高血压大鼠冠状动脉平滑肌细胞大电导钙激活钾通道的变化

目的研究在高血压背景下大电导钙激活钾(BK)通道的功能改变及其机制。方法用酶解消化方法分离12～16周龄自发性高血压大鼠(SHR)和WKY大鼠冠状动脉平滑肌细胞(CASMCS),采用膜片钳全细胞模式记录SHR和WKY大鼠CASMCSBK电流,SHR和WKY大鼠BKα亚基和β1亚基mRNA水平用实时定量取聚合酶链式反应和凝胶电泳测定,蛋白水平表达用免疫组化的方法测定。结果 SHR大鼠CASMCS(n=6)BK电流密度比WKY(n=7)高2.03±0.62(P0.01);在mRNA水平,BKβ1亚基表达SHR组明显高于WKY组5.534±1.03倍(n=4,P0.05),BKα亚基表达则无明显差异(1.266±0.12,n=4,P0.05);BKβ1亚基和α亚基的表达SHR大鼠高于WKY大鼠。结论 SHR大鼠CASMCSBK电流密度比WKY大鼠增大,在mRNA和蛋白水平上BKβ1亚基表达也比WKY大鼠增强,这种变化可能是机体在高血压时自我调节的结果 。     

大蒜素对自发性高血压大鼠肠系膜动脉平滑肌细胞钙电流的作用

目的:观察大蒜素(Gar)对自发性高血压大鼠(SHR)肠系膜动脉平滑肌细胞L-型钙电流(LCa,L)的影响。方法:利用双酶-两步法消化得到单个大鼠肠系膜动脉血管平滑肌细胞,用全细胞膜片钳记录钙电流。在细胞池中灌流含Gar的细胞外液,观察药物对LCa,L的作用和门控机制及门控动力学参数的改变。结果:1Gar对ICa,L的抑制效应呈浓度依赖性和电压依赖性特征。刺激电位0 mV时,200μmol/L Gar可使ICa,L峰值密度由(-8.4±0.4)pA/pF降低为(-6.1±0.3)pA/pF;2药物可使ICa,L半激活电压V1/2右移,半失活电压左移及失活后恢复动力学减慢等环节可减少通道的开放和重复开放,从而减少ICa,L峰值密度和窗口电流。结论:Gar可能通过减少细胞的钙电流发挥降压效应。     

依那普利对自发性高血压大鼠平滑肌细胞Gqα基因表达的影响

目的:(1)自发性高血压大鼠是否存在Gqα基因表达的异常;(2)ACEI抑制血管平滑肌的增殖是否与抑制Gqα基因表达有关.方法:采用半定量RT-PCR方法,观察SHR及WKY主动脉血管平滑肌细胞静止期及增殖期Gqα基因表达情况以及依那普利对Gqα基因表达的影响.结果:(1)静止期,SHR及WKY血管平滑肌细胞Gqα mRNA表达无明显差别;在10%小牛血清刺激下,SHR血管平滑肌细胞Gqα mRNA表达增加四倍(P<0.01);(2)依那普利对静止期SHR Gqα mRNA表达无明显作用,对小牛血清引起的Gqα mRNA表达的增加有明显抑制作用,且呈浓度及时间依赖性;(3)非特异性肽类AngⅡ受体阻制剂无此作用.结论:SHR的Gqα基因表达反应性增加,依那普利对高血压病的治疗除抑制ACE 外,通过对Gq蛋白基因表达的抑制也起一定的作用.     

