雌二醇对绝经后妇女肠系膜动脉平滑肌细胞BK 的激活作用

生理学报 Acta Physiologica Sinica , April 25, 2012, 64(2): 121–128https://www.wendangku.net/doc/c610872405.html,

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Received 2011-10-18 Accepted 2011-12-29

The work was supported by the National Natural Science Foundation of China (No. 30670763) and the Natural Science Fund from Department of Education, Sichuan Province, China (No. 10ZB123).

*

Corresponding author. Tel: +86-830-3160619; E-mail: wyangyan@https://www.wendangku.net/doc/c610872405.html,

β-雌二醇对绝经后妇女肠系膜动脉平滑肌细胞BK Ca 的激活作用

程 俊，曾晓荣，李鹏云，鲁婷婷，谭晓秋，文 静，杨 艳*

四川省泸州医学院心肌电生理学研究室，泸州医学院心肌电生理学省部共建教育部重点实验室，泸州 646000

摘 要：本文旨在研究雌激素对动脉平滑肌细胞大电导钙激活钾通道(large-conductance Ca 2+-activated potassium channels, BK Ca )的作用。取绝经后女性患者手术切除物中的肠系膜动脉，应用急性酶分离法分离出肠系膜动脉平滑肌细胞，按动脉平滑肌细胞的来源分为非高血压(non-hypertension, NH)组及原发性高血压病(essential hypertension, EH)组，采用单通道膜片钳技术研究β-雌二醇(β-estradiol, β-E 2)及其受体(ER)抑制剂ICI 182780对BK Ca 的作用。结果显示，肠系膜动脉平滑肌细胞BK Ca 通道的开放具有电压依赖性和钙依赖性，可被IbTX 所阻断。100 μmol/L β-E 2能够显著增加NH 和EH 组BK Ca 的通道开放概率(open probability, Po)，且β-E 2处理后NH 组BK Ca 的通道Po 显著大于EH 组。ICI 182780可显著抑制β-E 2对BK Ca 通道的增强作用。以上结果提示，β-E 2能够通过激活ER 显著增强绝经后妇女肠系膜动脉平滑肌细胞的BK Ca 功能，而高血压可以削弱β-E 2对动脉平滑肌细胞BK Ca 功能的增强作用。

关键词：大电导钙激活钾通道；β-雌二醇；雌激素受体；人肠系膜动脉平滑肌；高血压中图分类号：R331

β-estradiol activates BK Ca in mesenteric artery smooth muscle cells of post-menopause women

CHENG Jun, ZENG Xiao-Rong, LI Peng-Yun, LU Ting-Ting, TAN Xiao-Qiu, WEN Jing, YANG Yan *

Department of Electrophysiology, Institute of Cardiovasology, State Key Laboratory, Luzhou Medical College, Luzhou 646000, China Abstract: The aim of the present study was to study the effect of β-estradiol (β-E 2) on the large-conductance Ca 2+-activated potassium (BK Ca ) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of β-E 2 and ICI 182780 (a specific blocker of estrogen receptor) on BK Ca in the SMCs. The results showed the opening of BK Ca in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. β-E 2 (100 μmol/L) significantly increased open probability (Po) of BK Ca in both NH and EH groups. After β-E 2 treatment, NH group showed higher Po of BK Ca compared with EH group. ICI 182780 could inhibit the activating effect of β-E 2 on BK Ca in no matter NH or EH groups. These results suggest β-E 2 activates BK Ca in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of β-E 2 on BK Ca .

