尾加压素Ⅱ和血管紧张素Ⅱ对血管收缩的协同作用及其信号机制的研究

【作者】 王艳霞；

【导师】 朱依纯；

【作者基本信息】 复旦大学 ， 生理学， 2006， 博士

【摘要】 尾加压素Ⅱ（urotensin Ⅱ，UⅡ）最早是从硬骨鱼脊髓尾部分离出来的神经环肽，是迄今为止所知的最强的收缩血管的活性物质。UⅡ对血管的作用有解剖学差异和种属依赖性，并且在不同的生理病理状态下对血管的作用不一致。在心血管疾病中，如充血性心力衰竭，心肌梗塞，高血压，动脉粥样硬化等疾病中，血浆及局部组织中UⅡ含量升高，并且其它的一些生物活性物质，如血管紧张素Ⅱ（angiotensin Ⅱ，Ang Ⅱ），内皮素1（endothelin，ET-1），儿茶酚胺类物质（catecholamines）含量也升高，说明UⅡ和这些物质在疾病的发生发展中可能有相关性。目前已有研究报道UⅡ和oLDL、serotonin对平滑肌细胞的增殖具有协同作用，可以加速高血压或高脂血症病人粥样斑块的产生，加速疾病的发展。所以研究UⅡ与其它生物活性物质的相互作用，阐明相互作用的机制，对于进一步揭示UⅡ对心血管疾病的作用机制及治疗心血管疾病新药开发与研究都有重要的意义。 本研究采用体外张力测定等方法，观察UⅡ和Ang Ⅱ对血管收缩的协同作用。我们采用低剂量不引起明显的血管收缩的UⅡ和Ang Ⅱ观察两者对大鼠胸主动脉（去除内皮）的协同作用，并且探讨协同作用的机制。结果我们发现2nM、3 nM的Ang Ⅱ和1 nM的UⅡ对血管的收缩有协同作用，可以被GPR14阻断剂urantide和AT₁受体阻断剂Losartan阻断，也可以被PKC非特异性阻断剂chelerythrine阻断。我们进一步研究发现在协同作用的血管中磷酸化的PKCα/βⅡ亚型，磷酸化的MLC和磷酸化的PKC底物含量与对照组相比升高，而单独加入同等剂量的UⅡ和Ang Ⅱ对于磷酸化的PKCα/βⅡ，MLC和PKC底物没有作用。单独加入高剂量的UⅡ（10 nM）可以引起血管明显的收缩，而且可以激活血管中PKCα/βⅡ、PKCθ亚型以及PKC底物和MLC，也可以被GPR14阻断剂、PKC阻断剂chelerythrine阻断，说明PKC参与了UⅡ缩血管的作用，并且在UⅡ缩血管的不同阶段，是不同的PKC亚型起作用的。 为了进一步探讨PKC在UⅡ收缩血管中的作用，我们用钙离子阻断剂（Methoxyverapamil/thapsigargin）阻断细胞内外的钙离子，观察到UⅡ对血管的收缩作用比对照组减少约50％左右，并且可以被PKC阻断剂chelerythrine阻断。UⅡ对于17周龄自发性高血压大鼠（SHR，内皮破坏）主动脉的收缩作用强于对照组Wistar-Kyoto（WKY）大鼠，说明UⅡ在不同的病理状态下对血管的收缩反应不同，具体机制有待于进一步研究。另外，我们还在人和大鼠的平滑肌细胞上观察到UⅡ对细胞也有收缩作用，PKC通路也参与了细胞的收缩反应。 由于UⅡ在心血管疾病中有重要作用，为了观察UⅡ对心脏的作用，我们用免疫沉淀方法证实UⅡ受体GPR14（现已被命名为UT受体）蛋白主要存在左心室，为UⅡ对心脏的作用提供了直接的证据。另外，我们还观察到UⅡ不能引起心肌细胞肥大，可能是由于新生大鼠心肌细胞上UⅡ受体表达较少原因。最后我们构建了含有UⅡ受体GPR14的腺病毒载体，为以后研究GPR14的功能及UⅡ的作用提供基础。 总之，本实验首次观察到UⅡ和Ang Ⅱ通过PKC信号通路的串话对血管收缩有协同作用，PKCα/βⅡ可能参与了协同作用，并且不同PKC亚型在UⅡ缩血管的不同时段起不同的作用。UⅡ对SHR收缩作用强于WKY。这对于进一步揭示UⅡ在心血管疾病中的作用和其作用的机制将具有十分重要的意义。更多还原

