Apelin-13对大鼠心肌正性肌力作用的离子通道机制

发布时间：2018-06-02 04:41

本文选题：Apelin-13 + 正性肌力　；参考：《宁夏医科大学》2010年硕士论文

【摘要】：Apelin是Tatemoto等在1998年利用反向药理学方法从牛胃分泌物中提取出来的血管活性多肽,是G蛋白偶联受体APJ受体(putative receptor protein related to the angiotensinII receptor AT1)的内源性配体。Apelin基因位于X染色体的q25-26.1,包含3个外显子和2个内含子。Apelin的前体蛋白由77个氨基酸组成,可水解生成不同长度的活性肽段,如apelin-36、apelin-31、apelin-17、apelin-13等。在体内,Apelin及其受体APJ广泛分布在中枢与外周组织,如心脏、肺脏、肾脏、中枢神经系统、脂肪组织等,具有调节心血管系统稳态、水盐平衡、调节免疫反应等作用,是一种重要的生理调节肽。其中在心血管组织中, apelin具有增强心肌收缩力、舒张血管、降低血压等作用,提示该肽可能是一种重要的心血管调节肽。本工作以单个大鼠心室肌细胞为模型,应用全细胞膜片钳技术,观察apelin-13对大鼠心室肌细胞钠钙交换电流(I_(Na/Ca))、L型钙通道电流(I_(Ca,L))、ATP敏感钾通道电流(IK_(ATP))的影响,以进一步明确apelin对心脏正性肌力作用的离子电流机制。目的本实验旨在研究apelin-13对急性分离大鼠心室肌细胞正性肌力作用的离子通道机制。方法雄性SD大鼠(250-300 g),腹腔麻醉后断头处死,迅速取其心脏,以胶原酶溶液灌流心脏,待心脏基本变软后剪取心室部分,剪碎吹打使之分离成为单个心室肌细胞。使用全细胞膜片钳技术记录心室肌细胞的I_(Na/Ca)、I_(Ca,L)、IK_(ATP)。给药组以含不同浓度apelin-13的细胞外液灌流心室肌细胞5 min;对照组直接以细胞外液灌流5 min,记录并观察电流曲线的变化。结果 1.Apelin-13对大鼠心室肌细胞I_(Na/Ca)的影响Apelin-13对内向、外向I_(Na/Ca)均有增大作用。浓度为0.01 nmol/L、0.1 nmol/L、1 nmol/L的apelin-13使正向INaCa分别比给药前增大20±4%( P 0.05,n=8)、33±7%( P 0.01,n=8)、54±10%( P 0.01,n=8),使反向I_(Na/Ca)分别增大1±6%(P 0.05 ,n=8),7±2%(P 0.01,n=8),30±7%(P 0.01,n=8),对照组单纯给予细胞外液5 min后正、反向I_(Na/Ca)未分别减小5±4%和2±7%。 2.Apelin-13对大鼠心肌细胞I_(Ca,L)的影响Apelin-13可浓度依赖性增大大鼠心肌细胞I_(Ca,L),以apelin-13浓度分别为0.01 nmol/L、0.1 nmol/L、1 nmol/L的细胞外液灌流心肌细胞5 min后I_(Ca,L)分别增大3士3%、20士6%、45士9%,对照组由于rundown现象减小5士5%,0.1 nmol/L组、1 nmol/L组与对照组相比较有差异显著(P 0.01,n=8),0.01 nmol/L与对照组无显著差异(P 0.05,n=8)。1 nmol/L的apelin-13灌流5 min后IV曲线明显下移,但不改变I_(Ca,L)的电压依赖关系和反转电位。对照I_(Ca,L)激活曲线半激活电位是-19.43±2.76mV,斜率是4.76±0.33 mV,1 nmol/L apelin-13灌流5 min后半激活电位变为-18.93±3.05 mV,斜率变为5.21±0.38 mV,二者无明显区别(P 0.05, n=6)。对照I_(Ca,L)失活曲线半失活电位是-23.62±1.79mV,斜率是5.35±0.21 mV,1 nmol/L apelin-13灌流5 min后半失活电位变为-24.6±2.14 mV,斜率变为5.47±0.23,二者无明显区别(P 0.05, n=6)。上述结果表明apelin-13增大I_(Ca,L)的作用不是通过改变L型钙通道激活和失活的门控特点。 3.Apelin-13对IK_(ATP)的影响在由50 mV到-100 mV持续125 ms的斜坡刺激下记录心肌细胞IK_(ATP)。1 nmol/L、10 nmol/L的apelin-13不能使该电流增加,给予pinacidil后该电流明显增加,并且能被glibenelmaide阻断(说明该电流是IK_(ATP)),提示apelin-13不能使静息状态下心肌细胞的ATP敏感钾通道开放。结论本工作以单个大鼠心室肌细胞为模型,应用全细胞膜片钳技术,经研究发现,apelin-13可浓度依赖性地增大心室肌细胞的I_(Na/Ca)和I_(Ca,L),对ATP敏感钾通道无开放作用。上述结果表明,apelin-13对心脏正性肌力作用的离子通道电流机制可能与通过增大心肌细胞I_(Na/Ca)和I_(Ca,L)电流,从而增加心肌细胞的钙离子内流有关。
[Abstract]:Apelin is a vasoactive polypeptide extracted from bovine gastric secretions in 1998 by Tatemoto and so on. It is the endogenous ligand of the G protein coupled receptor APJ receptor (putative receptor protein related to the angiotensinII receptor), which contains 3 exons and 2 The precursor protein of intron.Apelin consists of 77 amino acids that can be hydrolyzed to produce active peptide segments of different lengths, such as apelin-36, apelin-31, apelin-17, apelin-13, etc. in vivo, Apelin and its receptor APJ are widely distributed in central and peripheral tissues, such as the heart, lungs, kidneys, central nervous system, adipose tissue, and so on, which regulate the cardiovascular system. The effect of homeostasis, water and salt balance and regulating immune response, is an important physiological regulation peptide. In cardiovascular tissue, Apelin has the effect of enhancing myocardial contractile force, diastolic blood vessel, lowering blood pressure and so on. It suggests that the peptide may be an important cardiovascular regulation peptide. Patch clamp technique was used to observe the effect of apelin-13 on sodium calcium exchange current (I_ (Na/Ca)), L type calcium channel current (I_ (Ca, L)), ATP sensitive potassium channel current (IK_ (ATP)) in ventricular myocytes of rats, in order to further clarify the ionic current mechanism of apelin on the positive inotropic action of the heart.