Key words: large-conductance calcium-activated potassium channel; β-estradiol; estrogen receptor; human mesenteric artery smooth

muscle; hypertension

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原发性高血压病(essential hypertension,EH)是严重危害人体健康的常见病，其发病机理尚不清楚。高血压病临床表现主要为小动脉与微动脉血管张力增加及血管对扩张的缓冲能力下降，而持续的动脉张力增加是高血压发展的关键因素。血管张力的调节主要是通过细胞膜Ca2+通道介导的Ca2+内流(voltage-dependent calcium-induced calcium currents, VDCCs)和胞内Ca2+库释放Ca2+。由于血管平滑肌的肌浆网较不发达，平滑肌的收缩主要依赖于VD-CCs。在这一过程中，平滑肌细胞膜的各种离子通道也起着重要的作用。钙激活钾通道(calcium-acti-vated potassium channels, K Ca)是血管平滑肌上最主要的外向电流通道，在控制平滑肌细胞的膜电位中起主导作用，其中大电导钙激活钾通道(large-con-ductance calcium-activated potassium channels, BK Ca)携带70%~80%的外向电流，是血管平滑肌上表达密度最高、最重要的K Ca通道[1]。BK Ca广泛存在于血管平滑肌，其开放使K+外流增加，细胞膜超极化，引起VDCCs关闭，减少胞外Ca2+内流，降低胞内Ca2+浓度，从而引起血管舒张。

EH存在着性别差异，流行病学资料表明绝经前妇女高血压的发病率明显低于同龄男性，但绝经后这种差别则不复存在[2]。研究显示，雌激素对于妇女正常心血管功能的维持和调节具有重要意义，其可通过功能性的雌激素受体(estradiol receptor, ER)调节血管平滑肌的结构和功能[3]。雌激素作为一种主要由卵巢分泌的女性激素，对电压依赖性钙通道、氯通道都具有阻断作用，而对BK Ca具有激活作用[4,5]。然而雌激素作用的细胞内机制，尚未明确阐释。目前人们普遍认为雌激素是通过基因组和非基因组两种方式作用于细胞[6]。我们猜想β-雌二醇(β-estradiol, β-E2)可能通过非基因组机制影响BK Ca功能，进而影响血压。为了验证这一猜想，本实验采用单通道膜片钳技术，观察了β-E2对绝经后妇女肠系膜动脉平滑肌细胞BK Ca电流的作用。

1材料和方法

1.1研究对象 标本取自于绝经后女性患者腹部手术切除物中肠系膜动脉细小分支，该切除物的使用得到了泸州医学院附属医院伦理委员会的许可。采用《中国高血压指南》(2005年修订版)中的高血压诊断标准(收缩压≥140 mmHg和/或舒张压≥90 mmHg)对患者资料进行分组。总样本数13例：非高血压(non-hypertension, NH)组7例(年龄56~68岁，血压90~135/60~85 mmHg)，原发性高血压病组(EH组)6例(年龄55~73岁，血压150~185/ 100~120 mmHg)，排除继发性高血压病及其它血管疾病。两组患者在术中探查均未见肠道血管病变。

1.2 主要试剂 木瓜蛋白酶，XI型胶原酶，透明质酸酶，白蛋白，K-aspartate，HEPES，EGTA，β-E2，ER抑制剂ICI 182780和IbTX均购自美国Sigma 公司；余为国产分析纯试剂。

1.3 主要仪器 膜片钳放大器(CEZ-2200, Nihon Kohden, Japan)；倒置相差显微镜(Axiovert135, Zeiss, Germany)；微管电极拉制仪(PC-10, Narishige, Ja-pan)；A/D-D/A转换器(Digidata-1200A, Axon Instru-ments, USA)；恒温水浴振荡器(GLD-30938, Germa-ny)。

1.4 实验溶液的配制 溶液I (mmol/L)：NaCl 127，KCl 5.9，MgCl2 1.2，葡萄糖 12，HEPES 10，CaCl2

2.4，pH用NaOH调至7.40。溶液II (mmol/L)：NaCl 127，KCl 5.9，MgCl2 1.2，葡萄糖 12，HEPES 10，pH用NaOH调至7.40。电极液(mmol/L)：K-aspartate 40，KCl 100，HEPES 10, CaCl2 1, pH用KOH调至7.40。浴液(mmol/L)：K-aspartate 100，KCl 40，HEPES 10, EGTA 1, CaCl2 (按实验设计加入)，pH用KOH调至7.40。酶液：酶I (mg/mL)：木瓜蛋白酶1.3，DTT 0.15，白蛋白1.7，用溶液II 溶解；酶II (mg/mL)：XI型胶原酶1.3，透明质酸酶 1.5，白蛋白1.7，用溶液II溶解。