【Abstract】 Urotensin II （U-II） was the first isopeptides characterized from the terminal organ of the urophusis and has been demonstrated to the most potent vasoconstrictor identified to date. The vasoconstrictor activity of U-II is dependent upon, not only the anatomical origin of the vessel studied, but also the species from which it was isolated and has differential roles in cardiovascular physiology and pathophysiology. The expression of U-II has been shown to be up-regulated in the plasma of patients with congestive heart failure as well as in infrated heart of the rat and hypertension and atherosclerotic lesions of human aorta. In cardiovascular disease, many vasocontrictors such as angiotensin II （Ang II）, endothelin （ET-1） catecholamines have shown to be up-regulated, which may consequently modulate the vasoactive effect of U-II. Recently, synergistic effects of U-II with mildly oxidized LDL and serotonin on vascular smooth muscle cell proliferation has been reported. And this may explain the relatively rapid progression of atherosclerosis in patients with hypertension and hypercholesterolemia. In conclusion, exploration of interactive effect and mechanism between U-II and other vasoactive peptides may have far-reaching influence on our understanding of the mechanism of cardiovascular remodeling and the development of new anti-remodeling agents.In present study, we observed a novel synergistic interaction between U-II and Ang II in organ bath study. We use low dose of U-II and Ang II which induced only a little response in endothelium-denuded rat thoracic aorta and explore the mechanism.We found that co-administration of U-II （1 nM） and Ang II （2 nM） exerted a significant contractile effect which was abrogated by either PKC inhibitor chelerythrine, U-II receptor ligand inhibitor urantide, or Ang II type 1 receptor antagonist Losartan. We further observed the synergistic effect was paralleled with increase PKC activity as well as phosphorylation of PKC isoforms alpha /beta II and MLC, whilst U-II and Ang II administered alone at these concentrations has no effect. U-II alone caused a dose-dependent increase in phosphorylation of PKC isoforms alpha /beta II and theta and MLC and PKC activity. U-II-induced constriction of aorta and MLC and PKC isoform phosphorylation were prevented by urantide and chelrythrine.To further analyze the PKC-sensitive component, hU-II-induced contraction was measured in the presence of the voltage-gated Ca²⁺ channel inhibitorMethoxyverapamil/thapsigargin（TSG/D600） suppress the agonist-induced rise in [Ca²⁺]_i. The TSG/D600-resistant component of hU-II-induced contraction was dose-dependently inhibited by chelerythrine. Moreover, U-II elicited a greater constrictor response of aortic rings isolated from 17-week-SHR （endothelium-denuded） compared to WKY rats. The contractile response to U-II was different in different pathophysiological disease. Moreover, we investigated the effect and mechanism in rat and human arterial vascular smooth muscle cells.Because U-II had important effect in cardiovascular disease, we investigated GPR 14 receptor （now named UT receptor） in the ventricle of normal rat heart with immunoprecipitation and Western blot analysis and the effects on cardiomyocytes. We also constructed recombinant adenovirus vector which containing U-II receptor （GPR14） and made a basis for further research.In conclusion, we first investigated U-II interacts synergistally with Ang II through a PKC-dependent cross-talk of the intracellular signaling pathways of these two peptides. Alpha /betaII may be the PKC isoform involved in the synergistic interaction between U-II and Ang II. Morover, different PKC isoforms contributes to the transient or long-lasting effect of hU-II-induced vasoconstriction. The contractile effect of U-II is more pronounced in arteries of SHR compared to WKY rats. This will have far-reaching influence on our understanding of the mechanism and effect of U-II in cardiovascular disease.更多还原