高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道变化及前列腺素E_1对通道活性的影响

目的　探讨高血压病患者肠系膜动脉平滑肌细胞钙激活钾 (KCa)变化及前列腺素 (PG)E1 对通道活性的影响。方法　用酶消化法获取血管平滑肌细胞 ,以膜片钳技术检测KCa通道的活性 ,通过Pclamp专用软件实时采样记录其平均开放时间 (To) ,平均关闭时间 (Tc) ,平均开放概率 (Po)等。然后分别加入不同浓度的Ca2 +(10 - 8、10 - 7、10 - 6mol L)及PGE1 (10、2 0、40、10 0、2 0 0、40 0nmol L)后 ,再观察上述参数变化。结果　高血压病组肠系膜动脉平滑肌细胞KCa通道活性显著高于非高血压组 ,加入Ca2 +后 ,两组KCa通道均被明显激活 ,与加Ca2 +前比较 ,非高血压组Po增加了 18.4倍 ,而高血压病组Po仅增加了 1.7倍。PGE1 可使高血压病患者肠系膜动脉平滑肌细胞KCa通道To延长 ,Tc缩短 ,Po增加。结论　高血压病患者肠系膜动脉平滑肌细胞KCa通道活性明显高于非高血压者 ,但对Ca2 +的敏感性降低 ,PGE1 可明显激活高血压病患者肠系膜动脉平滑肌细胞KCa通道。     

血管活性剂对高血压患者肠系膜动脉平滑肌细胞钙激活钾通道活性的调控作用

目的　探讨血管活性剂内皮素 1(ET 1)及前列腺素E1(PGE1)对高血压病患者肠系膜动脉平滑肌细胞钙激活钾 (KCa)通道活性的调控作用。方法　切取 2 1例老年高血压病患者及 18例老年非高血压者肠系膜动脉小分支节段 ,用酶消化法获取血管平滑肌细胞 ,以膜片钳技术检测KCa通道的活性 ,通过Pclamp专用软件实时采样记录其平均开放时间 (To) ,平均关闭时间 (Tc) ,平均开放概率 (Po)等。结果　 (1)高血压病患者KCa通道活性变化 ,于细胞内面向外膜片下 ,高血压病组与血压正常组比较 ,前者KCa通道Po增加 ,Tc延长 ,To缩短。在浴液中分别加入Ca2 + 后 ,两组KCa通道均被明显激活 ,与加Ca2 + 前比较 ,血压正常组Po的增加显著高于高血压病组 (P <0 0 0 1) ,说明高血压病组KCa通道对Ca2 + 的敏感性显著低于血压正常组。 (2 )两组溶液中加入ET 12～ 8× 10 9mol/L后 ,在内面向外式膜片下 ,血压正常组Po先增加后降低 ,Tc缩短 ,然而高血压病组患者肠系膜血管平滑肌KCa通道对ET 1则缺乏显著反应。 (3)溶液中加入PGE1后 ,在内面向外膜片下 ,高血压病患者血管平滑肌细胞KCa通道的通道电导增加 ,To显著延长 ,Tc显著缩短 ,Po显著增加 ,并呈浓度依赖性。结论　 (1)高血压病患者肠系膜动脉平滑肌细胞KCa通道活性明显高于非高血压者     

血管内皮源性舒张因子对血管平滑肌钙激活钾通道活性影响的对比研究

目的　探讨血管内皮源性舒张因子对高血压及非高血压患者血管平滑肌钙激活钾通道 (KCa)活性的影响。方法　选择 2 0 0 1～ 2 0 0 3年泸州医学院附属医院 2 1例高血压病患者及 18例非高血压者 ,切取肠系膜动脉小分支节段用酶消化法获取血管平滑肌细胞 ,以膜片钳技术检测KCa通道的活性 ,通过Pclamp专用软件实时采样记录其平均开放时间 (To)、平均关闭时间 (Tc)、平均开放概率 (Po)等。采用硝酸还原法测定血浆一氧化氮(NO)含量 ,用比色法测定血浆一氧化氮合成酶 (NOS)含量。结果　 (1)两组对象KCa通道活性变化 :于细胞内面向外膜片下 ,高血压病组与非高血压组比较 ,前者KCa通道Po增加 ,Tc延长、To缩短。在浴液中分别加入Ca2 +后 ,两组KCa通道均被明显激活 ,与加Ca2 + 前比较 ,非高血压组Po的增加显著高于高血压病组 (P <0 0 1) ,说明高血压病组KCa通道对Ca2 + 的敏感性显著低于血压正常组。 (2 )高血压患者血浆NO、NOS较非高血压组显著降低。 (3)非高血压组血浆NO与KCa通道Po、To呈正相关 ,与Tc呈负相关。高血压病组NO与KCa通道Po、To、Tc的相关性明显弱于非高血压组。结论　高血压病患者肠系膜动脉平滑肌KCa通道活性明显增高 ,血管内皮源性舒张因子对非高血压及高血压病患者血管平滑肌KCa通道均有激活作     

