兔不全梗阻性膀胱一氧化氮合酶神经和内皮素-1含量的改变及意义

目的　研究一氧化氮合酶 (NOS)神经、内皮素 (ET) - 1在兔不全梗阻性膀胱中的改变和意义。方法　运用 NADPH组织化学及放射免疫分析技术对 1 0只成年雄性新西兰白兔不全梗阻性膀胱及 1 0只同龄雄性新西兰白兔无梗阻性膀胱的兔血浆、尿液及膀胱平滑肌组织中 ET- 1和膀胱平滑肌中 NOS神经进行研究。结果　实验组兔膀胱体、膀胱颈部粘膜及膀胱体平滑肌 NOS神经均明显减少 (膀胱体粘膜为 P<0 .0 5,膀胱颈粘膜为 P<0 .0 1 ,膀胱体平滑肌为 P<0 .0 5) ;实验组较对照组血浆与尿液中 ET- 1含量均明显增高 (血浆 3 w组与血浆 6 w组均为 P<0 .0 5;尿液 3 w组与 6 w组均为 P<0 .0 0 1 ) ;实验组较对照组膀胱体部平滑肌组织 ET- 1含量明显升高 (P<0 .0 5)。结论　 NOS神经的减少和 ET- 1的上调对兔不全梗阻性膀胱的功能和结构变化具有一定作用 ,为梗阻性膀胱的病理生理变化提供了一个较为合理的解释。血浆、尿液中 ET- 1在兔不全梗阻性膀胱中的含量上调 ,并可作为膀胱出口梗阻诊断的重要参数。     

硫化氢对大鼠肝星状细胞增殖及Ca2+浓度的影响

目的 探讨硫化氢(H2S)对大鼠原代肝星状细胞(HsC)增殖及Ca2+浓度的影响及其作用机制.方法 大鼠肝星状原代细胞作为研究对象,将过氧化氢(H2O2)作用于大鼠原代HSC制造肝纤维化的氧化应激模型,用钙离子荧光探针Fluo-3/AM负载细胞,并在此基础上应用不同剂量的NaSH(H2S供体)和KATP通道抑制剂(格列本脲)对各组细胞进行干预,用激光扫描共聚焦显微镜(LSCM)和CCK-8的方法分别检测不同刺激条件对细胞内Ca2+浓度改变及细胞增殖情况的影响.结果 低浓度H2S(100μmo/L NaSH)明显降低HSC细胞内Ca2+浓度(P＜0.05),抑制细胞增殖;K离子通道阻断剂——格列本脲可阻断H2S的作用.高浓度H2S(1mmol/L NaSH)使HSC细胞内Ca2+浓度增加,促进细胞增殖.结论 低浓度H2S通过激活HSC细胞KATP通道,降低细胞内Ca2+浓度,从而抑制细胞增殖;高浓度H2S使HSC细胞内Ca2+浓度增加,促进细胞增殖.     

动脉粥样硬化兔主动脉平滑肌细胞钙激活钾通道变化及意义

目的　探讨动脉粥样硬化 (atherosclerosis,AS)兔主动脉平滑肌细胞钙激活钾 (Calcium activatedpotassium ,KCa)通道变化及意义。方法　 2 0只健康新西兰白兔随机分为AS组和对照组 ,每组 10只。用酶消化法获取单个主动脉平滑肌细胞 ,通过膜片钳记录技术检测AS兔主动脉平滑肌细胞KCa通道的活性 ,并在浴液中分别加入不同浓度的Ca2 + ,实时采样记录通道的开放概率 (Po)、平均开放时间 (To)、平均关闭时间 (Tc)和电流幅值 (Am) ,以加钙前作为对照组观察上述参数的变化。结果　AS组KCa通道的活性明显高于对照组。结论　AS血管平滑肌细胞KCa通道的活性明显增加 ,可能是AS血管发生代偿性扩张的机制之一。     

前列腺增生患者逼尿肌中碱性成纤维细胞生长因子的变化

目的探讨前列腺增生症患者膀胱逼尿肌中碱性成纤维细胞生长因子(bFGF)的表达及其意义。方法前列腺增生患者54例,分为逼尿肌稳定组(33例)和逼尿肌不稳定组(21例)；对照组15例为同期在我院因膀胱癌施行开放手术的男性患者。分别应用逆转录聚合酶链反应(RT-PCR)和免疫组化的方法检测各组逼尿肌中bFGFmRNA和bFGF蛋白的表达。结果稳定组和不稳定组bFGFmRNA的表达水平分别为0．5814±0．2362、0．6098±0．1829,明显高于对照组的0．3469±0．2986,差异有统计学意义(P＜0．05和0．01)；bFGF亦高于对照组。稳定组和不稳定组间差异无统计学意义(P＞0．05)结论前列腺增生患者致膀胱出口梗阻后膀胱逼尿肌细胞中bFGF表达水平升高,但与膀胱逼尿肌不稳定的发生无直接关系。     

