论文部分内容阅读
本文旨在研究血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对人肠系膜动脉平滑肌细胞大电导钙激活钾通道(large conductance calcium-activated potassium channels,BKCa)的影响,探讨AngⅡ在分子水平扩张血管的作用机制。采用单通道膜片钳及全细胞穿孔膜片钳技术观察人肠系膜动脉平滑肌细胞BKCa对AngⅡ的反应性;采用RT-PCR技术确定人肠系膜动脉上AngⅡ受体基因的表达情况。在细胞贴附式膜片下(Vm=+40mV),直接加入AngⅡ(100nmol/L),人肠系膜动脉平滑肌细胞BKCa的活性无明显变化;经AngⅡ1型受体(angiotensinⅡtype 1 receptor,AT1R)特异性阻断剂缬沙坦(10μmol/L)预处理后,再分别加入25、100和250nmol/L的AngⅡ,BKCa的开放概率明显增加,呈现出浓度依赖性。缬沙坦预处理后,当加入100nmol/LAngⅡ时,BKCa的开放概率由0.010±0.003增至0.039±0.015,平均关闭时间由(2729.5±808.6)ms减至(487.7±182.5)ms(n=11,P<0.05),但平均开放时间和电流幅值在给药前后无明显变化。BKCa被AngⅡ(100nmol/L)激活后,再加入AngⅡ2型受体(angiotensinⅡtype 2 receptor,AT2R)特异性阻断剂PD123,319,BKCa的活性受到抑制,开放概率由0.016±0.003减至0.004±0.001(n=5,P<0.05),而通道平均开放时间、平均关闭时间和电流幅值在给药前后均无明显变化。另外,将人肠系膜动脉平滑肌细胞经缬沙坦和PD123,319共同处理后再加入AngⅡ(100nmol/L),BKCa的活性无明显变化。在全细胞穿孔膜片钳下,平滑肌细胞经缬沙坦(10μmol/L)预处理后,在测试的电压范围内,AngⅡ(100nmol/L)对膜电位从60mV到+30mV时BKCa电流密度均无明显影响,而在+40、+50和+60mV时,BKCa电流密度分别从(9.03±2.23)pA/pF、(12.88±2.55)pA/pF和(17.26±2.84)pA/pF增加到(12.47±2.22)pA/pF、(18.71±2.51)pA/pF和(27.21±3.12)pA/pF(n=6,P<0.05)。经RT-PCR证实人肠系膜动脉上有AT1R和AT2R这两种AngⅡ受体基因的表达。上述结果提示,AngⅡ对经缬沙坦预处理的人肠系膜动脉平滑肌细胞BKCa有激活作用,且这种作用由AT2R所介导。
The aim of this study was to investigate the effect of angiotensinⅡ (AngⅡ) on large conductance calcium-activated potassium channels (BKCa) in human mesenteric artery smooth muscle cells and to explore the mechanism of angiotensin Ⅱ (AngⅡ) at the molecular level to dilate blood vessels. Single-channel patch-clamp and whole-cell perforated patch-clamp technique were used to observe the responsiveness of BKCa to AngⅡ in human mesenteric artery smooth muscle cells. The expression of AngⅡreceptor gene in human mesenteric artery was determined by RT-PCR. There was no significant change in the activity of BKCa in human mesenteric artery smooth muscle cells by direct addition of AngⅡ (100 nmol / L) under the cell attachment membrane (Vm = +40 mV). The specificity of the angiotensin Ⅱ type 1 receptor (AT1R) After the pretreatment with valsartan (10 μmol / L), the open probability of BKCa increased significantly with the concentration of AngⅡ at 25, 100 and 250 nmol / L, showing a concentration-dependent manner. After valsartan was pretreated, the open probability of BKCa was increased from 0.010 ± 0.003 to 0.039 ± 0.015 with the addition of 100nmol / LAngⅡ, and the average closure time decreased from (2729.5 ± 808.6) ms to (487.7 ± 182.5) ms (n = 11 , P <0.05), but the average open time and current amplitude did not change significantly before and after administration. Activation of BKCa by AngⅡ (100 nmol / L) followed by addition of angiotensin Ⅱ type 2 receptor (AT2R) -based PD123,319 inhibited the activity of BKCa and decreased the open probability from 0.016 ± 0.003 to 0.004 ± 0.001 (n = 5, P <0.05). However, there was no significant change in mean open time, mean close time and current amplitude before and after administration. In addition, the human mesenteric artery smooth muscle cells were treated with valsartan and PD123, 319 and then added AngII (100nmol / L), BKCa activity no significant change. Under the whole-cell perforated patch clamp, the current density of BKCa in smooth muscle cells pretreated with valsartan (10μmol / L) was higher than that of AngⅡ (100nmol / L) for membrane potential from 60mV to + 30mV (9.03 ± 2.23) pA / pF, (12.88 ± 2.55) pA / pF, and (17.26 ± 2.84) pA / pF at +40, +50 and +60 mV, respectively 12.47 ± 2.22) pA / pF, (18.71 ± 2.51) pA / pF and (27.21 ± 3.12) pA / pF (n = 6, P <0.05). The expression of two AngⅡ receptor genes, AT1R and AT2R, was confirmed by RT-PCR in human mesenteric artery. The above results suggest that AngⅡ can activate BKCa in human mesenteric artery smooth muscle cells pretreated with valsartan, and this effect is mediated by AT2R.