1.5 细胞分离 从腹部手术切除的肠管中小心取得含血管的组织块，置入4 °C溶液I中，剔除结缔组织，纵行剖开，剪成2 mm × 2 mm × 1 mm的组织条块。于酶液I中37 °C恒温水浴箱中振荡消化约9 min，再置于酶液II中消化约4 min，即可得到细胞膜完整光滑、折光性好的细胞，然后静置于4 °C冰箱中保存备用。

1.6 通道电流的记录 在室温下(20~25 °C)，用微电极操纵器控制微管电极浸入浴液，操纵微推进器使电极尖端贴附在细胞表面，在示波器上可见应答电流减少，施加10~20 cm H2O负压，形成高阻封接(阻抗高达10 GΩ以上)，即已经形成细胞贴附式膜片(cell-attached patch)。封接形成后，提起微管电极，在空气中暴露数秒，再放入浴槽中，即形成内面向外式膜片(inside-out patch)。

1.7 统计分析方法 实验数据以means ± SD表

程 俊等：β-雌二醇对绝经后妇女肠系膜动脉平滑肌细胞BK Ca 的激活作用

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示。采用OriginPro 8.0软件对数据进行配对t 检验和独立样本t 检验，P < 0.05时认为差异有统计学意义。

2 结果

2.1 BK Ca 通道的电导

在140 mmol/L 对称性高钾溶液([K +]o :[K +]i = 140:140)中，在inside-out patch 下，NH 组和EH 组平滑肌细胞膜BK Ca 通道电导值分别为(176.3 ± 21.1) pS (n = 7)和(182.9 ± 29.2) pS (n = 6)，两组细胞膜电导值之间无显著性差异。图1示NH 组和EH 组的电流-电压关系，两条直线的斜率即为BK Ca 通道的电导值。2.2 BK Ca 通道的电压依赖性

在cell-attached patch 和inside-out patch 下，NH 组(n = 7)和EH 组(n = 6)平滑肌细胞膜通道开放概率(open probability, Po)、开放时间(open time, To) 和幅度 (amplitude, Am)均呈膜电位依赖性增加；两组关闭时间(close time, Tc)均呈膜电位依赖性降低(表1、2，图2)。2.3 BK Ca 通道的钙依赖性

在inside-out patch 记录方式和+40 mV 膜电位条件下，NH 组和EH 组通道Po 、To 均呈[Ca 2+]i (1 × 10?8~1 × 10?5 mol/L)依赖性增大；两组Am 随[Ca 2+]i 的升高增加不明显；两组Tc 呈[Ca 2+]i 依赖性降低(表3)。当[Ca 2+]i 为1 × 10?8~1 × 10?5 mol/L 时，NH 组Po 显著高于EH 组(表3，图3)。当[Ca 2+]

i

图 1. Inside-out patch 下NH 组和EH 组的电流-电压关系

Fig. 1. The current-voltage (I -V ) relationships in NH and EH groups in inside-out patch. Means ± SD, n NH = 7, n EH = 6.

表1. Cell-attached patch 记录方式下NH 组和EH 组BK Ca 通道的电压依赖性

Table 1. The voltage dependence of BK Ca channels of NH and EH groups in cell-attatched patch

V m (mV) Group Am (pA) Po To (ms) Tc (ms)+10 NH 0.669 ± 0.025 0.003 ± 0.002 0.898 ± 0.054 2 316.731 ± 242.856 EH 0.651 ± 0.019 0.004 ± 0.001 0.912 ± 0.029 2 124.990 ± 220.203