雌激素对高血压人体肠系膜动脉平滑肌细胞大电导钙激活钾通道的作用研究

目的 研究雌激素（E）对非高血压（NH）及原发性高血压（EH）人体肠系膜动脉平滑肌细胞（HMASMC）大电导钙激活钾通道（BKCa channels）及自发性瞬时外向电流（STOCs）的作用,探讨雌激素在NH及EH下对该通道作用的差异性.方法 急性酶分离法分离获取单个HMASMC,采用全细胞穿孔膜片钳技术记录该细胞上的BKCa和STOCs.结果 雌激素可明显激活NH组HMASMC上的BKCa和STOCs,在测试电压范围内,雌激素使膜电位从0到＋60 mV时BKCa的电流密度均显著性增加,在0和＋60 mV时其电流密度分别从（1.95±0.39）pA/pF、（15.40±4.27）pA/pF增加到（2.81±0.84）pA/pF（P＜0.05,25例）、（26.55±6.24）pA/pF（P＜0.01,25例）,其中0 mV时增加了0.44倍,＋60 mV时增加了0.72倍;电位为-20 mV时STOCs的幅度和频率分别从（7.920±2.031）pA、（3.15±0.79）Hz增加到（12.92±3.41）pA（P＜0.05,25例）、（4.41±0.96）Hz（P＜0.01,25例）,其中幅度增加了0.63倍,频率增加了0.40倍.而EH组在测试的-60到＋50 mV电压范围,雌激素没有这种显著性激活作用,在0和＋60 mV时其电流密度分别从（1.34±0.43）pA/pF、（4.91±1.40）pA/pF增加到（1.53±0.55）pA/pF（P＞0.05,14例）、（8.04±2.0）pA/pF（P＜0.05,14例）,其中0 mV时增加了0.14倍,＋60 mV时增加了0.63倍;在电位为-20 mV时STOCs的幅度和频率分别从（5.39±1.93）pA、（0.75±0.37）Hz增加到（6.70±1.06）pA（P＞0.05,14例）、（2.34±0.98）Hz（P＜0.05,14例）,其中幅度增加了0.24倍,频率增加了2.12倍.结论 雌激素对NH组HMASMC上的BKCa和STOCs有明显的激活作用,而在EH组这种激活作用显著降低,由此推测EH组HMASMC对雌激素的反应性较NH组低,这种差异性将为雌激素合理应用于临床提供有力的实验依据. 关键词：高血压;雌激素;人体肠系膜动脉;平滑肌细胞;自发性瞬时外向电流;大电导钙激活钾通道     

大鼠冠状动脉平滑肌细胞的分离及电压依赖性钾通道电流特性的初步研究

目的建立一种稳定、高效的大鼠冠状动脉平滑肌细胞(CASMCs)的急性酶分离方法,并对其电压依赖性钾离子通道(KV)进行研究。方法采用"三步"(依次用3种分离酶液)急性酶分离方法分离大鼠CASMCs,并进行细胞鉴定,采用膜片钳记录KV电流。结果①本分离方法可以获得数量较多,活性较好的平滑肌细胞;②测试电位为+60 mV时,KV电流密度达到最大值34.35±2.53 pA/pF,加入4-氨基吡啶后电流密度降至3.55±0.47pA/pF。两组之间电流密度的差异具有显著性(n=5,P<0.05)。KV稳态激活曲线的半数激活电压V1/2=23.66±1.03 mV,曲线斜率因子k=14.44±0.94 mV。结论 "三步"法为使用膜片钳记录CASMCs的离子通道电流提供了较好的研究方法。     

大电导钙激活钾离子通道与糖尿病视网膜动脉病变

大电导钙激活钾离子通道,即BK。通道,最早于1981年在牛嗜铬细胞中发现,1991年其仪亚基从果蝇中克隆并表达出来,由于Ⅸ亚基位点上的突变与肌肉和神经元的钙激活钾电流相关,因此又称为dSlo通道,随后在哺乳动物、人和线虫上也克隆了出来。后续研究证明,BK。通道广泛分布于各种组织细胞中,犬及人平滑肌细胞中最多怛。     

