川芎嗪在豚鼠离体气管螺旋条中的作用

目的：研究川芎嗪在豚鼠离体气管螺旋条中的作用。方法：在浴槽内加入川芎嗪从10^-7 mol/L至10^-2 mol/L，观察气管螺旋条张力的变化，并检测浴槽中NO₂^-/NO₃^-含量，气管螺旋条组织cAMP、cGMP含量的变化。结果：加入川芎嗪气管螺旋条张力下降，与川芎嗪浓度呈剂量依赖性，未去皮组舒张度显著大于去皮组（P<0.01）。浴槽内NO₂^-/NO₃^-含量增加，未去皮组(2.3±0.37) mol/L升至(5.4±0.42) mol/L（P<0.05），去皮组(1.12±0.12) mol/L升至(2.29±012) mol/L（P<0 05）。气管螺旋条组织cAMP 含量增加，未去皮组(17.6±1.19) nmol/g protein升至(36.48±7.20) nmol/g protein（P<0.05） , 去皮组(8.20±2.30) nmol/g protein 升至(18.34±2.30) nmol/g protein（P<0.05），cGMP 含量增加，未去皮组(0.33±0.11) nmol/g protein 升至(0.67±0.27) nmol/g protein（P<0.05），去皮组(0.16±0.03) nmol/g protein 升至(0.33±0.16) nmol/g protein（P<0.05）。结论：川芎嗪有舒张气管螺旋条的作用。     

Cl-通道活动对自发性高血压大鼠血管张力的影响

目的：观察自发性高血压大鼠(SHR)血管平滑肌细胞Ca2+激活Cl-通道［ICl（Ca）］的活动。 方法： 测定离体肠系膜血管床灌注压和离体尾动脉肌条血管张力, 以其对去甲肾上腺素(NE)收缩反应的变化作为舒缩活动的指标。 结果： （1）SHR肠系膜动脉和尾动脉对NE收缩反应显著大于Wistar大鼠。（2）硝呋咪酸可显著抑制NE诱发的肠系膜动脉和尾动脉收缩反应，并具有浓度依赖性，SHR肠系膜动脉和尾动脉收缩活动受硝呋咪酸的抑制程度明显小于Wistar大鼠。（3）SHR肠系膜动脉对低氯缓冲液的反应显著大于Wistar大鼠。 结论： SHR肠系膜动脉和尾动脉血管平滑肌的Ca2+激活Cl-通道的活动增强，并导致血管对NE的反应性增高。这可能是在高血压的发生过程中引起和维持较高血管张力和外周血流阻力的因素之一。     

大电导钾通道对小鼠皮层神经元胞内游离钙和动作电位的影响

目的 探讨大电导钾通道（BK）对小鼠大脑皮层神经元胞内游离钙 ( [Ca2+]i） 和兴奋性的调节作用。方法 体外培养小鼠皮层神经元,用膜片钳技术观察BK特异性阻断剂IBERIOTOXIN对神经元 [Ca2+]i和动作电位频率的影响,用显微荧光测钙法观察IBERIOTOXIN对高钾条件下[Ca2+]i的影响。结果 生理状态下,IBERIOTOXIN灌流(100nmmol/L)对神经元细胞自发动作电位的频率、[Ca2+]i无显著影响;持续电刺激去极化后,IBERIOTOXIN灌流使动作电位频率增加,[Ca2+]i显著升高。高钾溶液(20mmol/L)引起神经元[Ca2+]i升高, 而IBERIOTOXIN灌流则使[Ca2+]i进一步显著升高。结论 BK对小鼠受持续去极化刺激或高钾条件时的神经元[Ca2+]i和兴奋性具有明显调节作用。     

缺O_2、低温和高CO_2对某些致喘介质致痉作用的影响

用20只成年豚鼠气管,制成多个“Z”形相连气管条,置入含Krebs-Henseleit液的器官浴中,以浴液温度为37℃,通5％CO_2和95％O_2为对照,观察缺O_2(通5％CO_2和95％N_2)、低温(浴液温度为12～15℃)或高CO_2(10％CO_2和90％O_2)对乙酰胆碱和组织胺致痉作用的影响并分析其机制。结果表明:缺O_2使平滑肌对乙酰胆碱和组织胺张力反应速度加快,总张力升高;低温使平滑肌对乙酰胆碱和组织胺张力反应速度减慢,总张力降低;高CO_2不影响平滑肌对组织胺或乙酰胆碱的反应;缺O_2、低温和高CO_2不影响异丙基肾上腺素对乙酰胆碱或组织胺所致痉挛的解痉作用。     