主动脉平滑肌钙激活钾通道的增龄变化

目的研究不同月龄大鼠主动脉平滑肌细胞钙激活钾通道的活性,探讨老化过程中血管平滑肌细胞钙激活钾通道的变化。方法取三组Wistar大鼠(分别为1月龄、6月龄、20月龄各20只)主动脉用酶消化法获得单个平滑肌细胞。以膜片钳技术检测细胞钙激活钾通道的活性,记录不同钳制电压下单通道电流的平均开放时间、平均关闭时间、平均开放概率、电流幅值,并绘制成电流—电压关系曲线。对比各月龄组Wistar大鼠主动脉平滑肌细胞钙激活钾通道活性。结果(1)各月龄组大鼠主动脉平滑肌细胞随着膜电位从+10 mV向+60 mV方向去极化,钙激活钾通道的电流强度逐渐加大,但加大幅度随月龄增加而降低。1月龄、6月龄和20月龄大鼠主动脉平滑肌细胞钙激活钾通道的电导值分别为192±47 ps、177±56 ps和163±35 ps,但差异无统计学意义(P>0.05)。(2)在内面向外式膜片上,20月龄和1月龄大鼠主动脉平滑肌细胞钙激活钾通道平均开放概率分别为0.009±0.001和0.015±0.004,两组比较具有显著性差异(P<0.01)。20月龄和1月龄大鼠主动脉平滑肌细胞钙激活钾通道平均关闭时间分别为2 260±653和2 512±185*,两组比较具有显著性差异(P<0.01)。(3)在同样的对称性高钾浴液中,加入不同浓度钙后,发现钙对各月龄组大鼠主动脉平滑肌细胞钙激活钾通道均有明显的激活作用,表现为平均开放概率逐渐加大,平均关闭时间逐渐缩短。但对各月龄相同钙浓度间比较发现,6月龄组平均开放概率比1月龄加大,而20月龄组平均开放概率和平均开放时间比6月龄和1月龄组均显著降低。结论大鼠主动脉平滑肌细胞钙激活钾通道的活性随着年龄增大而下降。     

原发性高血压患者血压参数与血管平滑肌细胞钙激活钾通道活性的相关性研究

目的:探讨原发性高血压患者血压参数与血管平滑肌细胞钙激活钾通道(KCa)活性的相关性。方法:切取21例原发性高血压患者(高血压组)及18例非高血压者(血压正常组)的肠系膜动脉小分支节段,用酶消化法获取血管平滑肌细胞,以膜片钳技术检测KCa通道的活性,通过Pclamp专用软件实时采样记录其平均开放时间(To),平均关闭时间(Tc),平均开放概率(Po)等。采用美国Meditech公司的ABPM-04动态血压监护仪,记录24h平均收缩压、24h平均舒张压、24h平均脉压、白昼血压、夜间血压、脉压变异性。结果:①两组血压参数:高血压组24h平均收缩压与平均舒张压、白昼平均收缩压与平均舒张压、夜间平均收缩压与平均舒张压、24h平均脉压、脉压变异性均显著高于血压正常组;②两组KCa通道活性变化:两组于细胞内面向外膜片下,不同浓度Ca2+较Ca2+浓度为0时Po、To增加,Tc缩短,有显著性差异;③高血压组血管平滑肌KCa活性与血压参数的相关性:24h平均舒张压、白昼及夜间平均舒张压、24h平均脉压升高时,Po显著增加,To显著延长,Tc显著缩短。但24h平均收缩压、白昼及夜间平均收缩压、脉压变异性变化与Po、To、Tc关系不明显。结论:原发性高血压患者肠系膜动脉平滑肌KCa通道活性明显高于非高血压者,但对Ca2+的敏感性降低。原发性高血压患者舒张压、     