+20

NH 0.712 ± 0.021**

0.008 ± 0.002** 3.868 ± 1.021** 1 026.956 ± 100.256**

EH 0.701 ± 0.020* 0.008 ± 0.003** 3.001 ± 1.009** 1 244.373 ± 98.661**

+30 NH 0.912 ± 0.058** 0.016 ± 0.002** 15.243 ± 5.213** 812.061 ± 20.994**

EH 0.848 ± 0.057* 0.017 ± 0.001** 16.189 ± 5.191** 799.901 ± 24.546**

+40 NH 1.512 ± 0.149** 0.022 ± 0.004** 22.409 ± 4.212** 461.990 ± 9.911**

EH 1.251 ± 0.216** 0.030 ± 0.007** 22.956 ± 5.772** 464.071 ± 8.979**

+50 NH 2.021 ± 0.073** 0.046 ± 0.008** 25.952 ± 6.124** 378.052 ± 10.454**

EH 1.928 ± 0.055** 0.043 ± 0.006** 25.343 ± 7.066** 383.837 ± 11.645*

+60 NH 2.601 ± 0.195** 0.068 ± 0.008** 30.948 ± 9.957** 293.687 ± 23.515**

EH 2.395 ± 0.226** 0.063 ± 0.003** 29.139 ± 10.013** 287.451 ± 25.120*

Means ± SD, n NH = 7, n EH = 6. *P < 0.05, **P < 0.01 vs the same group under +10 mV . V m: membrane voltage; Am: membrane ampli-tude; Po: open probability; To: open time; Tc: close time.为1 × 10?7~1 × 10?6 mol/L 时，NH 组To 显著高于EH 组；当[Ca 2+]i 为1 × 10?7~1 × 10?6 mol/L 时，NH 组Tc 显著低于EH 组(表3)。2.4 IbTX 对BK Ca 电流的阻断作用

在inside-out patch 下，加入200 nmol/L BK Ca 特异性阻断剂IbTX ，在+30 mV 膜电位时，膜片外向电流几乎完全被阻断(图4)。

2.5 β-E 2及ER 特异性抑制剂ICI 182780对BK Ca 通道的作用

在cell-attached patch 记录方式和+40 mV 膜电位条件下，100 μmol/L β-E 2使NH 组和EH 组BK Ca

生理学报Acta Physiologica Sinica, April 25, 2012, 64(2): 121–128 124

表2. Inside-out patch记录方式下NH组和EH组BK Ca通道的电压依赖性

Table 2. The voltage dependence of BK Ca channels of NH and EH groups in inside-out patch

V m (mV) Group Am (pA) Po To (ms) Tc (ms)

±±±±EH 1.535 ± 0.180 0.001 ± 0.000 0.922 ± 0.687 4 308.987 ± 667.909 +20 NH 3.722 ± 0.398* 0.002 ± 0.001** 2.907 ± 1.011* 3 809.756 ± 400.022* EH 3.761 ± 0.218* 0.002 ± 0.000** 2.898 ± 1.001* 3 923.690 ± 385.577* +30 NH 4.212 ± 0.307** 0.005 ± 0.001** 3.811 ± 1.342** 1 022.482 ± 189.709** EH 4.465 ± 0.513** 0.004 ± 0.001** 3.762 ± 1.633** 1 210.796 ± 198.582** +40 NH 5.381 ± 0.821** 0.007 ± 0.003** 23.561 ± 2.998** 728.395 ± 109.904** EH 5.184 ± 1.004** 0.005 ± 0.002** 22.119 ± 2.636** 750.233 ± 86.119**

+50 NH 6.278 ± 1.028** 0.030 ± 0.008** 33.211 ± 3.52** 320.005 ± 55.125** EH 6.081 ± 1.032** 0.031 ± 0.006** 30.414 ± 4.421** 402.564 ± 60.056**

+60 NH 7.182 ± 0.987* 0.052 ± 0.008** 55.327 ± 8.113* 83.298 ± 49.687* EH 7.279 ± 0.782* 0.049 ± 0.009** 51.250 ± 9.435* 73.056 ± 51.279* Means SD, NH = 21, EH = 15. ***

plitude; Po: open probability; To: open time; Tc: close time.

