雄激素对高血压患者肠系膜动脉血管BK_（Ca）作用的研究
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摘要
目的：高血压是危害人类健康的常见病,主要表现为小动脉张力增加,不能对抗增高的血管紧张度及对血管活性物质做出正常反应,出现持续血压增高。肠系膜动脉作为人体主要的内脏血管,在全身的动脉中所占比例最大,是形成外周阻力的主要血管,对循环中的活性物质敏感,对血压的调节具有重要意义。大电导钙激活钾通道(large-conductance calcium-activated potassium channels, BKCa)是血管平滑肌上主要的负反馈调节器,在血管的舒张调节中发挥着关键作用。雄激素(testosterone, TES)是男性体内重要的内分泌激素,具有广泛的生物学效应。心血管疾病,包括高血压,在男性的发病率明显高于同年龄段的绝经前女性。TES一度被认为是心血管疾病的危险因素。然而近期的研究表明TES有舒张血管等心血管保护作用,其作用可能与血管平滑肌细胞(vascular smooth muscle cells, VSMCs)上的K+通道有关。TES对人体血管的作用及其机制还不清楚,有待进一步研究。本实验通过运用血管环和全细胞膜片钳技术观察高血压患者肠系膜动脉血管平滑肌细胞BKCa电流及BKCa对TES的敏感性变化,探讨TES的作用机制及其与高血压之间的关系。方法：(1)血管环实验：于溶液Ⅰ中迅速分离出肠系膜动脉血管,将游离血管剪成长约0.3～0.4 cm的血管环,用两根银丝穿过动脉环并形成三角环状,固定在含95%O2和5%C02,37℃的浴槽中。血管环经过张力换能器、生物放大器与生理记录仪相连,采用Chart软件采集数据并进行分析。观察TES对HT组(高血压组)和NT组(正常血压组)肠系膜动脉血管环张力的影响。KCl检验血管环活性并引起预收缩。(2)膜片钳实验：将分离好的肠系膜动脉,纵向剖开剪成2 mm×2 mm的组织条块,置于酶液中进行两步消化。选取具有较强立体感,表面光滑的平滑肌细胞进行实验,采用全细胞穿孔膜片钳技术记录电流。电流信号经膜片钳放大器放大,滤波处理后输入计算机,经Clampex 10.1软件采集电流,Clampfit 10.1、MiniAnalysis 6.0软件系统进行数据分析。观察并比较高血压组和血压正常组BKCa宏观电流及自发瞬时外向电流(spontaneous transient outward currents, STOCs),观察TES对BKCa和STOCs的作用。结果：(1)TES对HT组和NT组肠系膜动脉血管环张力的影响：①与基础张力相比KCl(40 mM)分别增加NT组(n=10)和HT组(n=10)的张力2.00±0.49倍,2.26±0.91倍,两组间无明显差异(P>0.05)；②TES降低人肠系膜血管环张力,IbTX(100 nM)可对抗TES引起的张力降低(n=5)；③TES引起NT组(n=1 5)和HT组(n=12)血管舒张,分别舒张预收缩的74.67±24.24%,57.98±19.67%,HT组舒张的程度比NT组低(P<0.05)。(2)急性酶分离的肠系膜动脉平滑肌细胞BKCa电流的基本特性：①BKCa宏观电流具有明显的电压依赖性及外向整流的性质；STOCs的幅度和频率具有明显的电压依赖性,可以随机性的叠加在全细胞BKCa电流上(n=10)；②BKCa的特异性阻断剂Iberiotoxin(IbTX)(100nM)可阻断BKCa宏观电流及STOCs。(3)比较HT组与NT组BKCa宏观电流及STOCs:①NT组(n=12)与HT组(n=8)的电流密度分别为13.31±2.25 pA/pF(+60 mV)、6.80±4.93 pA/pF(+60 mV)。经t检验,P<0.05,HT组电流密度相比NT组呈降低趋势；②STOCs (-20 mV)幅值及频率在NT组(n=5)为61.26±2.75 pA、2.27±0.47 Hz,在HT组(n=5)为27.93±3.26 pA、1.52±0.28 Hz,HT组STOCs幅值和频率比NT组低(P<0.05)。(4)TES对BKCa电流及STOCs的作用：①TES可明显增加BKCa宏观电流。②TES增加BKCa电流密度(n=6)。加入TES前BKCa电流密度13.00±2.20pA／pF(+60 mV),之后24.53±4.61 pA/pF (+60mV)。经t检验,P<0.05。③TES明显增加STOCs的幅值和频率。结论：(1)人肠系膜动脉平滑肌细胞的BKCa宏观电流有明显的电压依赖性和外向整流特征；由BKCa介导的STOCs幅度和频率具有明显的电压依赖性及随机性。(2)高血压组的BKCa电流密度相比血压正常组呈降低趋势,STOCs幅值、频率比血压正常组低。(3)TES增加人肠系膜动脉平滑肌细胞的BKCa宏观电流和STOCs幅度和频率。(4)TES明显引起人肠系膜血管舒张,BKCa参与了TES舒张人肠系膜血管的机制。(5)高血压组血管对TES的舒张反应比血压正常组低。
Objective:Hypertension is one of high risk diseases displaying increased arterial tension, and failure of resisting higher vascular tone and making normal response to vasoactive substances. As one of the main human visceral vessels, mesenteric artery holds the largest proportion of vessels in the body and forms peripheral resistance. In addition, mesenteric artery is sensitive to vasoactive substances, and therefore has a significant effect on the regulation of the blood pressure. Large-conductance calcium-activated potassium channels (BKCa), the main negative feedback regulator in vascular smooth muscle, plays a key role in the relaxation. Testosterone (TES) is a steroid and sex hormone released by body's endocrine system, and has a wide range of biological effects. The incidence of cardiovascular diseases is higher in men than in pre-menopausal women. TES had been thought to be a risk factor for cardiovascular. However, several studies have shown that TES-induced vasorelaxation may be involved in many cardiovascular protective effects. The mechanisms of protective effects remain unclear; but the activation of K+ channels in vascular smooth muscle cells (VSMCs) is possible to be involved in these effects. In this study, vascular rings and whole-cell patch clamp techniques are used to examine the effects of TES on BKCa in mesenteric artery VSMCs of hyper-tension patients, and therefore to explore the potential role of TES during hypertension. Methods:(1)Vascular rings:The mesenteric artery was quickly detached