动脉粥样硬化兔血管平滑肌大电导钙激活钾通道的活性探讨

目的研究动脉粥样硬化(atherosclerosis,AS)时血管平滑肌细胞(Vascular Smooth Musele Cell,VSMC)大电导钙激活钾通道(Large conductance of calcium-activated potassium channel,BKca)的活性变化。方法3月龄新西兰兔随机分为实验组(AS组)和对照组(N组),每组10只。AS组喂高脂饲料以建立AS模型,对照组喂普通饲料,两组均喂养8周．采用单通道膜片钳技术测定VSMC的BKca的电流。结果随着电压和Ca~(2 )浓度的增加,AS组和对照组的通道开放概率(Po)逐渐增大。AS组的Po较对照组明显增大(P＜0．001)。在Ca~(2 )浓度为10~(-6)M时,AS组和对照组的Po分别增加(68．9±34．4)倍和(126．4±70．6)倍,差别有显著性意义(P＜0．05)。结论AS组VSMC的BKca活性增强。AS组VSMC的BKca的Ca~(2 )敏感性降低。     

脱氢表雄甾酮对慢性缺氧大鼠肺动脉平滑肌细胞钙激活性钾通道的作用

目的　研究钾通道开放剂脱氢表雄甾酮 (DHEA)对慢性缺氧大鼠肺动脉平滑肌细胞钙激活性钾通道 (KCa)的作用和缺氧性肺动脉高压的降压作用。方法　 50只Wistar大鼠随机分为对照组 (A组 ,10只 )和慢性缺氧组 (B组 ) ,B组又随机分为B1、B2 、B3 、B4 组 (每组各 10只 ) ,B组大鼠均以常压缺氧 3周建立大鼠慢性缺氧肺动脉高压模型。采用急性酶分离法分离得到大鼠肺动脉平滑肌细胞(SMCs)。应用膜片钳技术 ,在对称性高钾溶液中 ,于急性分离的大鼠肺动脉平滑肌细胞的内面向外式膜片 (inside outpatch)上 ,分离出KCa电流。比较A组和B1组KCa电流活性 ;观察DHEA对B1组KCa通道电流的激活作用。应用右心插管技术 ,测定给药前后B2 、B3 、B4 组大鼠平均肺动脉压 (mPAP)和平均体动脉压 (mSAP)等血流动力学指标。结果　B组大鼠肺动脉平滑肌细胞KCa活性比A组大鼠显著降低 (P <0 0 1)。DHEA可明显激活慢性缺氧所抑制的B1组大鼠肺动脉平滑肌细胞的KCa电流。给缺氧大鼠静脉注射DHEA可明显降低其mPAP(P <0 0 1) ,而对mSAP无明显作用 (P >0 0 5)。结论　缺氧对KCa通道的抑制作用在缺氧 3周大鼠缺氧性肺动脉高压发病中起着重要作用 ;DHEA可直接激活KCa活性而拮抗慢性缺氧对KCa的抑制作用 ;DHEA对大鼠慢性缺氧性肺动脉高压可产     