图 2. 人肠系膜动脉平滑肌细胞BK Ca电流

Fig. 2. The currents of BK Ca channels in human mesenteric artery smooth muscle cells．A, B: Representative BK Ca single channel current traces in EH group under different voltages (+10–+60 mV) in cell-attached (A) and inside-out (B) patches. C, D: Representa-tive BK Ca single channel current traces in NH group under different voltages (+10–+60 mV) in cell-attached (C) and inside-out (D) patches.

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表3. Inside-out 记录方式下NH 组和EH 组BK Ca 通道的Ca 2+依赖性

Table 3. The Ca 2+ dependence of BK Ca channels of NH and EH groups in inside-out patch

Ca 2+ (mol/L) Group Am (pA) Po

To (ms)

Tc (ms)

1 × 10?8 NH 8.381 ± 0.621 0.021 ± 0.014 5.05

2 ± 0.897 2 252.665 ± 521.770

EH 10.380 ± 1.004 0.005 ± 0.001## 4.547 ± 0.969 2 043.231 ± 380.5581 × 10?7

NH 8.412 ± 0.808 0.034 ± 0.023** 6.997 ± 1.292** 700.958 ± 112.868**

EH 10.311 ± 1.112 0.014 ± 0.004**## 6.005 ± 0.824**## 802.332 ± 227.019**##5 × 10?7

NH 8.351 ± 1.221 0.408 ± 0.288** 9.152 ± 1.168** 54.756 ± 29.010**

EH

9.533 ± 1.714 0.316 ± 0.299**# 8.812 ± 1.055**# 67.562 ± 34.978**#1 × 10?6

NH

8.924 ± 1.522 0.899 ± 0.025** 28.665 ± 5.341** 10.260 ± 8.384**

EH

10.382 ± 1.681 0.670 ± 0.147**# 22.906 ± 4.278**# 21.921 ± 2.351**#1 × 10?5 NH 9.222 ± 0.989 1.413 ± 0.182* 40.897 ± 5.122* 3.291 ± 1.021**

EH

10.012 ± 1.828 0.851 ± 0.153*# 38.774 ± 3.702* 4.214 ± 0.605**

Means ± SD, n NH = 4, n EH = 5. *P < 0.05, **P < 0.01 vs the same group under 1 × 10?8 mol/L Ca 2+. #P < 0.05, ##P < 0.01 vs the NH group. Am: membrane amplitude; Po: open probability; To: open time; Tc: close time.

图 3. 在inside-out patch 下记录人肠系膜动脉平滑肌细胞BK Ca 通道的Ca 2+依赖性

Fig. 3. The Ca 2+-dependence of BK Ca channels in human mesenteric artery smooth muscle cells of NH (A ) and EH (B ) groups in

inside-out patch.

通道的Po 均显著增加，此时EH 组Po 显著小于NH 组。加入β-E 2 15 min 后，再加入100 μmol/L ICI 182780, 15 min 后可观察到NH 组和EH 组BK Ca 通道的Po 均显著减小，此时两组Po 之间差异不明显(图5)。加入ICI 182780之后，再加入100 μmol/L 的β-E 2，15 min 后观察NH 组和EH 组BK Ca 通道Po 均无明显变化。

3 讨论

本实验结果显示，NH 组和EH 组BK Ca 通道的Po 、To 和Am 均呈膜电位依赖性增大，而两组Tc 呈膜电位依赖性降低，且NH 组BK Ca 活性高于

EH

图 4. 200 nmol/L IbTX 几乎可以完全阻断BK Ca 电流

Fig. 4. In outside-out patch, 200 nmol/L IbTX could almost completely block BK Ca at +30 mV .