一种气管条的制备及其主动张力定量记录法

本文在气管螺旋条法基础上加以改进,根据气管平滑肌的分布特点,在横行平滑肌丰富的气管背面软骨环缺口处每隔3～4个软骨环行一横切,切口长约为气管周径的1/3。在气管腹面及两侧面斜切,斜切口与相邻两横切口相连成,“Z”形,制成“Z”形气管条。本法简单易行,减少切断横行平滑肌,增加了肌条收缩反应幅度。 机械—电换能器与二道生理记录仪相连。在换能器上负重,记录曲线上升高度可根据校对电压换算成相应电压值,制成重量—电压关系曲线。将制成的“Z”形气管条一端固定于盛有克—亨氏(Krebs—Henseleit)营养液的器官浴槽中的通气管弯头上,另一端与换能器弹性应变梁相连。肌条收缩时产生主动张力拉动弹性应变梁。主动张力大小可根据描记曲线的高度和重量—电压关系曲线换算成重量来定量表示。     

海马去甲肾上腺素能及乙酰胆碱能系统损伤对大鼠类P3的影响

目的:借助P300研究在不同神经递质缺失状态下认知功能的改变。方法:手术切断大鼠左侧穹窿海马伞(FF)建立胆碱能系统损伤模型,向大鼠双侧背去甲肾上腺素束注射6-OHDA建立去甲肾上腺素能系统损伤模型,于建模前后对其进行迷宫检查及类P3潜伏期测定。结果:两种神经递质系统的失常均可造成大鼠类P3潜伏期显著长于对照组,并与迷宫测试指标(EN、TRT)成正相关关系,但在两个实验组之间未见有显著差异。结论:乙酰胆碱与去甲肾上腺素在P300的产生与整合机制中均占有重要地位。     

缺血后处理对抗大鼠离体心脏缺血再灌注损伤及其作用机制

目的： 研究缺血后处理（postconditioning）对抗大鼠离体心脏缺血再灌注损伤及其作用机制。方法：采用SD大鼠心肌缺血/再灌注模型，并于再灌注一开始即给予3次全心停灌30 s，再灌30 s处理作为缺血后预处理。记录心肌收缩功能指标，以Even’s blue-TTC法监测心肌梗死范围，并对心律失常严重程度进行定量分析。结果：缺血后处理组左室峰压（LVSP）、最大左室收缩速率（+dp/dt_max）以及心率明显高于缺血对照组。缺血后处理可明显缩小心肌梗死范围（22.97%±3.96% vs 缺血对照组 44.30%±13.61%，P<0.01）。观察复灌10 min时心律失常评分发现，缺血后处理组明显低于缺血对照组。缺血后处理组和缺血预处理组具有类似的心肌保护作用。5-HD组LVSP和+dp/dt_max低于缺血后处理组，心律失常评分增高，心肌梗死范围扩大。结论: 缺血后处理对大鼠缺血再灌注损伤具有心脏保护作用，其作用机制可能是部分通过激活线粒体ATP依赖性钾离子(mitoK_ATP)通道起作用。     

高钙对烧伤早期豚鼠离体心脏固有收缩性的影响

为了探讨烧伤早期心肌固有收缩力降低的机制,本实验观察了35％体表面积Ⅲ度烧伤后8小时,灌流豚鼠离体心脏对高钙溶液(含Cacl_2 3.2mmol/L台氏液)的反应及对Ca~(2+)的消耗量。结果表明,烧伤组在高钙液灌流情况下,左心室内压的±dp/dtmax、心室肌细胞动作电位幅度,最大去极化速率.复极化50％的时程缩短程度均低于对照组(P<0.05);对常钙或高钙灌流液中Ca~(2+)的交换量,烧伤组也显著低于对照组(P<0.01)。提示烧伤早期心肌细胞对Ca~(2+)的摄取、利用,心肌细胞电位对Ca~(2+)的反应均低于正常,这可能是此期心肌固有收缩性和舒张功能显著降低的重要原因之一。     