糖尿病大鼠膀胱组织中NGF、P75的表达及意义

成年雌性Sprague-Dawley（SD）大鼠35只,随机分成2组,正常对照组15只,糖尿病模型组20只。应用链脲佐菌素诱导。注射STZ72小时后测尾静脉血葡萄浓度。BG≥16.7mmol/L为模型成功。造模成功8周后进行实验,实验前1天测定代谢笼24小时尿量。实验切取完整膀胱和称量膀胱湿重。苏木素-伊红后,光镜下观察糖尿病大鼠膀胱逼尿肌的形态学变化。采用免疫组织化学方法检测膀胱逼尿肌细胞中NGF及P75的表达情况。结果体重较正常对照组增长缓慢,为（248.05±15.52g）vs（322.80±15.92g）;24小时尿量模型组大于正常对照组,为（88.31±7.22ml）vs（20.93±2.90ml）;膀胱湿重测定重于正常对照组,为（171.15±6.08mg）vs（135.53±5.33mg）。HE染色后光镜下观察逼尿肌结构表现,正常对照组：移形上皮细胞排列整齐,固有膜完整,逼尿肌细胞大小形态均一,肌纤维排列有序,神经元细胞排列整齐。糖尿病模型组：移形上皮细胞排列紊乱,固有膜充血水肿,粘膜下层有明显的嗜酸细胞浸润,逼尿肌肌细胞肥大,形态多样,肌束排列紊乱松散,神经元细胞排列紊乱松散,细胞水肿。免疫组化结果显示：正常对照组大鼠膀胱壁NGF主要分布在逼尿肌细胞浆内,阳性表达率为86.67%,而糖尿病模型组大鼠膀胱壁NGF也主要分布在逼尿肌细胞浆内,阳性表达率为35%,两组间有显著差异。同样,p75在膀胱壁黏膜下、固有膜和逼尿肌细胞内均有分布,和正常组相比,糖尿病组表达明显减少,差异具有统计学意义。结论NGF及其受体p75在STZ诱导的Ⅰ型糖尿病大鼠的膀胱逼尿肌细胞内表达均明显减少,NGF、P75在DC的发生发展中发挥了重要作用。     

Na+/Ca2+交换体α-2(807-844)抗体的制备及其对大鼠心肌细胞Na+/Ca2+交换电流的抑制作用

目的 在制备和纯化抗心肌Na+/Ca2+交换体α-2(807- 844)肽段抗体的基础上,观察抗体对大鼠心肌细胞Na+/Ca2+交换电流及L型钙电流(ICa)、瞬时外向钾电流(Ito)和内向整流钾电流(Ik1)的影响.方法 利用人工合成的α-2(807-844)多肽免疫兔制备抗α-2(807- 844)抗体,抗血清经Protein A亲和柱纯化,利用全细胞膜片钳技术观察纯化后抗体对心肌细胞Na+/Ca2+交换电流(INa/Ca)、L型钙电流(ICa)、瞬时外向钾电流(Ito)和内向整流钾电流(Ik1)的影响.结果 经主动免疫,抗Na+/Ca2+交换体α- 2(840-877)抗血清效价达1∶243000,纯化后抗α-2(840- 877)抗体浓度为7.21 mg/mL.纯化后的抗体在SDS - PAGE时出现清晰的2条带,其分子量分别为25 kD和50 kD左右.在1 nmol/L～104 nmol/L浓度范围内,抗α-2抗体对成年大鼠心肌细胞外向和内向Na+/Ca2+变换电流均表现为剂量依赖性的抑制作用；对Ito和Ik1则均未见明显影响.此外,104 nmol/L该抗体对L型钙电流亦具有抑制作用.通过氨基酸序列比对发现,α- 2(807 - 844)肽段与L型钙通道第2结构域孔环(697～730位氨基酸)序列相似度为23.7％,可能是抗体对L型钙通道出现交叉反应的原因.结论 在1 nmol/L～103 nmol/L浓度范围内,抗心肌Na+/Ca2+变换体α-2(807- 844)抗体可特异性抑制大鼠心肌Na+/Ca2+变换活动；对L型钙通道电流、瞬时外向钾通道电流和内向整流钾通道电流均未见明显影响.     