二十二碳六烯酸对大鼠冠状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)对大鼠冠状动脉平滑肌细胞(CASMCs)上大电导钙激活性钾通道(BKCa)的影响。方法采用酶消化法获得大鼠CASMCs,用膜片钳技术分别记录0,10,20,40,60和80μmol/LDHA对大鼠CASMCs上BKCa通道动力学的影响。结果在不同浓度DHA作用下,IBKCa和BKCa尾电流均呈浓度依赖性增加。IBKCa和BKCa尾电流I-V曲线均上移,对IBKCa稳态激活曲线无影响。在指令电压+150 mV,不同浓度DHA作用下,IBKCa电流密度分别为68.24±22.75,72.40±24.49,120.44±37.96,237.48±53.22,323.60±74.83和370.61±88.16pA/pF(P<0.05,n=20)。DHA对IBKCa激活的药物半效浓度为36.22±2.17μmol/L。在测试电压+90 mV,不同浓度DHA作用下,BKCa尾电流密度分别为91.02±13.52,100.23±17.34,224.02±38.76,369.19±65.39,511.39±82.77和700.14±96.64 pA/pF(P<0.05,n=20)。结论 DHA对全细胞BKCa有激活作用,对稳态激活曲线无影响。DHA对BKCa通道的激活作用可能是其舒张血管机制之一。     

依那普利对自发性高血压大鼠平滑肌细胞Gqα基因表达的影响

目的：（１）自发性高血压大鼠是否存在的Ｇｑα基因表达的异常;（２）ＡＣＥＩ抑制血管平滑肌的增殖是否与抑制Ｇｑα基因的表达有关。方法：采用半定量ＲＴ－ＰＣＲ方法,观察ＳＨＲ及ＷＫＹ主动脉血管平滑胸止期及增殖期Ｇｑα基因表达情况以及依依那普利对Ｇｑα基因表达的影响。结果：（１）静止期,ＳＨＲ及ＷＫＹ血管平滑肌细胞ＧａαｍＲＮＡ表达无明显差别;在１０％小牛血清刺激下,ＳＨＲ血管平滑肌细胞ＧｑαｍＲＮＡ表     

瘦素对自发性高血压大鼠血管平滑肌细胞增殖的影响

目的 探讨leptin对自发性高血压大鼠血管平滑肌细胞的增殖影响，并研究leptin b型受体在VSMC中的表达。方法 本文主要采用培养的自发性高血压大鼠（SHR）血管平滑肌细胞（VSMC），研究Leptin受体b(OB-Rb)的mRNA的表达，以及对平滑肌细胞增殖的影响。结果 OB－Rb的mRNA仅在SHR大鼠的平滑肌细胞表达，而没有在正常对照大鼠（WKY）的平滑肌细胞中表达。Leptin仅仅可以刺激SHR大鼠VSMC的DNA合成率的增加而对WKY大鼠的VSMC则无作用。同样，Leptin对VSMC细胞增殖的作用，也仅仅在SHR大鼠可以发现，而WKY大鼠则没有。结论 上述研究的结果显示；高血压时，Leptin是一种对血管平滑肌细胞的增殖因子，也许在高血压的发生，发展过程中起重要的作用。     

瘦素对自发性高血压大鼠血管平滑肌细胞增殖的影响

目的探讨leptin对自发性高血压大鼠血管平滑肌细胞的增殖影响,并研究leptin b型受体在VSMC中的表达.方法本文主要采用培养的自发性高血压大鼠(SHR)血管平滑肌细胞(VSMC),研究Leptin受体b(OB-Rb)的mRNA的表达,以及对平滑肌细胞增殖的影响.结果 OB-Rb的mRNA仅在SHR大鼠的平滑肌细胞表达,而没有在正常对照大鼠(WKY)的平滑肌细胞中表达.Leptin仅仅可以刺激SHR大鼠VSMC的DNA合成率的增加,而对WKY大鼠的VSMC则无作用.同样,Leptin对VSMC细胞增殖的作用,也仅仅在SHR大鼠可以发现,而WKY大鼠则没有.结论上述研究的结果显示;高血压时,Leptin是一种对血管平滑肌细胞的增殖因子,也许在高血压的发生、发展过程中起重要的作用.     

