大电导钙激活钾通道结构与功能调节及其在疾病中的作用

正大电导钙激活钾通道(big-conductance Ca~(2+)-activated K+channel,BK通道)广泛表达于各种组织中,其激活具有钙和电压依赖性。激活血管平滑肌细胞(vascular smooth muscle cell,VSMC)BK通道可引起膜电位超极化,舒张血管。BK通道由4个BK-α亚基和4个调节亚基组成,其中调节亚基包括β亚基(BK-β)或γ亚基(BK-γ)。研究表明,BK-β1亚基和     

大电导钙激活钾通道调节血管平滑肌的研究进展

正大电导钙激活钾通道(large conductance calciumactivated potassium channels,BK通道)因其电导大、对Ca~(2+)敏感性高而与其他钙激活钾通道明显相区别。随着免疫组织化学、放射性配体结合实验技术的发展,发现BK通道在平滑肌细胞、内皮细胞、神经细胞等细胞膜上均有表达[1-5],特别是血管平滑肌中,BK通道大量存在,并在病理生理过程中起着至关重要的作用,     

人肠系膜动脉平滑肌BKCa增龄变化的研究

目的研究肠系膜动脉平滑肌大电导钙激活钾通道的增龄变化及其意义。方法应用单通道膜片钳技术，在细胞贴附式和内面向外式模式下，检测不同年龄组通道的开放概率、电流幅值、平均开放时间和平均关闭时间。结果（1）在细胞贴附式膜片。老年组Po较中、青年组显著降低（P〈0．05）。（2）在内面向外式膜片，当浴液中Ca^2＋浓度为10^-6mol／L时，老年组的开放概率显著低于青年组（P〈O．05），而平均关闭时间显著高于青年组（P〈0．05）。结论老年人MASM细胞BKCa的活性下降，对胞内侧Ca^2＋敏感性降低，可能是老年人血管紧张性增加的原因之一。     

高胆固醇血症时大电导钙激活钾离子通道的改变及其调控机制

大电导钙激活钾通道(BK通道)在血管平滑肌细胞上分布极为广泛,参与血管张力调节、神经兴奋、神经递质释放和缺血再灌注损伤等多种重要生理和病理活动。高胆固醇血症时BK通道的功能和表达会发生变化,可能与质膜双分子层性质改变、胆固醇-蛋白直接作用或细胞分子信号通路传导等密切相关。     

冠状动脉平滑肌细胞大电导钙离子激活钾通道电流的特点

目的探讨正常大鼠冠状动脉平滑肌细胞大电导钙离子激活钾通道（BK通道）电流的特点,为研究疾病状况下冠状动脉平滑肌细胞BK通道电流异常变化提供正常对照。方法酶消化法分离大鼠冠状动脉平滑肌细胞;采用不同阻滞剂,对冠状动脉血管平滑肌细胞上钾通道进行鉴定;采用全细胞和单通道膜片钳实验技术分别记录冠状动脉平滑肌细胞BK通道电流,计算开放幅度和电导,观察BK通道电压敏感性和钙敏感性及加入特异性BK通道阻滞剂IBTX后BK通道电流的变化。结果正常冠状动脉平滑肌细胞BK通道电流约占总钾离子流65％±4％（t／,=12）,BK通道电导为（258±42）pS（n=6）,在刺激电位150mV时,电流密度为（275±40）pA／pF（n=8）;在电极外液钙离子浓度为1μmol／L,刺激电位为0、20、40、60、80、100、120、140和160mV条件下,BK通道开放概率（NP0）分别为0、0．0002、0．0016±0．0005、0．0283±0．0081、0．05694±0．0102、0．3533±0．0514、1．4922±0．1578、2．5975±0．3632和4．6041±0．7834（P〈0．05,n=5）;在刺激电位60mV,电极外液钙离子浓度为0、0．001、0．01、0．1、1、10、50和100μmol／L条件下,BK通道NP。分别为0、0．0001、0．0031±0．0008、0．0042±0．0090、0．0808±0．0105、0．7591±0．1274、2．7242±0．4612和3．2366±0．5728（P〈0．05,n=6）。结论BK通道广泛分布于冠状动脉平滑肌细胞上,具有电压敏感性和钙敏感性,对冠状动脉血管张力调节起重要作用。     