ALA-PDT诱导SW480细胞凋亡和胞内Ca2+浓度变化的关系

目的:探讨ALA-PDT诱导人结肠癌细胞SW480凋亡和游离钙浓度变化关系.方法:将SW480细胞分为四组:空白对照组、激光照射照组、ALA组和ALA-PDT组,用DNA片段分析和TUNEL法检测细胞凋亡;用激光共聚焦显微镜观测各组细胞内游离钙离子浓度的变化.结果:ALA-PDT组的人结肠癌细胞在1、2h有大量的DNA片段,TUNEL法显示ALA-PDT组的人结肠癌细胞在PDT后30minAI为25.26±5.04%,PDT后60minAI为50.45%±7.85%,均高于其他3组(AI均<10%,P<0.01);激光共聚焦结果为ALA-PDT组细胞内游离钙离子浓度在20min达高峰(荧光强度:185.40±18.90),与10min(荧光强度:100.00±19.83)相比有显著差异(P<0.01),之后又逐渐下降.结论:细胞内Ca2 浓度的逐渐增加在PDT诱导的细胞凋亡过程中可能起着重要作用.     

横纹肌肌浆网Ca2+释放通道的调节

横纹肌细胞内Ca2+转运的中心环节是肌浆网Ca2+释放通道(RyR)。RyR是565 kDa蛋白分子组成的四聚体,其位于胞浆的部分可与离子、小分子和蛋白相结合,并有磷酸化、氧化与亚硝基化位点,跨肌浆网膜部分形成Ca2+通道。在心肌与骨骼肌,因离子、小分子与蛋白-蛋白的相互作用,以及蛋白修饰等调节机制的差别,使得经细胞膜L-型Ca2+通道介导的RyR开放机制并不相同,对RyR通道关闭的调节也不相同。但是,心肌与骨骼肌中的Calstabins在RyR的多个结构域上存在结合位点,从而通过影响RyR结构域的相互作用,便可调节其空间构象而改变RyR通道的功能。因此,基因突变改变RyR的空间构象可导致遗传性心肌病与肌病,药物JTV519则通过稳定RyR的空间构象而防治心律失常,对抗骨骼肌疲劳。     

心房颤动时心房肌电重构与Ca2+调控异常的研究进展

心房颤动 (AF)时心房肌电重构在 AF的发生和发展中起至关重要的作用 ,直接和间接证据表明频率相关的细胞内 Ca2 超负荷是电重构时心房肌电生理特性改变的始动因素 ,细胞内 Ca2 的平衡取决于细胞外 Ca2 通过 L 型 Ca2 通道进入细胞内和肌浆网对 Ca2 的释放、摄取和储存。有限的动物实验和临床资料表明 ,快速心房起搏和持续性 AF时 ,型 Ca2 通道和肌浆网 Ca2 调控蛋白信使核糖核酸 (m RNA)和蛋白质表达下调 ,是 AF电重构的分子生物学机制     

蜂胶诱导喉鳞癌Hep-2细胞凋亡及机制探讨

采用体外细胞培养实验方法,应用四甲基偶氮唑蓝法、流式细胞仪检测、共聚焦显微镜技术,观察蜂胶对喉鳞癌Hep-2细胞的抑制率、凋亡率的影响及细胞内Ca2 变化.细胞抑制率随用药时间的延长、剂量增加而增大.蜂胶能诱导喉鳞癌细胞凋亡,非特异性地阻滞Hep-2细胞周期的全过程.细胞内Ca2 随药物浓度的增加而升高(P<0.05).认为蜂胶有诱导Hep-2细胞凋亡的作用,其机制可能与细胞内Ca2 变化有关.     

Ca2+ channel subtypes and pharmacology in the kidney

A large body of evidence has accrued indicating that voltage-gated Ca(2+) channel subtypes, including L-, T-, N-, and P/Q-type, are present within renal vascular and tubular tissues, and the blockade of these Ca(2+) channels produces diverse actions on renal microcirculation. Because nifedipine acts exclusively on L-type Ca(2+) channels, the observation that nifedipine predominantly dilates afferent arterioles implicates intrarenal heterogeneity in the distribution of L-type Ca(2+) channels and suggests that it potentially causes glomerular hypertension. In contrast, recently developed Ca(2+) channel blockers (CCBs), including mibefradil and efonidipine, exert blocking action on L-type and T-type Ca(2+) channels and elicit vasodilation of afferent and efferent arterioles, which suggests the presence of T-type Ca(2+) channels in both arterioles and the distinct impact on intraglomerular pressure. Recently, aldosterone has been established as an aggravating factor in kidney disease, and T-type Ca(2+) channels mediate aldosterone release as well as its effect on renal efferent arteriolar tone. Furthermore, T-type CCBs are reported to exert inhibitory action on inflammatory process and renin secretion. Similarly, N-type Ca(2+) channels are present in nerve terminals, and the inhibition of neurotransmitter release by N-type CCBs (eg, cilnidipine) elicits dilation of afferent and efferent arterioles and reduces glomerular pressure. Collectively, the kidney is endowed with a variety of Ca(2+) channel subtypes, and the inhibition of these channels by their specific CCBs leads to variable impact on renal microcirculation. Furthermore, multifaceted activity of CCBs on T- and N-type Ca(2+) channels may offer additive benefits through nonhemodynamic mechanisms in the progression of chronic kidney disease.     