Objective to study the ion channel mechanism of apelin-13 on the positive inotropic effect of acute isolated ventricular myocytes in rats.
Methods the male SD rats (250-300 g) were killed after the abdominal anesthesia, and the heart was taken quickly. The heart was perfused with collagenase solution. After the heart became soft, the ventricular part was cut to separate into single ventricular myocytes. The whole cell patch clamp technique was used to record the I_ (Na/Ca) of ventricular myocytes, I_ (Ca, L), and IK_ (ATP). The extracellular fluid of different concentrations of apelin-13 Perfused Ventricular Myocytes 5 min; the control group was perfused directly with extracellular fluid for 5 min, recording and observing the changes of current curve.
Result
The effect of 1.Apelin-13 on I_ (Na/Ca) in ventricular myocytes of rats increased Apelin-13 to introverted and extroverted I_ (Na/Ca). The concentration of 0.01 nmol/L, 0.1 nmol/L, and 1 nmol/L apelin-13 made the positive INaCa 20 + 4% (P 0.05, 0.01, 54), 54 + 10% (54 + 0.01,), respectively. (n=8), 7 + 2% (P 0.01, n=8), 30 + 7% (P 0.01, n=8), the control group only given extracellular fluid 5 min, the reverse I_ (Na/Ca) did not decrease 5 5 4% and 2 2 7%. respectively.
The effect of 2.Apelin-13 on rat cardiac myocyte I_ (Ca, L), Apelin-13 could increase the concentration dependent I_ (Ca, L) of rat cardiac myocytes, and the concentration of apelin-13 was 0.01 nmol/L, 0.1 nmol/L, and 1 nmol/L was 3%, 20 6%, 45 9%, respectively. Group l/L, the 1 nmol/L group was significantly different from the control group (P 0.01, n=8), and there was no significant difference between the 0.01 nmol/L and the control group (P 0.05, n=8).1 nmol/L apelin-13 perfusion 5 min after the IV curve obviously moved down, but did not change the voltage dependence and reverse potential. .76 + 0.33 mV, 1 nmol/L apelin-13 perfusion 5 min half activation potential changed to -18.93 + 3.05 mV, the slope changed to 5.21 + 0.38 mV, and there was no obvious difference between two (P 0.05, n=6). The changes were 5.47 + 0.23, and there was no obvious difference between the two (P 0.05, n=6). The results showed that the effect of apelin-13 increasing I_ (Ca, L) was not by changing the activation and inactivation of the L type calcium channel.
The effect of 3.Apelin-13 on IK_ (ATP) was recorded from 50 mV to -100 mV sustained by the slope of 125 ms, and IK_ (ATP).1 nmol/L was recorded. The current of 10 nmol/L could not increase the current. The ATP sensitive potassium channel of cardiac myocytes is open.
Conclusion a single rat ventricular myocyte was used as a model, and the whole cell patch clamp technique was applied. It was found that apelin-13 could increase the concentration of I_ (Na/Ca) and I_ (Ca, L) in ventricular myocytes in a concentration dependent manner, and had no open effect on ATP sensitive potassium channels. The results showed that the ion channel current mechanism of apelin-13 for positive inotropic action of the heart was possible. It can be related to increasing the calcium influx of cardiac myocytes by increasing the I_ (Na/Ca) and I_ (Ca, L) currents.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33

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