖Ｃａ￣（２＋）通道（ＰＤＣ）和受体操纵Ｃａ￣（２＋）通道（ＲＯＣ）都有阻滞作用，并以西红花的阻滞作用最强；而银杏叶对细胞膜Ｃａ￣（２＋）通道无阻滞作用。     

THE CURRENT DENSITY DEPENDENCE OF ACTIVATION ENERGY IN HIGH－Tc SUPERCONDUCTOR HgBa_2Ca_2Cu_3O_（8＋x） IN LOW CURRENT DENSITY

ＴＨＥＣＵＲＲＥＮＴＤＥＮＳＩＴＹＤＥＰＥＮＤＥＮＣＥＯＦＡＣＴＩＶＡＴＩＯＮＥＮＥＲＧＹＩＮＨＩＧＨ－ＴｃＳＵＰＥＲＣＯＮＤＵＣＴＯＲＨｇＢａ_２Ｃａ_２Ｃｕ_３Ｏ_（８＋ｘ）ＩＮＬＯＷＣＵＲＲＥＮＴＤＥＮＳＩＴＹＴＨＥＣＵＲＲＥＮＴＤＥＮＳＩＴＹＤＥ...     

当归注射液对兔主动脉平滑肌细胞中增殖细胞核抗原表达的影响

目的：观察当归注射液对平滑肌细胞（ Ｓ Ｍ Ｃ） 中增殖细胞核抗原（ Ｐ Ｃ Ｎ Ａ） 表达的影响。方法：用免疫组织化学 Ｌ Ｓ Ａ Ｂ 法检测培养的兔主动脉 Ｓ Ｍ Ｃ 中 Ｐ Ｃ Ｎ Ａ 的表达,同时测定培养液中超氧化物歧化酶（ Ｓ Ｏ Ｄ） 、脂质过氧化物（ Ｌ Ｐ Ｏ） 、前列环素（ Ｐ Ｇ Ｉ２） 及环磷酸腺苷（ｃ Ａ Ｍ Ｐ） 的含量。结果：当归注射液能增加 Ｓ Ｏ Ｄ 活性,降低 Ｌ Ｐ Ｏ 和升高 Ｐ Ｇ Ｉ２ 、ｃ Ａ Ｍ Ｐ 水平,抑制 Ｓ Ｍ Ｃ 中 Ｐ Ｃ Ｎ Ａ 的表达（ Ｐ＜ ００５ ～００１） 。结论：当归有抑制 Ｓ Ｍ Ｃ 增殖的作用。     

当归注射液对兔主动脉平滑肌细胞中增殖细胞核抗？…

目的：观察当归注射液地平滑肌细胞（ＳＭＣ）中增殖细胞核抗原（ＰＣＮＡ）表达的影响。方法：Ｅ和免疫组织化学ＬＳＡＢ法检测培养的兔主动脉ＳＭＣ中ＰＣＮＡ的表达,同时测定培养液中超氧化物歧化酶（ＳＯＤ）、脂质过氧化物（ＬＰＯ）、前列环素（ＰＧＩ２）及环磷酸腺苷的含量。结果：当归注射液能增加ＳＯＤ活性,降低ＬＰＯ和升高ＰＧＩ２、ＣＡＭＰ水平,抑制ＳＭＣ中ＰＣＮＡ的表达。结论：当归有抑制ＳＭＣ增殖的作用。     

血管段孵育和原代血管平滑肌细胞培养的比较

探讨微血管段孵育方法能否与原代平滑肌细胞培养一样检测蛋白表达,以用于初步的基础研究,解决原代培养耗时、不易存活的问题。分离大鼠肠系膜动脉三级以下分支,使用胶原酶和木瓜蛋白酶混合消化单个血管平滑肌细胞,获取的平滑肌细胞采用含20%胎牛血清的DMEM培养基进行培养。培养的平滑肌细胞经特异性的α-actin进行免疫组化鉴定;血管段孵育是在无菌条件下分离大鼠肠系膜动脉三级以下分支,培养基孵育72 h。分别使用不同浓度的尼氟灭酸(niflumic acid,NFA)孵育原代培养的平滑肌细胞和肠系膜三级分支血管段24 h,观察平滑肌细胞连接蛋白43(connexin43,Cx43)的变化。形态学和免疫组织化学鉴定表明,培养的原代细胞为血管平滑肌细胞。不同浓度NFA处理原代平滑肌细胞能够使Cx43表达量呈浓度依赖性下降,相对于对照组具有统计学意义(P0.01);不同浓度NFA处理血管段能够使Cx43的表达量也呈浓度依赖性下降,相对于对照组具有统计学意义(P0.01)。由此可知,观察平滑肌细胞特异性蛋白Cx43的表达情况,使用血管段孵育的方法检测结果与细胞培养的结果相似。血管段孵育简单易行,可以作为类似实验的初步研究。     