二十二碳六烯酸对大鼠肺动脉平滑肌细胞大电导钙激活性钾通道的影响

 目的： 探讨二十二碳六烯酸（DHA）对大鼠肺动脉平滑肌细胞（PASMCs）大电导钙激活性钾离子通道（BKCa）的作用。方法：采用酶解法获得单个活性良好的PASMC,PASMCs的BKCa电流变化采用全细胞膜片钳技术记录,PASMCs胞浆内游离钙离子浓度(［Ca2+］i)采用激光共聚焦显微镜技术测定。结果：DHA 0.01 μmol/L对BKCa无显著激活作用;0.1、1、10 μmol/L DHA可显著激活BKCa。不同浓度的DHA（0、0.1、1、10 μmol/L）在指令电压为+60 mV时,BKCa电流密度分别为(30.5±6.5) pA/pF、(59.4±5.8) pA/pF、(87.2±4.3) pA/pF和(117.3±7.1) pA/pF (P<0.01)。急性缺氧在指令电压为+60 mV时,BKCa电流密度从(32.7±8.5) pA/pF降低至(11.9±5.8) pA/pF (P<0.01)。DHA (10 μmol/L) 能明显逆转急性缺氧对BKCa的抑制作用（P<0.01）,同时DHA触发PASMCs内［Ca2+］i的增加,最大增加速率为（71.9±4.1）%（P<0.01）。结论：DHA通过增加PASMCs［Ca2+］i激活BKCa,逆转急性缺氧对BKCa的抑制作用,具有舒张肺血管的作用。     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes

The interaction of Ba²⁺ and TEA with Ca²⁺-activated K⁺ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 m external diameter). Ba²⁺ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba²⁺ into the channel and was reduced by increasing K⁺ ion concentration on the outside of the membrane. These results are consistent with Ba²⁺ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.     

UTP对猪冠状动脉平滑肌细胞BK_(Ca)通道作用机制的研究

目的:利用膜片钳技术,研究UTP对急性酶分离的猪冠状动脉平滑肌细胞上BKCa通道的作用,探讨生理条件下UTP调节BKCa通道的机制。方法:应用Cell-attached技术记录急性酶分离的猪冠状动脉平滑肌细胞上BKCa电流。用pCLAMP9.0软件系统进行数据采集及分析。结果:在Cell-attached膜片上,80μM UTP可明显激活BKCa通道(P0.05,n=21)。PLC抑制剂U73122可抑制UTP对BKCa的激活作用(P0.05,n=5)。PKC激动剂PMA(10nM),可使BKCa通道开放概率明显降低(P0.05,n=9)。UTP(80μM)预处理细胞之后,IP3抑制剂2APB(80μM),可使BKCa通道开放受抑(P0.05,n=6)。结论:在Cell-attached膜片上,UTP能激活猪冠脉平滑肌细胞BKCa通道。PLC信使转导通路中的IP3介导的胞内Ca2+释放是UTP激活BKCa通道的重要途径。     

Characterization of ouabain-induced noradrenaline and acetylcholine release from in situ cardiac autonomic nerve endings

