生理学报Acta Physiologica Sinica, April 25, 2003, 55(2): 187-190
Received 2002-07-29
Accepted 2002-12-30
This work was supported by the National Natural Science Foundation of China (No.39970327).
*Corresponding author. Tel: +86-29-3374574; E-mail: mz_zhu@fmmu.edu.cn
研究论文
血管钠肽对离体人乳内动脉的舒张作用
于军1, 朱妙章1, *, 韦耿泽1, 陈宝莹2, 吕顺艳1, 康云帆3, 郭海涛1, 马恒1,董明清4
1第四军医大学生理学教研室, 2唐都医院放射科, 3西京医院心血管外科中心, 4病理生理学教研室, 西安 710032
摘要: 为了研究血管钠肽(VNP)对人乳内动脉(human intramammary artery, HIMA)的舒张作用及其机制, 采用离体血管灌流的方法, 观察VNP对内皮完整和去内皮HIMA的舒张作用, 以及HS-142-1、TEA、8-Br-cGMP和镁蓝(MB)对这一过程的影响。实验中观察到, VNP (0.0001-1 μmol/L)可引起剂量依赖性的舒张效应, 且无内皮依赖性; 8-Br-cGMP (0.1-1000 μmol/L)也可引起剂量依赖性的血管舒张效应。钠尿肽鸟苷酸环化酶(guanylate cyclase, GC)受体的特异性阻断剂HS-142-1 (20 μmol/L)使VNP舒张HIMA的作用几乎完全消失。MB是GC的抑制剂, 10 μmol/L的MB不但使VNP舒张HIMA的作用完全消失, 而且可增强HIMA对去甲肾上腺素(NE)产生的收缩反应。钙激活钾通道(KCa)的阻断剂 TEA(1mmol/L)可减弱(但是不完全阻断)VNP的舒血管作用。上述结果表明, VNP对HIMA具有不依赖内皮的舒张作用; 此作用是通过作用于平滑肌细胞的钠尿肽GC受体, 引起细胞内的cGMP水平升高实现的, 并且与KCa有关。
关键词: 血管钠肽 ; 人乳内动脉; cGMP; TEA; 镁蓝
中图分类号: Q463; R33
Vasorelaxing role
of vasonatrin peptide in human
intramammary artery in
vitro
YU Jun1, ZHU Miao-Zhang1,*, WEI Geng-Ze1, CHEN Bao-Ying2, LU Shun-Yan1, KANG Yun-Fan3,
GUO Hai-Tao1, MA Heng1, DONG Ming-Qing4
1Department of Physiology, 2Radiology Department of Tangdu Hospital, 3 Cardiovascular Surgery Center of Xijing Hospital, and 4Department of Pathophysiology, Fourth Military Medical University, Xi′an 710032
Abstract: The purpose of this study was to investigate the vasorelaxing effect of vasonatrin peptide (VNP) on human intramammary artery (HIMA).The vasorelaxing effect of VNP on HIMA was measured by means of perfusion in vitro. The effects of HS-142-1, TEA, 8-Br-cGMP and methylene blue (MB) were also observed. It was found that VNP caused a concentration-dependent relaxation in HIMA which was independent of the endothelium. 8-Br-cGMP (0.1-1000 μmol/L) also caused a concentration-dependent relaxation in HIMA. The vasorelaxing effect of VNP disappeared in the presence of HS-142-1 (20 μmol/L), an antagonist of the natriuretic peptide guanylate cyclase (GC) receptor. MB (10 μmol/L), an inhibitor of GC, not only blocked completely the relaxation of HIMA, but also enhanced the vascular contraction induced by norepinephrine. TEA (1mmol/L), an antagonist of calcium activated potassium channels (KCa), reduced but not completely blocked the vasorelaxing effect of VNP. These findings suggest that VNP can relax HIMA, which is independent of the endothelium. This effect is possibly achieved by the binding of VNP with the natriuretic peptide GC receptors in the smooth muscle cells (SMCs), leading to an increase in intracellular cGMP level. Moreover, the vasorelaxing effect of VNP is associated with KCa.