生理学报 Acta Physiologica Sinica , April 25, 2012, 64(2): 121–128

126组。虽然Liu 等[7]的结果显示自发性高血压大鼠(spontaneouly hypertensive rats, SHRs) BK Ca 通道功能强于正常血压的大鼠，但是他们认为出现该现象的原因是BK Ca 通道的表达增多，而不是单个通道的电导增加或是开放的几率增加，而且BK Ca 通道功能增强也有可能是继发于血压的升高。BK Ca 通道功能的增强可以通过治疗高血压治疗得到逆转[8]。由此可见，本文的结果和Liu 等[7]的结果并行不悖，可以相互补充。

本实验结果显示，BK Ca 通道具有明显的Ca 2+依赖性，且NH 组Po 增加幅度大于EH 组，说明NH 组对Ca 2+的敏感性高于EH 组，这与Zhou 等[9]的报道一致。由于BK Ca 通道β1亚单位可增加α亚单位的Ca 2+

敏感性，因而我们推测高血压状态下

图 5. 在cell-attached patch 下NH 组和EH 组绝经后妇女肠系膜动脉平滑肌细胞BK Ca 通道对β-E 2和ICI 182780的反应

Fig. 5. Representative BK Ca single channel current traces of NH (A ) and EH (B ) groups with β-E 2 alone or in combination with ICI 182780. C : The open probability (Po) of BK Ca in NH and EH groups in cell-attached patch. Means ± SD, n NH = 7, n EH = 6. **P < 0.01 vs control. #P < 0.05.

BK Ca 对Ca 2+敏感性降低的具体机制可能是α、β1亚单位本身发生变异和/或其耦联中断所致[10]。

绝经后高血压病是最常见的女性心脏病的危险因素，尤其是动脉压随年龄变化明显的女性，伴随着雌激素水平的变化，心血管疾病的危险度随之上升。绝经后女性患高血压的机制目前尚未完全明确，但研究证实，雌激素水平下降，可能在绝经后妇女高血压的发病中起重要作用。有文献报道，长期应用激素替代疗法(hormone replacement therapy, HRT)对患有高血压的绝经后妇女疗效尚不确切，血压可受雌激素应用的类型、剂量、方式及时序等因素影响[11,12]，如Fisman 等[13]的实验结果显示舌下服用雌激素可降低高血压病人的血压，而对绝经后正常血压女性不起作用。不过也有实验结果显示在绝经

程俊等：β-雌二醇对绝经后妇女肠系膜动脉平滑肌细胞BK Ca的激活作用127

后正常血压女性中，皮下注射雌激素比口服更能降低血压[14]。一般认为，雌激素的快速扩血管作用是主要由雌激素对Ca2+通道和K+通道的作用所介导[4,5]。Thomas等[15]证明雌激素抑制Ca2+内流产生的快速血管松弛效应不依赖ER，而是依赖Ca2+。但Prakash 等[16]的研究结果显示，雌激素通过ER介导机制促进Ca2+外流，减少冠脉平滑肌细胞内的Ca2+浓度。动物实验还证实，超过生理剂量的雌激素能直接阻滞心血管平滑肌Ca2+通道使血管扩张[17]。但高血压病条件下雌激素是否通过ER对血管平滑肌BK Ca 起作用，尚未见相关报道。本实验结果显示，β-E2可激活肠系膜动脉平滑肌细胞BK Ca电流，而ICI 182780部分抑制此效应，表明β-E2部分通过ER来增加BK Ca的开放。和我们的结果相似，White等[18]的结果显示β-E2在没有内皮素的参与下能够直接激活人冠脉平滑肌细胞BK Ca，并且是通过cGMP/ PKG信号转导途径；Han等[19]的结果显示ER对人冠脉平滑肌细胞K+通道起调节作用；Bakir等[20]通过在体应用ICI 182780证实雌激素的血管舒张反应需依赖ER，而ER表达下调或ER被敲除的小鼠中，雌激素的舒血管效应也被抑制[21,22]。

本实验结果还显示EH组BK Ca对β-E2的反应比NH组低，表明高血压病患者血管平滑肌BK Ca 调节能力下降。目前出现这种结果的原因还无法确定，我们下一步将运用分子生物学技术深入研究血管平滑肌BK Ca、ER、β-E2和EH之间的关系，也为HRT应用于临床绝经后女性高血压患者的治疗提供实验依据。

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