碱性环境和高磷条件下大鼠胸主动脉平滑肌细胞成骨样表型转化中钙激活钾通道mRNA的表达

目的观察碱性环境中高磷诱导大鼠胸主动脉平滑肌细胞中电导钙激活钾通道(KCa3.1)与大电导钙激活钾通道(KCa1.1)表达的变化,以及探究钙激活钾通道与大鼠胸主动脉平滑肌细胞表型转化之间的关系。方法采用组织块贴壁法培养原代大鼠主动脉平滑肌细胞,利用10 mmol/Lβ-甘油磷酸钠制备血管平滑肌细胞钙化模型。使用HCl和Na HCO3调节培养基p H值。细胞随机分为5组:正常p H 7.4组、高磷p H 7.4组、高磷p H 7.7组、高磷p H 8.0组、TRAM-34干预组,共培养4天。用逆转录-聚合酶链反应检测各组细胞中KCa3.1、KCa1.1α、KCa1.1β、Runt相关转录因子2(Runx2)和平滑肌22α(SM22α)表达。结果与正常p H 7.4组相比,高磷组Runx2水平明显升高,且随着p H升高而表达量增加(P0.05);高磷组SM22α水平明显下降,且随着p H升高而表达量减少(P0.05)。与正常p H 7.4组相比,高磷p H 7.4组KCa3.1表达升高(P0.05),KCa1.1α表达下降(P0.05)。在高磷组中,随着p H升高KCa3.1、KCa1.1α表达量增加(P0.05)。在同一组中KCa3.1表达高于KCa1.1α(P0.05)。KCa1.1β表达在3个高磷组间未见统计学差异(P0.05)。与高磷p H 8.0组相比,TRAM-34干预组Runx2mRNA水平明显下降(P0.05),SM22αmRNA水平明显上升(P0.05)。相关分析显示,KCa3.1表达与Runx2表达呈正相关(r=0.945,P0.01),与SM22α表达呈负相关(r=-0.926,P0.01);在正常p H 7.4组、高磷p H 7.4组中KCa1.1α表达与Runx2表达呈负相关(r=-0.746,P=0.029),与SM22α表达呈正相关(r=0.971,P=0.002);在高磷p H 7.7组、高磷p H 8.0组中KCa1.1α表达与Runx2表达呈正相关(r=0.805,P=0.002),与SM22α表达呈负相关(r=-0.806,P=0.005);KCa1.1β表达与Runx2、SM22α表达不相关(r=0.414,P=0.356;r=-0.155,P=0.714)。结论碱性环境中平滑肌细胞钙激活钾通道表达参与高磷诱导的大鼠胸主动脉平滑肌细胞的表型转化。     

槲皮素对人肠系膜动脉平滑肌细胞钙激活钾通道的激活作用

目的:观察槲皮素对人肠系膜动脉平滑肌细胞钙激活钾通道(KCa)的影响,以探讨槲皮素在分子水平扩张血管的作用机制.方法:选取需择期行腹部手术并且血压正常的患者14例,取肠系膜动脉小分支,用急性酶消化法获得单个肠系膜动脉平滑肌细胞.然后应用单通道细胞膜片钳技术观察不同浓度槲皮素对肠系膜动脉平滑肌细胞KCa通道开放概率(NPo)、电流幅度值(Am)、平均开放时间(To)、平均关闭时间(Tc)等各指标的影响.结果:在内面向外式方式下,随着浴液中槲皮素浓度的增高,通道NPo明显增高:槲皮素浓度100μmol/L时,NPo由用药前(即0 μmol/L)的0.028 62±0.011 99增高到0.147 03±0.058 17(P＜0.01);通道Am、To变化不大(P＞0.05);而通道Tc明显缩短:由用药前(477.650±376.650)ms缩短到(112.643±114.365)ms(P＜0.01).结论:槲皮素能直接激活人类血管平滑肌细胞KCa通道而实现降血压效应.     

高血压病患者动脉平滑肌细胞钙激活钾通道变化及意义

目的　研究高血压病 (essential hypertension,EH )患者肠系膜动脉平滑肌 (mesenteric arterial sm oothmuscle,MASM)细胞钙激活钾通道 (Ca2 + activated K+ channel,KCa)改变及意义。方法　利用膜片钳单通道记录技术记录胞内和胞外 Ca2 +对无 EH家族史的正常血压者 (Nn 组 ) ,有 EH家族史的正常血压者 (Nf组 ) ,无 EH家族史的 EH患者 (En 组 ) ,有 EH家族史的 EH患者 (Ef组 )肠系膜动脉平滑肌 (MASM)细胞 KCa开放概率 (Po) ,平均开放时间 (To) ,平均关闭时间 (Tc)及电流幅值 (Am)的影响。结果　 (1)人动脉平滑肌 KCa开放具有明显的电压和 Ca2 +依赖性 ,其电导值大。 (2 )胞内 Ca2 + 浓度≥ 5× 10 - 7m ol/ L时 ,En 组、Ef 组 MASM细胞 KCa的 Po 分别低于 Nn 组、Nf组 ,To 缩短。(3)胞外 Ca2 +浓度≥ 1.8× 10 - 3m ol/ L 时 ,En 组、Ef 组 MASM细胞 KCa的 Po 分别高于 Nn 组、Nf组 ,To延长 ,Tc缩短。结论　 (1) EH患者 MASM细胞 KCa对胞内 Ca2 + 敏感性降低 ,这可能是促进高血压发生的机制之一。(2 ) EH患者 MASM细胞对胞外 Ca2 +敏感性增加 ,可能与胞外 Ca2 +内流增加导致胞内 Ca2 +增加有关     