Key words: vasonatrin peptide; human intramammary artery; cGMP; TEA; methylene blue
血管钠肽(VNP)是一种由27个氨基酸残基组成的人工合成多肽, 它对大鼠主动脉、肺动脉具有显著的舒张作用, 可以降低慢性低氧性大鼠肺动脉高压[1-4]。 而VNP对人动脉的舒张作用还未见报道。本实验以心脏搭桥手术采用的乳内动脉为研究对象, 探讨VNP的作用及机制, 旨为VNP的临床应用提供理论依据。
1材料和方法
1.1 材料、药品与仪器
人乳内动脉(human intramammary artery, HIMA) 征得供者或供者家属的同意, 由西京医院心血管外科取材自5例行冠脉旁路搭桥术的患者, 年龄45-67岁 (55±8); 男 3例, 女2例。TEA(盐酸四乙铵, tetraethylammonium chloride, TEA-Cl)、镁蓝(methylene blue, MB)、8-Br-cGMP、去甲肾上腺素(norepinephrine, NE)均为美国Sigma公司产品。血管钠肽(VNP)由上海生物化学研究所提供。HS-142-1为美国WEI Chi-Ming 教授惠赠。RM-6280多道智能生理记录及分析处理系统由第四军医大学生物医学工程系研制。
1.2 方法
1.2.1 主要药物配制
VNP用生理盐水配制成10 mmol/L的原液, 实验前用Kreb′s 液稀释成工作液。8-Br-cGMP直接用Kreb′s 液配成工作液(0.1-1000 μmol/L)。MB用生理盐水配成10 mmol/L的原液, 实验时加入Kreb′s 液, 工作浓度10 μmol/L。TEA用生理盐水配成1 mol/L的原液, 实验时加入Kreb′s 液, 工作浓度为1 mmol/L。HS-142-1直接用Kreb′s 液配成20 μmol/L。Kreb′s 液成分(mmol/L): NaCl 118、 KCl 4.75、 CaCl2 2.54、 KH2PO4 1.19、 MgSO4 1.19、 NaHCO3 25和glucose 10, 充以含95%的O2和5%的CO2的混合气, 用HCl调pH至7.2-7.4。
1.2.2 离体血管环灌流实验
将游离的HIMA放入到预冷、并充以95% O2和5% CO2 的Kreb′s 液中, 去除血管上的脂肪及结缔组织, 将动脉剪成2-3 mm的小段, 然后将血管环套在不锈钢小钩上, 放入37℃、 充以95% O2和5% CO2 的Kreb′s 液中, 并连接于张力传感器上。给血管环加0.5 g的前负荷, 在Kreb′s 液中平衡至少120 min以上。 在此期间, 孵育血管环的液体每15 min换1次, 并使前负荷维持在0.5 g。加入10 μmol/L的NE检测标本活性(收缩幅度小于500 mg者弃去), 然后冲洗, 使血管张力恢复到基线。稳定30 min后重复给予NE, 待血管收缩达到稳定值, 逐步增加VNP的浓度(0.0001-1 μmol/L)。去内皮采用细钢丝插入血管腔中滚动完成。用10 μmol/L的ACh对血管环是否有舒张反应作为判断血管内皮是否完整的标准。
于军等: 血管钠肽对离体人乳内动脉的舒张作用生理学报Acta Physiol. Sin., April 25, 2003, 55(2): 187-1901.2.3 统计学处理
血管环的例数n等于提供血管的病人数。以NE对血管环收缩的最大效应为100%, 各项数据以相对于NE所引起反应的%(mean±S)表示。制作累积浓度-舒张反应曲线, 求出最大舒张反应值(Rmax)和血管最大舒张的半数有效浓度(EC50)。统计学处理采用组间t检验, P<0.05为差异显著。
2结果
2.1 VNP对人乳内动脉的作用
NE使保留内皮或去内皮的HIMA收缩后, VNP (0.0001-1 μmol/L)可引起剂量依赖性的舒张效应, 且无内皮依赖性(图1)。VNP的浓度为1 μmol/L时Rmax=(88±6)%, EC50=7.5±0.4 (-lg mol/L)。
图1.VNP对人乳内动脉舒张的量效关系
Fig. 1.Dose-relaxation relationship of VNP in human intramammary artery rings with and without endothelium. Vascular rings were pre-contracted with 1 μmol/L NE to generate force. Once a stable contraction was obtained, VNP was added to a bath at cumulative concentrations of 0.0001-1 μmol/L. Data are expressed as percent of relaxation from maximal contraction induced by 1 μmol/L NE. n=5.
2.28-Br-cGMP对人乳内动脉的作用
NE使HIMA收缩后, 8-Br-cGMP (0.1-1000 μmol/L)可引起剂量依赖性的舒张效应(图2), 8-Br-cGMP的浓度为1000 μmol/L时Rmax=(90±8)%, EC50=4.7±0.3 (-lg mol/L)。提示: 8-Br-cGMP对HIMA可以产生类似于VNP的舒张作用。因为8-Br-cGMP是一种具有膜透过性的cGMP类似物, 说明VNP的舒血管作用可能与升高细胞内cGMP水平有关。
图2. 8-Br-cGMP对人乳内动脉舒张的量效关系
Fig. 2. Dose-relaxation relationship of 8-Br-cGMP in human intramammary artery rings with and without endothelium. Vascular rings were pre-contracted with 1 μmol/L NE to generate force. Once a stable contraction was obtained, NE was added to a bath with cumulative concentrations of 0.1-1000 μmol/L. Data are expressed as percent of relaxation from maximal contraction induced by 1 μmol/L NE. n=5.
