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
发布时间：2018-08-25 19:57
【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AT_1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。
[Abstract]:Urotensin II (U II) is the first neurocyclic peptide isolated from the caudal part of the spinal cord of the skeletal fish. It is the most potent vasoconstrictor known to date. The effects of U II on blood vessels are anatomically different and species-dependent, and have different effects on blood vessels under different physiological and pathological conditions. In diseases such as congestive heart failure, myocardial infarction, hypertension, and atherosclerosis, U II levels in plasma and local tissues are elevated, as are other bioactive substances, such as angiotensin II (Ang II), endothelin 1 (ET-1), and catecholamines, said Dr. It has been reported that UII and oLDL, serotonin have synergistic effects on the proliferation of smooth muscle cells, which can accelerate the formation of atherosclerotic plaques in patients with hypertension or hyperlipidemia, and accelerate the development of disease. To clarify the mechanism of interaction is of great significance for further revealing the mechanism of action of UII on cardiovascular diseases and the development and research of new drugs for the treatment of cardiovascular diseases.
In this study, the synergistic effect of U II and Ang II on vasoconstriction was observed by in vitro tension measurement. The synergistic effect of U II and Ang II on thoracic aorta (endothelium removal) in rats was observed by low dose of non-obvious vasoconstriction U II and Ang II, and the mechanism of synergistic effect was explored. UII of nM has synergistic effects on vasoconstriction, which can be blocked by urantide, a GPR14 blocker, and Losartan, a AT1 receptor blocker, or by chelerythrine, a PKC nonspecific blocker. We further investigated the phosphorylated PKC alpha/beta II subtype, phosphorylated MLC, and phosphorylated PKC substrate content in synergistic vessels. Compared with the control group, the phosphorylated PKC alpha/beta II, MLC and PKC substrates did not respond to the same doses of U II and Ang II alone. High doses of U II (10 nM) alone could induce significant vasoconstriction and activate PKC alpha/beta II, PKC theta subtypes, PKC substrates and MLC, and could also be blocked by GPR14, PKC. The blockade of the blocker chelerythrine indicated that PKC participated in the vasoconstriction of UII and played a role in different subtypes of PKC at different stages of UII vasoconstriction.
In order to further explore the role of PKC in U II vasoconstriction, we blocked intracellular and extracellular calcium ions with a calcium blocker (methoxyverapamil/thapsigargin). We observed that the contractile effect of U II on blood vessels was about 50% less than that of the control group, and could be blocked by the PKC blocker chelerythrine. UII could be blocked by a calcium blocker for 17-week-old spontaneous hypertension. The contraction of aorta in rats (SHR, endothelial destruction) was stronger than that in Wistar-Kyoto (WKY) rats, indicating that the contractile response of UII to blood vessels was different under different pathological conditions, and the specific mechanism needed further study. It participates in cell contractile response.
Because U II plays an important role in cardiovascular disease, in order to observe the effect of U II on the heart, we used immunoprecipitation method to confirm that U II receptor GPR14 (now named UT receptor) protein exists mainly in the left side.
In addition, we also observed that U II could not induce cardiomyocyte hypertrophy, possibly due to the low expression of U II receptor in neonatal rat cardiomyocytes. Finally, we constructed an adenovirus vector containing U II receptor GPR14, which could be used to study the function of GPR14 and the effect of U II. For the foundation.
In conclusion, we observed for the first time that U II and Ang II have synergistic effects on vasoconstriction via PKC signaling pathway. PKC alpha/beta II may be involved in the synergistic effect, and different PKC subtypes play different roles in different periods of U II vasoconstriction. The function and the mechanism of its action will be of great significance.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R33
[Abstract]:Urotensin II (U II) is the first neurocyclic peptide isolated from the caudal part of the spinal cord of the skeletal fish. It is the most potent vasoconstrictor known to date. The effects of U II on blood vessels are anatomically different and species-dependent, and have different effects on blood vessels under different physiological and pathological conditions. In diseases such as congestive heart failure, myocardial infarction, hypertension, and atherosclerosis, U II levels in plasma and local tissues are elevated, as are other bioactive substances, such as angiotensin II (Ang II), endothelin 1 (ET-1), and catecholamines, said Dr. It has been reported that UII and oLDL, serotonin have synergistic effects on the proliferation of smooth muscle cells, which can accelerate the formation of atherosclerotic plaques in patients with hypertension or hyperlipidemia, and accelerate the development of disease. To clarify the mechanism of interaction is of great significance for further revealing the mechanism of action of UII on cardiovascular diseases and the development and research of new drugs for the treatment of cardiovascular diseases.
In this study, the synergistic effect of U II and Ang II on vasoconstriction was observed by in vitro tension measurement. The synergistic effect of U II and Ang II on thoracic aorta (endothelium removal) in rats was observed by low dose of non-obvious vasoconstriction U II and Ang II, and the mechanism of synergistic effect was explored. UII of nM has synergistic effects on vasoconstriction, which can be blocked by urantide, a GPR14 blocker, and Losartan, a AT1 receptor blocker, or by chelerythrine, a PKC nonspecific blocker. We further investigated the phosphorylated PKC alpha/beta II subtype, phosphorylated MLC, and phosphorylated PKC substrate content in synergistic vessels. Compared with the control group, the phosphorylated PKC alpha/beta II, MLC and PKC substrates did not respond to the same doses of U II and Ang II alone. High doses of U II (10 nM) alone could induce significant vasoconstriction and activate PKC alpha/beta II, PKC theta subtypes, PKC substrates and MLC, and could also be blocked by GPR14, PKC. The blockade of the blocker chelerythrine indicated that PKC participated in the vasoconstriction of UII and played a role in different subtypes of PKC at different stages of UII vasoconstriction.
In order to further explore the role of PKC in U II vasoconstriction, we blocked intracellular and extracellular calcium ions with a calcium blocker (methoxyverapamil/thapsigargin). We observed that the contractile effect of U II on blood vessels was about 50% less than that of the control group, and could be blocked by the PKC blocker chelerythrine. UII could be blocked by a calcium blocker for 17-week-old spontaneous hypertension. The contraction of aorta in rats (SHR, endothelial destruction) was stronger than that in Wistar-Kyoto (WKY) rats, indicating that the contractile response of UII to blood vessels was different under different pathological conditions, and the specific mechanism needed further study. It participates in cell contractile response.
Because U II plays an important role in cardiovascular disease, in order to observe the effect of U II on the heart, we used immunoprecipitation method to confirm that U II receptor GPR14 (now named UT receptor) protein exists mainly in the left side.
In addition, we also observed that U II could not induce cardiomyocyte hypertrophy, possibly due to the low expression of U II receptor in neonatal rat cardiomyocytes. Finally, we constructed an adenovirus vector containing U II receptor GPR14, which could be used to study the function of GPR14 and the effect of U II. For the foundation.
In conclusion, we observed for the first time that U II and Ang II have synergistic effects on vasoconstriction via PKC signaling pathway. PKC alpha/beta II may be involved in the synergistic effect, and different PKC subtypes play different roles in different periods of U II vasoconstriction. The function and the mechanism of its action will be of great significance.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R33
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