in solution I, the free artery was cut into vascular rings (0.3-0.4 cm). Vascular rings were suspended with two silver wires and fixed in a 37℃chamber with 95%O2 and 5%CO2. Vascular rings were linked to electro-physiolograph with tension transducer and biological amplifier. Observe the effects of TES on the tension of mesenteric artery vascular rings between hypertension group (HT) and normotension group (NT). KCl was used to test activity of vascular rings and cause contraction. (2)Patch clamp:The free mesenteric artery was cut into small segments (2 mm×2 mm) and then transferred to enzymatic dissociation solution for incubation. Single smooth muscle cells were obtained by two-step enzyme digestion at 37℃. Chosen the smooth muscle cells stereo and slick for experiment. The currents were recorded by a whole-cell, amphotericin-perforated configuration of patch-clamp techniques. The currents were amplified and filtered by patch clamp amplifier, and the digital datas were recorded by a Lenovo-compatible personal computer running Clampex 10.1 software and further analyzed by Clampfit 10.1 and MiniAnalysis6.0 software. The difference between HT and NT in BKCa macroscopic currents and spontaneous transient outward currents (STOCs) was observed and compared. Results:(1)The effects of TES on the tension of mesenteric artery vascular rings in HT and NT:①The increased tension by KCl (40 mM) was 2.26±0.91 in HT (n=10) and 2.00±0.49 in NT (n=10), indicated no significant difference between two groups (P>0.05);②The tension of mesenteric artery vascular rings was decreased by treating with TES, and IbTX (100 hM) could partly reverse it (n=5);③The decreased tension by TES in HT (57.98±19.67%, n=12) was lower than that in NT (74.67±24.24%, n=15) (P<0.05). (2)The features of BKCa currents in mesenteric artery smooth muscle cells:①The BKCa macroscopic currents were voltage-dependent and outwardly rectifying; The amplitude and frequency of STOCs were also voltage-dependent; STOCs were superimposed stochastically on the whole-cell BKCa macroscopic currents (n=10);②The BKCa macroscopic currents and STOCs were suppressed by 100 nM Iberiotoxin (IbTX), a specific BKCa blocker. (3) Comparisons of BKCa macroscopic currents and STOCs between HT and NT:①The current density of BKCa was 6.80±4.93 pA/pF (+60 mV) in HT (n=8) and 13.30±2.25 pA/pF (+60 mV) in NT (n=12). The statistical test shows that the current density in HT is lower than the one in NT (P<0.05);②The amplitude and frequency of STOCs (-20 mV) in HT (27.93±3.26 pA、1.52±0.28 Hz, n=5) were lower than that in NT (61.26±2.75 pA、2.27±0.47 Hz, n=5) (P<0.05). (4)The effects of TES on BKCa and STOCs:①The BKCa macroscopic currents were increased after added TES in an identical cell;②The current density of BKCa was 13.00±2.20 pA/pF (+60 mV), changed to 24.53±4.61 pA/pF (+60 mV) after added TES. The current density of BKCa was increased by TES (n=6) (P<0.05);③The amplitude and frequency of STOCs increased after added TES in an identical cell. Conclusions:(1)The BKCa macroscopic currents of mesenteric artery VSMCs were voltage-dependent and outwardly rectifying; STOCs can be induced by BKCa, and therefore the amplitude and frequency of STOCs were voltage-dependent and stochastic. (2)By compared with NT, the current density of BKCa as well as the amplitude and frequency of STOCs were significantly lower in HT. (3)Both the BKCa macroscopic currents of mesenteric artery VSMCs and the amplitude and frequency of STOCs were increased after treated with TES. (4)BKCa was involved in the mechanisms of TES-induced relaxation in mesenteric artery. (5)The response of TES in HT was lower than that in NT.
