尾加压素Ⅱ对心脏的作用研究

【作者】 龚惠；

【导师】 朱依纯； 姚泰；

【作者基本信息】 复旦大学 ， 生理学， 2005， 博士

【摘要】 尾加压素Ⅱ是最早从鱼的尾垂体分离出来的一种多肽,最近哺乳动物包括人的尾加压素Ⅱ已经被克隆出来。研究表明,尾加压素Ⅱ在心血管系统有重要作用,它曾被认为是最强的缩血管物质之一。对于不同种属的动物,静脉注射尾加压素Ⅱ产生的心血管反应有很大的差异,甚至对于同一种属的动物,报道也并不一致。尾加压素Ⅱ能增加人右心房肌的收缩力,但对左心室心肌的直接作用还有待阐明。尾加压素Ⅱ受体GPR14分布比较广泛,包括心血管组织,中枢和内分泌组织等。人们通过RT-PCR和配体受体结合实验证实GPR14在心血管的表达,但对于GPR14蛋白在心脏的表达以及它在心脏中分布的具体的细胞类型还缺乏直接的证据。另外,尾加压素Ⅱ可能会介导心肌细胞的肥大,在一些慢性缺氧或充血性心衰情况,尾加压素Ⅱ和其受体出现上调,推测尾加压素Ⅱ可能参与心血管的重构过程。一氧化氮具有舒张血管,保护心肌的作用。它参与尾加压素Ⅱ对心血管作用的调节。硫化氢是最近受到关注的具有生物活性的气体小分子,能抑制心肌收缩,舒张血管。但它是否参与尾加压素Ⅱ对心血管的作用,还需要进一步的研究。 我们主要通过免疫组化和免疫荧光双标的方法观察GPR14在心脏中的表达,结果表明,GPR14主要表达于正常大鼠左心室的心肌细胞上,在心脏的其他部分包括心房,右心室和主动脉瓣膜没有观察到GPR14蛋白的阳性表达;在心脏中其他的细胞包括冠脉血管的内皮和平滑肌细胞上未见GPR14的表达。我们利用Western blot和逆转录—聚合酶链的方法进一步证实GPR14蛋白和基因确实表达在左心室。我们又通过免疫组化的方法观察GPR14蛋白在大鼠心梗后3天和2周梗塞区边缘的表达,结果表明,与假手术组相比,GPR14在表达的量和表达的细胞类型上,均没有出现明显的变化。在房间隔缺损病人的右心房,我们观察到GPR14受体的表达,主要在靠近心外膜的心肌组织;而在右心耳,GPR14蛋白的表达与血管分布有关。 为了从功能学上进一步证实尾加压素Ⅱ受体确实表达在左心室的心肌上,我们观察了尾加压素Ⅱ对左室乳头肌的作用,结果提示,尾加压素增加左室乳头肌的收缩强度,但并不改变其他的收缩参数,包括收缩舒张速率,收缩潜伏期,达到最大收缩张力的时间以及舒张到一半的时间。我们还观察了给麻醉大鼠静脉注射尾加压素Ⅱ引起的心血管效应,主要引起左室收缩压,股动脉收缩压和舒张压,心率的下降;心肌收缩力(dp/dt)和心肌舒张能力(-dp/dt)的减弱;左室舒张压的升高:提示尾加压素Ⅱ可能对心脏具有抑制作用。我们推测在整体实验中,尾加压素Ⅱ可能通过调节其他物质如一氧化氮和硫化氢等气体小分子更多还原

【Abstract】 Urotensin II, a cyclic neuropeptide initially isolated from the urophysis of teleost fish, has recently been cloned in several mammalian species including human. Recently it has been shown to play a role via its receptor GPR14 in the cardiovascular system of the mammalian. It has been recognized as one of most potent vasoconstrictors identified to date. Systemic administration of human urotensin II induced various cardiovascular responses in different species. A stimulant effect of urotensin II in isolated human right atrial trabeculae had been reported. While little is known about its putative direct effect on the left ventricle. GPR14 is widely expressed in many tissues such as cardiovascular tissues, central nervous system and endocrine tissues. GPR14 has been observed in the rat heart by ligand binding assay and reverse transcripticn-polymerase chain reaction. However, the cellular distribution of the GPR14 protein remains to be further clarified. Moreover, urotensin II has been suggested to be a mediator of cardiomyocyte hypertrophy. Both It and its receptor have been shown to be up-regulated in the heart in chronic hypoxia and congestive heart failure, suggesting a possible role of urotensin II in cardiac remodeling. Nitric oxide （NO） has been recognized as an important regulator in the cardiovascular system. NO induces blood vessel relaxation and inotropic effects in the myocardium. Urotensin II has been reported to induce cardiovascular effects via the NOS（nitric oxide synthase） /NO system. Hydrogen sulfide （H2S） is new biological gas molecule endogenously generated from cysteine in a reaction catalysed by cystathionine P -synthase（CBS） and/or cystathionine- Y -lyase. It has recently been discovered play an important role in the cardiovascular system. The role of H₂S in urotensin II effects remains to be explored.In the present study, we investigated the cellular distribution of GPR14 protein in rat heart by using immunohistochemistry and confocal microscopic immunofluorescence double staining with antipeptide polyclonal antibodies against GPR14 and cell type markers for myocytes and endothelial cells. In paraffin-embedded heart sections, positive immunohistochemical staining was observed in the left ventricle but not in the right ventricule, atria and aortic valve. Immunofluorescence double staining revealed the cardiac myocyte as the only cell type expressing GPR14 protein in frozen heart sections as well as in isolated cardiac myocytes. There was no visible signal for GPR14 in intramyocardial coronary arteries更多还原