Functional dependence of Ca(2+)-activated K+ current on L- and N-type Ca2+ channels: differences between chicken sympathetic and parasympathetic neurons suggest different regulatory mechanisms.

The influx of Ca2+ ions controls many important processes in excitable cells, including the regulation of the gating of Ca(2+)-activated K+ channels (the current IK[Ca]). Various IK[Ca] channels contribute to the regulation of the action-potential waveform, the repetitive discharge of spikes, and the secretion of neurotransmitters. It is thought that large-conductance IK[Ca] channels must be closely colocalized with Ca2+ channels (ICa) to be gated by Ca2+ influx. We now report that IK[Ca] channels can be preferentially colocalized with pharmacologically distinct subtypes of voltage-activated Ca2+ channel and that this occurs differently in embryonic chicken sympathetic and parasympathetic neurons. The effects of various dihydropyridines and omega-conotoxin on voltage-activated Ca2+ currents (ICa) and Ca(2+)-activated K+ currents (IK[Ca]) were examined by using perforated-patch whole-cell recordings from embryonic chicken ciliary and sympathetic ganglion neurons. Application of nifedipine or omega-conotoxin each caused a 40-60% reduction in ICa, whereas application of S-(-)-BAY K 8644 potentiated ICa in ciliary ganglion neurons. But application of omega-conotoxin had little or no effect on IK[Ca], whereas nifedipine and S-(-)-BAY K 8644 inhibited and potentiated IK[Ca], respectively. These results indicate that IK[Ca] channels are preferentially coupled to L-type, but not to N-type, Ca2+ channels on chicken ciliary ganglion neurons. Chicken sympathetic neurons also express dihydropyridine-sensitive and omega-conotoxin-sensitive components of ICa. However, in those cells, application of omega-conotoxin caused a 40-60% reduction in IK[Ca], whereas nifedipine reduced IK[Ca] but only in a subpopulation of cells. Therefore, IK[Ca] in sympathetic neurons is either coupled to N-type Ca2+ channels or is not selectively coupled to a single Ca(2+)-channel subtype. The preferential coupling of IK[Ca] channels with distinct ICa subtypes may be part of a mechanism to allow for selective modulation of neurotransmitter release. Preferential coupling may also be important for the differentiation and development of vertebrate neurons.     

K+ and Ca2+ channel activity and cytosolic [Ca2+] in oxygen-sensing tissues.

Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changes in oxygen tension (P(O2)). We review here the modifications of K+ and Ca2+ channel activity and the resulting changes in cytosolic [Ca2+] induced by low P(O2) in glomus cells and arterial smooth muscle which are well known examples of O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces a reduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine and other neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulating cytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduit pulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanism whereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensing molecules or redox modification by reactive chemical species.     

Preconditioning delays Ca2+-induced mitochondrial permeability transition

OBJECTIVE: We investigated whether ischemic preconditioning (PC) may modify mitochondrial permeability transition (MPT) pore opening. METHODS: In protocol 1, New Zealand White rabbits underwent either no intervention (sham group) or 10 min of ischemia followed by 5 min of reperfusion, preceded (PC) or not (C; control) by one episode of 5 min of ischemia and 5 min of reperfusion. Rabbits were pretreated by either saline or the MPT pore inhibitor cyclosporin A (CsA), or its non-immunosuppressive derivative Cs29 (10 mg/kg, IV bolus). Hearts were harvested and mitochondria isolated for further assessment of Ca(2+)-induced MPT using a Ca(2+)-sensitive micro-electrode. In protocol 2, C and PC hearts underwent 30 min of ischemia and 4 h of reperfusion. They were pretreated either by saline, CsA or Cs29, as in protocol 1. Infarct size was assessed by triphenyltetrazolium, and apoptosis by TUNEL staining. RESULTS: In protocol 1, the Ca(2+) overload required to induce MPT pore opening was significantly higher in PC than in C hearts. CsA and Cs29 significantly increased the Ca(2+) overload required for MPT pore opening. In protocol 2, mean infarct size averaged 25% of the risk region in CsA/Cs29 treated hearts versus 15% in PC and 55% in controls (P<0.05 vs. C, P=ns vs. PC). Cardiomyocyte apoptosis was significantly reduced by PC and cyclosporin treatment with a mean apoptotic index of less than 2% in either group versus more than 11% in controls. CONCLUSION: This suggests that delayed opening of MPT pore may play a major role in ischemic PC.     

Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.