大电导钙离子激活钾通道在心血管疾病中的研究进展

冠状动脉平滑肌细胞膜上存在许多大电导钙离子激活钾（BKCa）通道,在维持细胞正常生理活动中起重要作用。研究发现当细胞膜去极化或/（和）细胞内钙离子增加时,BKCa通道激活,开放增加,钾离子外流,细胞膜超极化,血管舒张。而在高血压、糖尿病、缺氧、心力衰竭和老化等许多病理情况下,BKCa通道功能发生改变,从而影响对血管功能的调节。本文主要综述近年来BK通道在心血管疾病中的研究进展。     

心脏兴奋收缩偶联

在参与心脏活动的离子中，Ca^2＋被认为可能是最重要的。人体内存在一种促使心脏收缩舒张的关键机制，这一机制即为兴奋收缩偶联。充分了解Ca^2＋如何在各种细胞器间的转运并引发兴奋收缩偶联，对于深入理解心脏功能的生理基础具有十分重要的意义。心脏兴奋收缩偶联是指由心肌电兴奋开始到心脏收缩并泵血这一过程。在心电活动中，Ca^2＋作为普遍存在的第二信使，起着关键的作用。它能够直接兴奋肌丝，从而引起心脏收缩。心肌Ca^2＋失调是引起心脏收缩功能障碍和心律失常等病理生理状态的关键原因。在心肌动作电位中，Ca^2＋通过去极化兴奋Ca^2＋进入细胞形成Ca^2＋内流（ICa），参与心电活动周期中平台期的形成。Ca^2＋内流会触发肌浆网（SR）释放Ca^2＋。这种Ca^2＋的内流和释放，使得细胞内Ca^2＋浓度迅速升高，随后Ca^2＋与肌纤蛋白-肌钙蛋白C结合，引发收缩机制。若是心脏发生舒张活动，[Ca^2＋]i必须下降，从而使得Ca^2＋由肌钙蛋白上释放出来。这一过程需将Ca^2＋转运出胞浆，主要通过以下四条途径来实现：①肌浆网（SR）Ca^2＋-ATPase；②肌纤维膜Na^＋-Ca^2＋交换（NCX）；③肌纤维膜Ca^2＋-ATPase；④线粒体Ca^2＋单向转运。本文将着重论述心肌细胞Ca^2＋转运体系及其调节机制。     

钙信号转导与急性胰腺炎

急性胰腺炎（AP）是胰腺腺泡细胞内胰蛋白酶原激活引起细胞自溶的过程。它的早期病理改变包括：胰腺腺泡细胞内酶原激活，细胞骨架裂解，分泌极性丧失，酶原空泡形成。这些病理改变均以细胞内Ca^2＋浓度异常增高为前提，应用Ca^2＋螯合剂能够抑制Ca^2＋浓度增高和胰蛋白酶原的激活。[第一段]     

镁离子对原代培养猪冠状动脉平滑肌细胞钙激活钾通道的影响

目的：应用膜片钳单通道技术研究镁离子(Mg^2 )对原代培养猪冠状动脉平精肌细胞钙澈活钾通道(KCa)的影响.方法：选择培养5天后的单个棱形平精肌细胞，用二步拉镐经热抛光尖端直径约1～2gμm，充灌液体后阻抗约5～7MΩ的微吸管进行高阻封接，在对称性高钾溶液下采用内面向外式膜片记录电流。单通道电流信号经膜片钳放大器放大，滤波(3KHz)后，经12位A／D，D／A转换器在pClamp6．0．2专用软件支持下输入并储存在计算机内，然后进行分析处理。结果：猪冠状动脉平滑肌细胞KCa单通道电导值为135±15pS，[Ca^2 ]i为10^-7mol，膜电位为-40mV，向膜内侧液加入Mg^2 至1和4mmol，通道活度(NPO)显著增加;并发现随着[M^2 ]i从0．5mmol依次增加至14mmol，Mg^2 对KCa通道可产生浓度依赖性激活作用；而且用一定浓度的EGTA把[Ca^2 ]螯合到很小时(<10^-9 mol)，再向膜内侧加入Mg^2 (4mmol)仍对通道有激活作甩。结论：[Mg^2 ]i对KCa通道可产生浓度依赖性激活作用，且在膜内侧相对无Ca^2-的情况下仍可激活KCa通道，这可能是Mg^2 舒张血管平滑肌的作用机制之一。     