梗阻性不稳定膀胱逼尿肌细胞内Ca2+及N型Ca2+通道的变化及意义

目的对Ca2 和N型Ca2 通道在兔不全梗阻性不稳定膀胱逼尿肌细胞中的变化进行研究。方法成年同龄雄性新西兰白兔30只,随机分两组,15只中膀胱出口不全梗阻8w尿流动力学证实为不稳定膀胱者为实验组,15只为对照组(成年雄性新西兰白兔仅仅手术游离兔膀胱颈而不做结扎梗阻为对照组)。采用急性酶法分离及传代培养的方法获得单个膀胱逼尿肌细胞,采用激光共聚焦显微镜观察模型膀胱逼尿肌细胞静态Ca2 浓度;运用共聚焦显微镜及免疫组织化学方法观察N型Ca2 通道在梗阻性不稳定膀胱逼尿肌细胞中的变化。结果静息状态下不全梗阻性不稳定膀胱组逼尿肌细胞内游离钙离子浓度明显增高(Ca2 超负荷);共聚焦显微镜及免疫组化均证实不稳定膀胱逼尿肌细胞膜之N钙离子通道数量明显增多。结论膀胱逼尿肌细胞Ca2 及其N型Ca2 通道病理性改变是出现不稳定膀胱的重要因素。     

Mechanical properties of tracheal smooth muscle are impaired in the rabbit with experimental cardiac pressure overload

STUDY OBJECTIVES/DESIGN: Impaired function of striated and arterial smooth muscle is known to occur in humans and animals with various forms of cardiac diseases, but limited information is available on the mechanical behavior of airway smooth muscle. We tested the hypothesis that the baseline mechanical properties of tracheal smooth muscle (TSM) were impaired at an early stage of cardiac overload. ANIMALS: We used a model of cardiac hypertrophy induced by surgical abdominal aortic stenosis (AS) in adult rabbits. Twelve animals with AS and 8 sham-operated control rabbits were studied 12 weeks after surgery. In rabbits with AS, the heart weight/body weight ratio was higher than in control rabbits (2.36 +/- 0.43 g/kg vs 1.98 +/- 0.20 g/kg, p < 0.05) [mean +/- SD], attesting to moderate cardiac hypertrophy. No clinical signs of congestive heart failure were observed. MEASUREMENTS: Isolated TSM strips were electrically stimulated at 37 degrees C, 2.5 mM [Ca(2+)](0), against 8 to 10 load levels, from zero load to full isometry. Force-velocity relationship was elicited using the conventional afterloaded isotonic method. RESULTS: Peak isometric tension was lower in rabbits with AS than in control rabbits (25 +/- 11 mN/mm(2) vs 34 +/- 14 mN/mm(2), p < 0.05), whereas maximum unloaded shortening velocity, maximum extent of muscle shortening, and relaxation parameters did not differ between groups. The curvature of the force-velocity relationship (which reflects the myothermal economy of force generation) and peak mechanical efficiency were lower in rabbits with AS than in control rabbits. CONCLUSIONS: These results indicate that the contraction of isolated rabbit TSM was less powerful and less economical in cardiac hypertrophy, attesting to early impairment of the mechanical properties of TSM during cardiac overload.     

Guanosine 5''-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Ca2+-activated K+ channels (PKCa channels) account for the predominant K+ permeability of many types of smooth muscle cells. When activated, they oppose depolarization due to Na+ and Ca2+ channel activity. Several vasodilatory agents that increase intracellular cGMP levels (e.g., nitroprusside, adenosine, and atrial natriuretic factor) enhance the activity of these high-conductance PKCa channels in on-cell patches of bovine aortic smooth muscle cells. In addition, dibutyryl-cGMP (1.0 mM) causes a similar increase in channel activity. To pursue the mechanism of channel modulation by these agents, a series of guanine and adenine nucleotides were evaluated by using inside-out excised patches. Whereas cAMP, AMP, ADP, and ATP were ineffective, all of the corresponding guanine nucleotides potentiated PKCa channel activity when tested at a high concentration (500 microM). However, only GMP consistently enhanced channel activity in the 1-100 microM range by increasing the percent open time and frequency of opening of these channels over a wide range of potentials and Ca2+ levels without affecting single-channel conductance. Thus, GMP is a potent modulator of PKCa channels and it, rather than cGMP, may mediate the action of the vasodilators examined in this study.     