Aim: Although ouabain modulates autonomic nerve ending function, it is uncertain whether ouabain‐induced releasing mechanism differs between in vivo sympathetic and parasympathetic nerve endings. Using cardiac dialysis, we examined how ouabain induces neurotransmitter release from autonomic nerve ending. Methods: Dialysis probe was implanted in left ventricle, and dialysate noradrenaline (NA) or acetylcholine (ACh) levels in the anaesthetized cats were measured as indices of neurotransmitter release from post‐ganglionic autonomic nerve endings. Results: Locally applied ouabain (100 μm ) increased in dialysate NA or ACh levels. The ouabain‐induced increases in NA levels remained unaffected by cardiac sympathetic denervation and tetrodotoxin (Na⁺ channel blocker, TTX), but the ouabain‐induced increases in ACh levels were attenuated by TTX. The ouabain‐induced increases in NA levels were suppressed by pretreatment with desipramine (NA transport blocker) and augmented by reserpine (vesicle NA transport blocker). In contrast, the ouabain‐induced increases in ACh levels remained unaffected by pretreatment with hemicholinium‐3 (choline transport blocker) but suppressed by vesamicol (vesicle ACh transport blocker). The ouabain‐induced increases in NA levels were suppressed by pretreatment with ω‐conotoxin GVIA (N‐type Ca²⁺ channel blocker), verapamil (L‐type Ca²⁺ channel blocker) and TMB‐8 (intracellular Ca²⁺ antagonist). The ouabain‐induced increases in ACh levels were suppressed by pretreatment with ω‐conotoxin MVIIC (P/Q‐type Ca²⁺ channel blocker), and TMB‐8. Conclusions: Ouabain‐induced NA release is attributable to the mechanisms of regional exocytosis and/or carrier‐mediated outward transport of NA, from stored NA vesicle and/or axoplasma, respectively, while the ouabain‐induced ACh release is attributable to the mechanism of exocytosis, which is triggered by regional depolarization. At both sympathetic and parasympathetic nerve endings, the regional exocytosis is because of opening of calcium channels and intracellular calcium mobilization.     

Kv在正常与被动致敏人气道平滑肌张力调控中的作用

目的：探讨电压依赖性延迟整流钾通道（Kv）对正常与体外致敏人气道平滑肌（HASM）的张力调控作用及其机制。 方法： 采用肌张力试验、逆转录聚合酶链反应（RT-PCR）和免疫细胞化学等技术，观察钾通道阻断剂对正常与哮喘患者血清致敏HASM张力、细胞Kv的mRNA和蛋白质表达。 结果： Kv 阻断剂4-氨基吡啶（4-AP）可引起正常HASM肌环产生浓度依赖性收缩反应，达到最大效应一半所需浓度的负对数值（pD2）为2.09±0.09，人哮喘血清致敏组为2.44±0.16，差异显著（P＜0.01）；但最大反应强度（Emax）分别为正常组（24.0±6.4）g/g，人哮喘血清致敏组（31.8±7.3）g/g，差异无显著（P＞0.05）。在培养的HASM细胞上，Kv1.2、Kv1.3和 Kv1.5基因mRNA均有表达；人哮喘血清致敏的HASM细胞Kv1.5基因mRNA（P＜0.01）和蛋白质（P＜0.01）的表达均下降。 结论： 体外被动致敏HASM细胞Kv功能较正常下调，致平滑肌兴奋性增高，该变化可能主要与Kv1.5基因相关。     

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

Effect of biologically active substances (acetylcholine,histamine, and bradykinin) on depolarized smooth muscle of the taenia coli

Experiments on isolated strips of the guinea pig taenia coil showed that compound D-600, which blocks calcium channels, depresses the contractile response of depolarized smooth muscle to administration of acetylcholine (5·10^–6), histamine (2·10^–6), and bradykinin (10^–5 g/ml). Besides depressing the effect of biologically active substances, compound D-600 also depresses the potassium contracture. It is postulated that the biologically active substances activate the flow of Ca⁺⁺ ions through the surface membrane but not the discharge of Ca⁺⁺ from the sarcoplasmic reticulum.Department of Physiology, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR the late A. A. Vishnevskii). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 12–14, January, 1975.     

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach ofBufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K⁺ selective. The probability of the channel being open (P _o) increased when [Ca²⁺]_i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca²⁺-activated K⁺ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot ofP _o versus membrane potential followed the Boltzman relationship. Increasing [Ca²⁺]_i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity ofP _o residing in the mean closed time. These results were verified with a series of experiments carried out at lowP _o (<0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NP _o) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.     

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

Calcium (Ca(2+)) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca(2+) permeable channels, Ca(2+) pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca(2+) levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca(2+) signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca(2+) channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca(2+) release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca(2+) signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca(2+) channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca(2+) release channels, pumps and Ca(2+)-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2a pump and reciprocally regulate isoforms of the ca(2+)/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca(2+) signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca(2+) signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.