长期使用咪达普利对心肌梗死引起的充血性心力衰竭的死亡率、心脏功能和基因表达的影响

尽管咪达普利在治疗充血性心力衰竭中的作用被普遍承认，作为一种血管紧张素转化酶抑制剂，它通过改善血液动力学参数而发挥作用，但是它对于肌纤维膜（SL）和肌浆网蛋白（SR）的基因表达的影响还没有完全搞清楚。在本研究中，我们检测了长期应用咪达普利对患者死亡率、心脏功能和基因表达的影响，后者包括肌纤维膜Na^＋／K^＋ATP通道、Na^＋／Ca^2＋交换器、肌浆网蛋白Ca^2＋泵ATP酶、Ca^2＋释放通道以及充血性心力衰竭中心肌梗死引起的受体磷酸蛋白和集钙蛋白。在大鼠中，心力衰竭是由于左侧冠状动脉堵塞导致心肌梗死而继发引起的，使用咪达普利进行治疗开始于手术后的第三周末并持续进行37周。     

Altered erythrocyte Na+ + K+ pump in adolescent obesity

The number of $\text{Na}\text{K}$ pump units and the cation transport activity of the pump were measured in erythrocytes from two etiologically different groups of obese adolescents and a group of normal controls. There was a significant reduction in the number of pump units, as measured by saturation ouabain binding, in erythrocytes from adolescents with idiopathic, early onset obesity. Individuals whose obesity developed subsequent to the appearance of a variety of hypothalamic lesions showed no reduction in the red cell complement of $\text{Na}\text{K}$ pump when compared to controls and the cation transport activity of their cells was higher than both the controls and the subjects with idiopathic obesity. These results support data obtained in adults that reduced red cell $\text{Na}\text{K}$ pump levels are seen in a group of individuals with idiopathic obesity. They further suggest that such reductions are not likely to be secondary to the obese state per se.     

Transport of Mg2+ by Na+-Mg2+ exchange

Intracellular free Mg(2+) concentration is maintained at low levels by active extrusion from the cells. One of postulated mechanisms is the Na(+)-Mg(2+) exchange, which extrudes Mg(2+) in exchange with Na(+) influx. Although the Na(+)-Mg(2+) exchange activity has been reported in many types of cell, including neurons, details of molecular mechanisms are only poorly understood. In this chapter, we briefly will review our current knowledge on [1] stoichiometry of the Na(+)-Mg(2+) exchange, [2] interaction between the Na(+)-Ca(2+) exchange and the Na(+)-Mg(2+) exchange, [3] molecular biology of the Na(+)-Mg(2+) exchanger.     

Difference between human red blood cell Na+-Li+ countertransport and renal Na+-H+ exchange

Several laboratories have reported that the activities of sodium-lithium countertransport are increased in red blood cells from patients with essential hypertension. Based on the many similarities between this transport system and the renal sodium-proton exchanger, a hypothesis has been put forth in the literature that increased red blood cell sodium-lithium countertransport activity may be a marker for increased sodium-proton exchange activity in the renal proximal tubule. The present studies were designed to test the hypothesis that sodium-lithium countertransport in red blood cells from humans or rabbits is mediated by the same transport mechanism that mediates sodium-proton exchange in the renal brush border from those species. Similar to what has been reported for the rabbit, the present studies show that an amiloride-sensitive sodium-proton exchanger is present in human renal brush border vesicles. However, Na+-Li+ countertransport in human and rabbit red blood cells, assayed under several different conditions, was not inhibited by amiloride. In agreement with what has been reported for humans, the present studies show that extracellular proton-stimulated sodium efflux is inhibited by amiloride in rabbit red blood cells. These data demonstrate a difference (amiloride sensitivity) between the red blood cell sodium-lithium countertransporter and the renal brush border sodium-proton exchanger in humans and rabbits. These experiments detract from the hypothesis that increased red blood cell sodium-lithium countertransport activity in patients with essential hypertension is a marker for increased sodium-proton exchange activity in the renal brush border.     

Red blood cell Li+-Na+ countertransport, Na+-K+ cotransport, and the hemodynamics of hypertension

Red blood cell Li+-Na+ countertransport and Na+-K+ cotransport activities, home blood pressure, invasive systemic hemodynamics, and limb venous compliance were measured in 65 white men (23 normotensive, 22 borderline hypertensive, and 20 mild essential hypertensive subjects). Li+-Na+ countertransport activity was positively and significantly correlated with subject-determined home systolic blood pressure (r = 0.31, p less than 0.02) and with directly measured systolic (r = 0.29, p less than 0.02) and diastolic (r = 0.27, p less than 0.03) blood pressures in the hemodynamic laboratory, independent of potential confounding variables. Analysis of the hemodynamic determinants of blood pressure revealed a significant positive correlation of countertransport with vascular resistance (r = 0.30, p less than 0.02) but not with cardiac output or cardiac index. High red blood cell Na+-K+ cotransport activity was not independently associated with hypertension or with a characteristic hemodynamic pattern but was related to decreased venous compliance. Red blood cell Li+-Na+ countertransport deserves further study as a marker for the genetic substrate of human essential hypertension. Red cell Na+-K+ cotransport may be altered secondarily by factors related to high blood pressure and seems to be a valid marker for abnormalities of the venous system in hypertension.     

Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.     

Na+-Li+ countertransport in essential hypertension

Several studies on Na+-Li+ countertransport have reported higher rates in essential hypertensive than in normotensives, with a distribution pattern which is dependent on racial and ethnic background. However, it is not well established whether this abnormality in Na+ transport is associated with an abnormal clinical setting. In the present study we have performed a kinetic analysis of the interaction of the Na+-Li+ countertransport system with internal Na+ in erythrocytes from a sample of 72 essential hypertensives and 30 normotensive controls. A significant increase in mean values of the maximal rate of Li+-stimulated Na+ efflux (Vmax; 375.1 +/- 23.8 versus 213.7 +/- 8.5 mumol/l cells per h; mean +/- s.e.m.; Mann-Whitney test: U = 500; P less than 0.0001), as well as in the apparent affinity constant for internal Na+ (KNa; 10.03 +/- 0.08 versus 6 +/- 0.4 mmol/l cells; Mann-Whitney test: U = 718; P less than 0.0079), were observed in essential hypertensives with respect to normotensives. Using the 95% confidence interval of Vmax in normotensives as the normal range, 29 (40.3%) of the essential hypertensives exhibited values above the normal upper limit. The maximal rate (Vmax) and the internal Na+ content required for half-maximal stimulation (K50%) of Na+-K+ ATPase and outward Na+-K+ cotransport, and the rate constant of Na+ leak (KPNa) in this subset were similar to the values observed in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells

Rapamycin is an immunosuppressive compound that is currently used to prevent acute graft rejection in humans. In addition, rapamycin has been shown to allow operational tolerance in murine models. However, a direct effect of rapamycin on T regulatory (Tr) cells, which play a key role in induction and maintenance of peripheral tolerance, has not been demonstrated so far. Here, we provide new evidence that rapamycin selectively expands the murine naturally occurring CD4(+)CD25(+)FoxP3(+) Tr cells in vitro. These expanded Tr cells suppress proliferation of syngeneic T cells in vitro and prevent allograft rejection in vivo. Interestingly, rapamycin does not block activation-induced cell death and proliferation of CD4(+) T cells in vitro. Based on this new mode of action, rapamycin can be used to expand CD4(+)CD25(+)FoxP3(+) Tr cells for ex vivo cellular therapy in T-cell-mediated diseases.     

Red cells from the original JAL+ proband are also DAK+ and STEM+

Background The low‐prevalence Rh antigen, JAL, was named after the index case, Mr. J. Allen. Based on reactivity of seven multi‐specific sera with his RBCs, it was apparent that they express at least one additional low‐prevalence antigen. The purpose of this study was to investigate the other low‐prevalence antigen(s) on J. Allen’s RBCs. Methods Blood samples and reagents were from our collections. Hemagglutination and DNA analyses were performed by standard methods. Results Our DNA analyses confirmed the presence of RHCE*ceS(340T) in J. Allen and revealed the presence of RHCE*ceBI (ce 48C, 712G, 818T, 1132G) and RHD*DOL (509T, 667T). RBCs from J. Allen were agglutinated by anti‐JAL, anti‐STEM, and anti‐DAK. Two of the reactive multi‐specific sera reported in the original paper reacted with RBCs from J. Allen, and with RBCs from four other people with RHCE*ceBI, including the original STEM+ index case (P. Stemper) but not with RBCs with the DIIIa, DAK+ phenotype. We conclude that they contain anti‐STEM. Conclusion J.Allen’s RBCs express the low‐prevalence Rh antigens, JAL, V/VS (extremely weakly), STEM, and DAK. The presence of JAL on the variant Rhce, RhceJAL (16Cys, 114Trp, 245Val), STEM on the variant Rhce, RhceBI (16Cys, 238Val, 273Val, 378Val), and DAK on the variant RhD (170Thr, 223Val), encoded by RHD*DOL in trans to RHCE*ceBI is consistent with expression of these antigens. When J. Allen RBCs are used to detect and identify an anti‐JAL, it is important to remember that they also express STEM and DAK.