Objective:Hypertension is one of high risk diseases displaying increased arterial tension, and failure of resisting higher vascular tone and making normal response to vasoactive substances. As one of the main human visceral vessels, mesenteric artery holds the largest proportion of vessels in the body and forms peripheral resistance. In addition, mesenteric artery is sensitive to vasoactive substances, and therefore has a significant effect on the regulation of the blood pressure. Large-conductance calcium-activated potassium channels (BKCa), the main negative feedback regulator in vascular smooth muscle, plays a key role in the relaxation. Testosterone (TES) is a steroid and sex hormone released by body's endocrine system, and has a wide range of biological effects. The incidence of cardiovascular diseases is higher in men than in pre-menopausal women. TES had been thought to be a risk factor for cardiovascular. However, several studies have shown that TES-induced vasorelaxation may be involved in many cardiovascular protective effects. The mechanisms of protective effects remain unclear; but the activation of K+ channels in vascular smooth muscle cells (VSMCs) is possible to be involved in these effects. In this study, vascular rings and whole-cell patch clamp techniques are used to examine the effects of TES on BKCa in mesenteric artery VSMCs of hyper-tension patients, and therefore to explore the potential role of TES during hypertension. Methods:(1)Vascular rings:The mesenteric artery was quickly detached in solution I, the free artery was cut into vascular rings (0.3-0.4 cm). Vascular rings were suspended with two silver wires and fixed in a 37℃chamber with 95%O2 and 5%CO2. Vascular rings were linked to electro-physiolograph with tension transducer and biological amplifier. Observe the effects of TES on the tension of mesenteric artery vascular rings between hypertension group (HT) and normotension group (NT). KCl was used to test activity of vascular rings and cause contraction. (2)Patch clamp:The free mesenteric artery was cut into small segments (2 mm×2 mm) and then transferred to enzymatic dissociation solution for incubation. Single smooth muscle cells were obtained by two-step enzyme digestion at 37℃. Chosen the smooth muscle cells stereo and slick for experiment. The currents were recorded by a whole-cell, amphotericin-perforated configuration of patch-clamp techniques. The currents were amplified and filtered by patch clamp amplifier, and the digital datas were recorded by a Lenovo-compatible personal computer running Clampex 10.1 software and further analyzed by Clampfit 10.1 and MiniAnalysis6.0 software. The difference between HT and NT in BKCa macroscopic currents and spontaneous transient outward currents (STOCs) was observed and compared. Results:(1)The effects of TES on the tension of mesenteric artery vascular rings in HT and NT:①The increased tension by KCl (40 mM) was 2.26±0.91 in HT (n=10) and 2.00±0.49 in NT (n=10), indicated no significant difference between two groups (P>0.05);②The tension of mesenteric artery vascular rings was decreased by treating with TES, and IbTX (100 hM) could partly reverse it (n=5);③The decreased tension by TES in HT (57.98±19.67%, n=12) was lower than that in NT (74.67±24.24%, n=15) (P<0.05). (2)The features of BKCa currents in mesenteric artery smooth muscle cells:①The BKCa macroscopic currents were voltage-dependent and outwardly rectifying; The amplitude and frequency of STOCs were also voltage-dependent; STOCs were superimposed stochastically on the whole-cell BKCa macroscopic currents (n=10);②The BKCa macroscopic currents and STOCs were suppressed by 100 nM Iberiotoxin (IbTX), a specific BKCa blocker. (3) Comparisons of BKCa macroscopic currents and STOCs between HT and NT:①The current density of BKCa was 6.80±4.93 pA/pF (+60 mV) in HT (n=8) and 13.30±2.25 pA/pF (+60 mV) in NT (n=12). The statistical test shows that the current density in HT is lower than the one in NT (P<0.05);②The amplitude and frequency of STOCs (-20 mV) in HT (27.93±3.26 pA、1.52±0.28 Hz, n=5) were lower than that in NT (61.26±2.75 pA、2.27±0.47 Hz, n=5) (P<0.05). (4)The effects of TES on BKCa and STOCs:①The BKCa macroscopic currents were increased after added TES in an identical cell;②The current density of BKCa was 13.00±2.20 pA/pF (+60 mV), changed to 24.53±4.61 pA/pF (+60 mV) after added TES. The current density of BKCa was increased by TES (n=6) (P<0.05);③The amplitude and frequency of STOCs increased after added TES in an identical cell. Conclusions:(1)The BKCa macroscopic currents of mesenteric artery VSMCs were voltage-dependent and outwardly rectifying; STOCs can be induced by BKCa, and therefore the amplitude and frequency of STOCs were voltage-dependent and stochastic. (2)By compared with NT, the current density of BKCa as well as the amplitude and frequency of STOCs were significantly lower in HT. (3)Both the BKCa macroscopic currents of mesenteric artery VSMCs and the amplitude and frequency of STOCs were increased after treated with TES. (4)BKCa was involved in the mechanisms of TES-induced relaxation in mesenteric artery. (5)The response of TES in HT was lower than that in NT.
引文
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