糖尿病冠状动脉平滑肌细胞大电导钙离子激活钾通道开放概率及蛋白表达的变化

目的研究糖尿病对冠状动脉平滑肌细胞大电导钙离子激活钾通道(BK通道)开放概率(NP0)及蛋白表达的影响,探讨糖尿病冠状动脉损伤的分子机制。方法采用链脲霉素腹腔内注射建立糖尿病大鼠动物模型,成功建立20只糖尿病大鼠动物模型作为糖尿病组;对照组(n=20)相应注射生理盐水。酶消化法分离冠状动脉平滑肌细胞,单通道膜片钳实验技术记录大鼠冠状动脉平滑肌细胞BK通道电流;采用Western blot实验方法测定大鼠冠状动脉平滑肌细胞BK通道亚基的蛋白表达。结果在电极外液钙离子浓度为1μmol/L,刺激电位为0,20,40,60,80,100和120 mV条件下,糖尿病组冠状动脉平滑肌细胞BK通道NP0明显低于对照组(P<0.05)。如刺激电位为120 mV时,对照组和糖尿病组大鼠冠状动脉平滑肌细胞BK通道NP0分别为1.210 5±0.048 1(n=5)和0.5217±0.1346(n=5);与对照组比较,糖尿病组BK通道α亚基蛋白表达无差异(P>0.05),但β1亚基蛋白表达下降了63%(P<0.05)。结论糖尿病冠状动脉平滑肌细胞BK通道β1亚基表达下调、BK通道NP0降低可能是糖尿病冠状动脉功能损伤的重要原因。     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

钙池操纵的钙通道与支气管哮喘气道平滑肌功能调控

钙信号是调控气道平滑肌细胞功能的重要机制。细胞内钙离子浓度受肌浆网内钙释放和胞外钙内流的双重调控。钙池操纵的钙通道（SOC）是哮喘气道平滑肌细胞外钙内流的重要机制,在哮喘气道高反应性和气道重塑中具有重要作用。近年来,通过分子生物学方法,已经发现了SOC相关调控分子STIM1和通道组成分子Orail,为深入研究气道平滑肌SOC的结构和功能关系,以及在哮喘防治中的作用提供了基础。本文就S0c及其与气道平滑肌功能的关系作一综述。     

Isolation from porcine cardiac muscle of a Ca2+-activated protease that partially degrades myofibrils

A protein fraction displaying Ca²⁺-activated proteolytic activity has been isolated from porcine cardiac muscle. The crude enzyme was purified approximately 2000 fold by isoelectric precipitation followed by gel permeation chromatography and by ion exchange chromatography. The partially purified enzyme exhibited optimal activity against either cardiac myofibril or casein substrates between pH 7.5 and 8.0, and in the presence of 1 mm Ca²⁺ and at least 2 mm 2-mercaptoethanol. The enzyme removes Z-discs from skeletal and cardiac myofibrils and also removes the density from intercalated discs of cardiac myofibrils. The enzyme hydrolyzes troponin-T and troponin-I of both cardiac and skeletal muscle myofibrils in vitro. In its proteolytic effect on either cardiac or skeletal myofibrils and in all other properties examined, the Ca²⁺-activated, cardiac protease is similar to a Ca²⁺-activated protease (CAF) recently purified from porcine skeletal muscle (Dayton, W. R., Reville, W. J., Goll, D. E. and Stromer, M. H. (1976) Biochemistry15, 2159–2167). It is possible that the Ca²⁺-activated, cardiac protease plays a role in degradation of myofibrils in injured myocardial cells.     

普鲁卡因胺、利多卡因和普罗帕酮对豚鼠心室肌细胞Na^＋／Ca^2＋交换电流的抑制作用

目的利用膜片钳技术观察Ⅰ类抗心律失常药物普鲁卡因胺、利多卡因、普罗帕酮对Na+/Ca2+交换电流的直接作用.方法采用胶原酶消化的成年大鼠单个心室肌细胞及全细胞膜片钳技术,记录Na+/Ca2+交换电流并观察药物对它的影响.结果3种药物对Na+/Ca2+交换电流的抑制均呈剂量依赖性,但抑制程度不同,其中普鲁卡因胺抑制作用最强.50、100μM的普鲁卡因胺分别使外向Na+/Ca2+交换电流从对照值(181±22)pA降低至(125±19)、(109±20)pA,内向电流由对照值(172±18)pA分别降低至(137±13)、(121±12)pA;50、100μM的利多卡因使外向电流从对照值(170±15)pA分别降低至(139±15)、(127±10)pA,内向电流由对照值(165±15)p/A分别降至(142±16)、(129±20)pA;50、100μM的普罗帕酮使外向电流由对照值(160±23)pA分别降至(130±27)、(112±26)pA,内向电流由对照值(169±13)pA分别降至(143±13)、(134±14)pA.普鲁卡因胺、普罗帕酮对外向电流的抑制大于内向电流,而利多卡因对内、外向电流的抑制差异无显著性.结论Ⅰ类抗心律失常药物对心室肌细胞Na+/Ca2+交换电流具有直接抑制作用,且抑制程度不同.     