粒细胞集落刺激因子与动脉粥样硬化对外周血内皮祖细胞数量的影响

目的观察粒细胞集落刺激因子（granuloeyte colony-stimulating factor,G-CSF）与动脉粥样硬化（atherosclerosis,AS）对外周血内皮祖细胞（EPC）的影响。方法将32只雄性新西兰白兔随机分为G组（重组人粒细胞集落刺激因子rhG-CSF50彬d）、G＋AS组（rhG-CSF 50μg/d、高脂饲料）、AS组（高脂饲料）及对照组,每组各8只。4组动物分别于实验前及第1、4、8、12周采血,培养7天后,用荧光显微镜观察鉴定FITC-UEA-1和Dil-acLDL双染色阳性细胞为正在分化的EPC;细胞培养3天后,通过流式细胞仪计数各组PE-CD34、FITC-CD133双阳性细胞为EPC;第12周测血清一氧化氮、血脂水平并做主动脉斑块分析。结果实验前,各组EPC含量均很低;用G-CSF治疗1周,G组及G＋AS组EPC迅速升高（与用药前比较增加了约13倍,P〈0．001）,继续给药G组EPC维持在一个较高水平（第1、4、8、12周比较P〉0．05）;给予高脂饮食后,G＋AS组EPC数量逐渐下降（第4、8周与对照组比较P〈0．001、P〈0．001;第12周与对照组比较P=0．326）;对照组EPC一直处于低水平,AS组EPC数量较对照组低,但两组各周比较P〉0．05。经过12周的高脂喂养,G＋AS组及AS组主动脉均形成了动脉粥样硬化斑块,但G＋AS组斑块面积、斑块／内膜面积均低于AS组（P〈0．01）。第12周G＋AS组及AS组一氧化氮含量均低于G组和对照组（P〈0．001）。结论EPC与动脉粥样硬化关系密切,AS损害内皮,减少EPC,G-CSF对EPC有动员作用,能够增加外周血EPC数量,因而对血管有保护作用,抑制AS进展。     

Reduction of large-conductance Ca²(+) -activated K(+) channel with compensatory increase of nitric oxide in insulin resistant rats

Cardiovascular disease prevalence and mortality are both increased by insulin resistance, hypertension, and atherosclerosis. The large-conductance Ca(2+)-activated K(+) channel (BK(Ca)) plays a pivotal role in the diastolic function of vascular smooth muscle cells. However, the role of this channel in insulin resistance remains unknown. Male Sprague-Dawley rats were randomly divided into an insulin resistant group and control group. We investigated the BK(Ca) current and subunit expression in myocytes from aortas and mesenteric arteries by Western blot, real-time PCR and the whole-cell patch-clamp methods. BK(Ca) current was decreased in smooth muscle cells in insulin resistant rats, compared with that in control group. Peak BK(Ca) current at + 60 mV was significantly decreased after iberiotoxin (IBTX) perfusion at 100 nmol/L (64.2 ± 4.7 versus 20.3 ± 3.5% in thoracic aortas and 65.6 ± 6.2 versus 29.3 ± 3.9% in mesenteric arteries, both p < 0.01). However, there was no significant difference in BK(Ca) alpha subunit between the two groups, both at the level of mRNA and protein. BK(Ca) beta 1 subunit expression in aortas and mesenteric arteries from the insulin resistant group was lower than in those from control group. The plasma level of nitric oxide was higher in the insulin resistant group than in the control group. Our results demonstrated that the BK(Ca) channel is decreased both in macrovessels and microvessels in insulin resistant rats. These impairments may be related to the down-regulation of β1 subunit expression and compensatory increase in plasma nitric oxide levels.     

Calcium modulation of vascular smooth muscle ATP-sensitive K(+) channels: role of protein phosphatase-2B

ATP-sensitive K(+) (K(ATP)) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP](i)) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that K(ATP) channels are insensitive to Ca(2+), although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca(2+) on whole-cell K(ATP) current in rat aortic smooth muscle cells recorded in a physiological [ATP](i) and K(+) gradient. Under control recording conditions, cells had a resting potential of approximately -40 mV when bathed in 1.8 mmol/L Ca(2+). The K(ATP) channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP](i) from 3 to 0.1 mmol/L hyperpolarized cells to approximately -60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca(2+) levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca(2+)](i) showed that K(ATP) current was maximally activated at 10 nmol/L [Ca(2+)](i) and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca(2+)-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by approximately 20 to 25 mV. We report for the first time that K(ATP) channels can be modulated by Ca(2+) at physiological [ATP](i) and conclude that modulation occurs via protein phosphatase type 2B.     