Proposed mechanism of cholinergic action in smooth muscle

An increased turnover of phosphatidate and phosphatidyl inositol has been found in many tissues where hormones or neurotransmitters are postulated to raise Ca2+ influx, for example in smooth muscle. However, the relationship between changes in phospholipid metabolism and changes in Ca2+ permeability was unknown. Following recent reports on the interactions of Ca2+ with phosphatidic acid in membranes and artificial systems, we investigated the hypothesis that phosphatidate accumulation mediates the action of cholinergic and other stimuli on Ca2+ influx. We report here that synthesis and accumulation of phosphatidate was accelerated in smooth muscle cells stimulated by carbamylcholine with a similar time course to that of contraction. This alteration in phosphatidate metabolism does not seem to result from an increase in intracellular Ca2+ or depolarisation of the cell membrane. Furthermore, submicromolar concentrations of phosphatidate rapidly produce contractions of isolated smooth muscle cells. These results support the contention that cholinergic-induced changes in membrane Ca2+ permeability in smooth muscle could be mediated by phosphatidate accumulation.     

2-APB对Wistar大鼠脑微动脉平滑肌细胞间缝隙连接的抑制作用

为探讨2-APB对Wistar大鼠脑微动脉平滑肌细胞间缝隙连接的作用。采用全细胞膜片钳技术,观察2-APB对Wistar大鼠脑微动脉段上平滑肌细胞膜电容(Cinput)、膜电导(Ginput)和膜电阻(Rinput)的影响。结果显示,应用2-APB后Wistar大鼠脑动脉段平滑肌细胞的Cinput、Ginput减小或Rinput增大。当2-APB浓度≥100μmol/L时Wistar大鼠脑微动脉段上平滑肌细胞的Cinput、Ginput分别从81±18 pF和3.5±0.43 nS降至10.5±1.3pF(n=7,P0.01)和0.35±0.03nS(n=7,P0.01),这与单个平滑肌细胞十分接近。由此可知,2-APB可以浓度依赖性的抑制Wistar大鼠脑微动脉平滑肌细胞间缝隙连接。     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Glucose-induced Ca2+ signals in rat pancreatic β cells

Using microfluorometry to assay intracellular Ca2+ , the influences of varied factors on glucose induced Ca22+ signals, such as glucose-induced initial decline phase (GIDP), Ca2+ oscillation, and Ca2+ release from internal stores, were investigated in single rat pancreatic β cells. Glucose was able to evoke GIDP even at non-stimulus concentration (5 mol/L), which is insufficient to induce Ca2+ spikes. GIDP was dependent on neither membrane depo larization nor extraeellular Ca2+ . However, GIDP was inhibited by thapsigargin, indicating a dependence on Ca2+ up take by Ca22+ stores. The glucose-induced calcium oscillation was inhibited when external Ca2+ was removed. However, thapsigargin could not block the Ca2+ oscillation. These results suggest that maintenance of Ca22+ oscillation requires ex tracellular Ca2+ but not Ca2+ stores. Glucose was able to evoke Ca2+ signals even in the absence of external Ca2+ . The glucose-induced Ca2+ release from intracellular Ca2+ stores was blocked by TTX. However, TTX had no effect on high K--induced Ca2+ store release, suggesting that membrane depolarization can directly release Ca2+ from some internal Ca2+ stores in β cells.     

Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Inhibition Mechanism of Emodin on Rabbit Vascular Smooth Muscle Cells Proliferation

ＩｎｔｒｏｄｕｃｔｉｏｎＥｍｏｄｉｎｉｓｏｎｅｏｆｔｈｅａｎｔｈｒａｑｕｉｎｏｎｅｃｏｍｐｏｕｎｄｓ[1] ,ｗｈｉｃｈｅｘｉｓｔｓｉｎｍａｎｙｍｅｄｉｃａｌｐｌａｎｔｓｓｕｃｈａｓｒａｄｉｘ ｐｏｌｙｇｏｎｉｍｕｌｔｉｆｌｏｒｉ,ｒｈｉｚｏｍａｐｏｌｙｇｏｎｉ,ｅｔｃ .Ｅｍｏｄｉｎｉｓａｎｉｍｐｏｒｔａｎｔｃｏｍｐｏｕｎｄｉｎｍｅｄｉｃｉｎｅ .Ｉｔｈａｓｍａｎｙｂｉｏｌｏｇｉｃａｌａｃｔｉｖｉｔｉｅｓｓｕｃｈａｓａｎｔｉ ｉｎｆｅｃｔｉｏｎ ,ｄｅｃｒｅａｓｉｎｇｂｌｏｏｄｌｉｐｉｄｓｃｏｎｔｅｎｔ ,ｅ…     

血管内皮细胞与平滑肌细胞间的信息传递

血管内皮细胞和血管平滑肌细胞之间存在着肌内皮间缝隙连接，进行电和化学的信息传递，以协调血管的舒缩活动。电信号可以从内皮细胞到平滑肌细胞进行传递，相反，也可以从平滑肌细胞到内皮细胞进行传递。内皮源性超极化因子、乙酰胆碱、缓激肽、第二信使等物质亦可引起内皮细胞或，和平滑肌细胞细胞膜的超极化或去极化，参与血管内皮细胞与平滑肌细胞间的信息传递。     

Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation

Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.