Na+ and Mg2+ contents in smooth muscle cells in spontaneously hypertensive rats.

Whereas in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content and exchange in vascular smooth muscle cells. Therefore in aortic smooth muscle cells from 10 spontaneously hypertensive rats (SHR) of the Münster strain and 10 normotensive Wistar-Kyoto rats (WKY) aged 8 to 10 months, the intracellular magnesium and sodium content was measured. Electron-probe X-ray microanalysis was used to determine intracellular Mg2+ and Na+ concentrations in aortic cryosections 3 microm thick. The magnesium ion content was 0.90 +/- 0.15 g/kg dry weight in SHR versus 1.15 +/- 0.10 g/kg dry weight in WKY (means +/- SD, P < .05). Vascular smooth muscle sodium ion content was 6.66 +/- 0.39 g/kg dry weight in WKY and 12.61 +/- 0.91 g/kg dry weight in SHR (P < .01). Aortic smooth muscle cells from SHR are characterized by markedly lowered intracellular magnesium ion content and increased sodium ion concentrations in animals 8 to 10 months old, compared with normotensive cells. The results may be due to genetically determined disturbances in transmembrane magnesium and sodium ion transport.     

四磨汤对胃窦平滑肌的影响及钙通道在其中的作用

目的研究四磨汤对SD大鼠离体胃窦纵、环行平滑肌条收缩活动的影响及钙通道在其中的作用。方法正常SD大鼠离体胃窦纵、环行平滑肌条置于盛有Krebs液的组织浴槽中,记录其等长收缩活动,观察四磨汤累积量加入(1μL、5μL、25μL、50μL、100μL、150μL、200μL)对大鼠胃窦纵、环行平滑肌条收缩活动的影响,以及钙通道阻断剂硝苯地平(30 nmol/L)对四磨汤引起的胃窦平滑肌条收缩活动的影响。结果四磨汤剂量依赖性引起大鼠胃窦纵、环行平滑肌条收缩活动增加,硝苯地平可部分阻断四磨汤对胃窦纵、环行平滑肌条的兴奋作用;硝苯地平对于四磨汤诱导的纵行肌兴奋的抑制效应明显强于环行肌。结论四磨汤对大鼠胃窦平滑肌的收缩活动具有明显的兴奋作用,这一兴奋作用部分通过钙通道介导,纵行肌较环行肌更多的依赖于外钙内流。     

Potassium relaxation of vascular smooth muscle from spontaneously hypertensive rats.

Helical strips of tail artery from spontaneously hypertensive (SHR) and Kyoto Wistar normotensive rats (WKY) were observed to relax in response to potassium after contraction induced by norepinephrine in potassium-free solution. Helical strips from SHR consistently showed greater relaxation in response to the addition of potassium than did those from WKY. The amplitude of potassium-induced relaxation is believed to be an index of the activity of electrogenic sodium-potassium transport and hence of sodium-potassium ATPase. Thus, the sodium-potassium ATPase activity of SHR vascular smooth muscle is increased as compared to WKY. This interpretation is supported by the observation that ouabain eliminated potassium-induced relaxation in both SHR and WKY strips. Potassium-induced relaxation in SHR was more sensitive to ouabain inhibition than in WKY. Relaxation induced by potassium in SHR and WKY strips was also shown to vary with: (1) the length of incubation in potassium-free solution, and (2) the concentration of potassium added back. The results of these experiments on potassium-induced relaxation serve as evidence that SHR have either an increased intrinsic sodium-potassium ATPase activity, or an enzyme activity that has been stimulated to a greater degree by an elevated intracellular sodium concentration which resulted from the incubation in potassium-free solution.