2.3 HS-142-1对VNP舒张人乳内动脉的影响
HS-142-1 (20 μmol/L)可以使VNP舒张HIMA的作用几乎完全消失(图3)。因为HS-142-1是钠尿肽鸟苷酸环化酶(guanylate cyclase, GC )受体的特异性阻断剂, 说明VNP舒张人乳内动脉是经过钠尿肽GC受体介导的, 进一步证明了VNP的舒血管作用可能与升高细胞内cGMP水平有关。
2.4 MB对VNP舒张人乳内动脉的影响
MB是GC的抑制剂, 10 μmol/L的MB不但可以使VNP舒张HIMA的作用完全消失, 而且可以增强HIMA对NE产生的收缩反应(图3), 提示在没有加VNP时细胞内有活性较低的GC, VNP与其受体结合后经过信号转导使GC活性增加。这就证实cGMP 是VNP舒血管作用过程中必不可少的信号分子。
2.5 TEA对VNP舒张人乳内动脉的影响
钙激活钾通道(KCa )的阻断剂 TEA(1mmol/L)可以减弱VNP的舒血管作用, 使VNP的浓度为1 μmol/L时Rmax降低为(54±5)%, 但是没有完全阻断VNP的舒血管作用(图3)。
图3.HS-142-1、 TEA 和 MB对VNP舒张人乳内动脉效应的影响
Fig. 3.Effects of HS-142-1, TEA and MB on the relaxation of human intramammary artery induced by VNP. Vascular rings were pre-contracted with 1 μmol/L NE to generate force. Once a stable contraction was obtained, VNP was added to a bath with cumulative concentrations of 0.0001-1 μmol/L. Some of the vascular rings were pretreated with HS-142-1 (20 μmol/L), TEA (1mmol/L) and MB (10 μmol/L) respectively before adding VNP. Data are expressed as percent of relaxation from maximal contraction induced by 1 μmol/L NE. n=5, *P<0.05, **P<0.01 vs vascular rings in the absence of HS-142-1, TEA and MB.
3讨论
VNP是CNP和ANP的嵌和体,
它是在CNP的羧基端加上ANP羧基端的5个氨基酸残基形成的27肽[5、6]。钠尿肽受体(natriuretic peptide receptors,
NPRs)有两类: 功能性受体和清除受体(NPR-C)。其中功能性受体与GC偶联, 故又称为GC受体。钠尿肽和GC受体结合可以激活受体本身的GC活性, 催化GTP转变为cGMP, cGMP依赖性蛋白激酶(PKG)活化, 再激活相关的蛋白激酶发挥生理效应[7]。
本研究发现:VNP对HIMA的舒张作用可以被GC受体的阻断剂HS-142-1阻断,
说明VNP对HIMA的舒张作用经过GC受体介导。并且8-Br-cGMP (一种具有膜透过性的cGMP类似物)可以模拟VNP的作用, 而GC的抑制剂MB可以完全阻断VNP对HIMA的舒张作用,
提示cGMP介导VNP的扩血管作用。我们以往的工作发现: VNP可以升高培养的肺动脉血管平滑肌细胞(VSMCs)、心肌细胞和心脏成纤维细胞内的cGMP水平[8-10],
腹腔注射VNP可以升高大鼠的血浆cGMP水平[3], 这与本研究的结论是一致的。
PKG舒张血管的可能机制为: (1) 抑制L-型钙通道, 减少钙内流, 增加钙泵对钙的摄取, 从而降低细胞内游离的钙离子浓度; (2)直接作用于收缩蛋白而使血管舒张; (3)在某些血管组织中, PKG还可特异性抑制磷酸二酯酶活性, 阻止cAMP降解而加强cAMP介导的舒张作用[11]。其中L-型钙通道具有电压依赖性, 其活性受细胞膜电位影响。PKG可以通过影响钾通道的活性改变细胞膜电位, 从而影响L-型钙通道。K+的通透性是调节VSMCs膜电位的主要因素, 钾通道开放导致K+外流增加, 细胞膜产生超极化, 从而抑制电压依赖性钙通道开放, 导致VSMCs舒张。KCa在VSMCs膜上密度高、电导较大, 因此它对膜电位影响较大, 在血管平滑肌张力的调节中起重要的作用[12]。本研究发现TEA (KCa阻断剂)可以减弱但不能完全阻断VNP的舒血管作用。说明VNP的舒血管作用与KCa有关, 但是必然还有其他机制存在。
总之, VNP对HIMA具有不依赖内皮的舒张作用。它直接作用于VSMCs的钠尿肽GC受体, 升高细胞内的cGMP水平, 增强PKG的活性。PKG发挥作用的机制之一是通过激活KCa使VSMCs超极化, 导致血管舒张, 而其它的可能机制还有待于进一步研究。
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