大鼠动脉粥样硬化斑块中T淋巴细胞亚群及Kv1.3通道蛋白的表达

目的探究Kv1.3通道蛋白与动脉粥样硬化(AS)模型中活化的T淋巴细胞间的关系。方法 Wistar雄性大鼠24只,随机分为对照组(n=10)和AS组(n=14),采用高脂饲料喂养方法建立AS模型。分别于实验开始前,实验第8周,实验第12周观察各组大鼠体重变化。于第12周处死大鼠前取血,检测血清中总胆固醇(TC)、低密度脂蛋白(LDL-L)、高密度脂蛋白(HDL-L)和甘油三酯(TG)的水平。通过病理HE染色及免疫组织化学方法观察AS斑块内T淋巴细胞亚群分布和Kv1.3通道蛋白表达的改变。结果 AS组体重、TC、LDL-C较对照组明显升高(P均<0.05);HDL-C和TG两组无差异。AS组主动脉管壁可见明显斑块形成,对照组血管壁各层的组织结构正常。AS组动脉斑块部位内膜下及中膜层可见CD4+与CD8+T淋巴细胞聚集,以CD4+T淋巴细胞聚集为主,在病变部位Kv1.3通道蛋白表达增加。对照组血管内膜、中膜中未见T淋巴细胞聚集及KV1.3通道蛋白的表达。结论 Kv1.3通道可能在调节AS斑块部T淋巴细胞亚群的激活中起着重要作用。     

Effect of hypercholesterolaemia on voltage-operated calcium channel currents in rabbit arterial smooth muscle cells

Cholesterol is a major component of cell membranes and influences membrane fluidity. Watanabe heritable hyperpercholesterolaemic rabbits (WHHL) possess defective receptors for low density lipoprotein leading to increased plasma cholesterol, accumulation of cholesterol in the arterial wall and atherosclerosis. In this study calcium channel currents (IBa) were compared using conventional whole cell voltage clamp techniques in ear artery cells isolated from control New Zealand White rabbits (NZ) with those from WHHL. IBa were larger in cells isolated from NZ than from WHHL, however cell capacitance was also greater in NZ cells. Consequently, there was no significant difference in current density between NZ and WHHL cells either in the absence of drug or in the presence of the calcium channel agonist (+)202 791. Current voltage-relationships, kinetics of fast inactivation and steady-state inactivation of IBa also did not differ significantly between WHHL and NZ. These findings suggest that hypercholesterolaemia in WHHL has no direct effect on calcium channel current density or voltage-modulation in arterial smooth muscle cells.     

Down-regulation of L-type calcium channels in inflamed circular smooth muscle cells of the canine colon

BACKGROUND & AIMS: Circular smooth muscle phasic contractions and tone are suppressed during colonic inflammation, but the contributing factors are poorly understood. This study investigated if the expression level of voltage-gated long-lasting (L-type) Ca(2+) channel protein and functional Ca(2+) channel current are down-regulated in the circular muscle cells of the inflamed canine colon. METHODS: L-type Ca(2+) channel expression was compared between normal and inflamed smooth muscle cells by Western immunoblots using an antibody directed against the pore-forming alpha 1C-subunit, and patch-clamp methods were used to evaluate Ca(2+) channel current density. RESULTS: The expression of the L-type Ca(2+) channel protein was significantly reduced in inflamed compared with normal circular smooth muscle cell membranes, and this finding was associated with suppressed levels of Ca(2+) channel current in patch-clamped cells. The L-type Ca(2+) channel current in normal and inflamed cells increased proportionately in response to Bay K 8644, but the maximal current density was still lower in the inflamed cells. Acetylcholine increased the L-type Ca(2+) channel current in normal but not in inflamed cells. CONCLUSIONS: The expression level of L-type Ca(2+) channels is down-regulated in the circular smooth muscle cell membranes of the inflamed colon, which may result in reduced Ca(2+) influx. The functional and pharmacologic properties of the channels seem normal. Although some Ca(2+) channels are still present in the inflamed cells, acetylcholine does not activate these channels, which may be caused by additional upstream defects in the receptor signaling cascade. The down-regulation of L-type Ca(2+) channel expression may suppress circular smooth muscle contractions in the inflamed colon and contribute to the abnormalities in motility and digestion observed during inflammatory disorders.