一氧化氮（NO）和內皮源性超極化因子（EDHFs）是內皮衍生的血管舒張因子兩大類。 EETs是構成EDHFs的主要類型，這是由花生四烯酸通過細胞色素P450 （CYP）表氧化酶的催化活性得到。雖然這兩個EET和NO誘導血管舒張，從而降低血壓，許多報告表明，NO對EET引起的血管舒張起抑製作用。然而，不管它的重要性，有關一氧化氮對EETs的抑制作用的機理尚未完全了解。 / 在本研究中，我調查了一氧化氮對EET的負調控。通過膜電位和動脈張力測量，我們發現， 11,12-EET可引起內皮剝脫豬冠狀動脈平滑肌細胞膜超極化和血管舒張。該反應被S-亞硝基-N-乙酰青黴胺（SNAP）和8-Br-cGMP，一個NO的供體和cGMP的膜穿透物類似物，分別抑制。 SNAP和8-Br-cGMP對11,12-EET引起的細胞膜超極化和血管舒張的抑製作用被羥鈷胺，一氧化氮清除劑; ODQ ，鳥苷酸環化酶抑製劑;和KT5823 ，蛋白激酶G（PKG）抑製劑逆轉。 SNAP和8-Br-cGMP對EET反應的抑製作用也被過度供應外源性激酶底物， TAT-TRPC1S¹⁷²和TAT -TRPC1T³¹³廢除。羥鈷胺，ODQ， KT5823， TAT -TRPC1，和TAT -scrambled獨自使用不影響11,12-EET引起的細胞膜超極化和血管舒張作用。然而，獨自使用14，15-EEZE（EET的拮抗劑）抑制了11,12-EET的作用。 此外，磷酸化試驗表明， PKG可以直接在Ser172和Thr313位點磷酸化TRPC1 。此外，TRPV4 ， TRPC1 ，或KCa1.1被選擇性地抑制時，11,12-EET未能引起細胞膜超極化和血管舒張。免疫共沉澱研究表明， TRPV4 ， TRPC1和KCa1.1物理上彼此相關聯。 / 以上結果表明，NO-cGMP-PKG通路可通過TRPC1的磷酸化來抑制11,12- EETs在冠狀動脈血管平滑肌細胞上的作用。此外，TRPV4，TRPC1和KCa1.1參與11,12-EET誘導平滑肌超極化和血管舒張，他們可能互相關聯。從本研究的結果表明，NO和cGMP可通過PKG-介導的TRPC1的磷酸化，抑製EET誘導的平滑肌超極化和血管舒張。 / Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHFs) are two main classes of endothelium-derived vascular relaxant factors. EETs constitute a major type of EDHFs, which are derived from arachidonic acids via the catalytic activity of cytochrome P450 (CYP) epoxygenases. Although both EET and NO induce vascular relaxation, thus reduce blood pressure, numerous reports demonstrated that NO exerts an inhibitory action on EET-induced vascular relaxation. However, despite of its importance, the mechanisms related to the inhibitory effects of NO on EETs are incompletely understood. / In the present study, I investigated the scheme for negative regulation of NO on EET action. Through measurements of membrane potential and arterial tension, we showed that 11,12-EET could induce membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. The responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, a NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, a NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-Br-cGMP on EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with excessive supply of exogenous PKG substrates, TAT-TRPC1S¹⁷² and TAT-TRPC1T³¹³. Hydroxocobalamin, ODQ, KT5823, TAT-TRPC1 and TAT-scrambled alone has no effect on 11,12-EET-induced membrane hyperpolarization and vascular relaxation. However, 14,15-EEZE (a selective EET antagonist) alone inhibits the action of 11,12-EET. Furthermore, phosphorylation assay was performed and it demonstrated that PKG could directly phosphorylate TRPC1 at Ser¹⁷² and Thr³¹³. In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1 and KCa1.1 physically associated with each other in smooth muscle cells. / Taking together, our findings demonstrated that the NO-cGMP-PKG pathway may act through the phosphorylation of TRPC1 to inhibit the action of 11,12-EETs in coronary arterial smooth muscle cells. Furthermore, TRPV4, TRPC1 and KCa1.1 are critically involved in the 11,12-EET-induced smooth muscle hyperpolarization and relaxation and that they may physically associate with each other. The results from this study demonstrated that NO and cGMP could lead to PKG-mediated phosphorylation of TRPC1, resulting in an inhibition of EET-induced smooth muscle hyperpolarization and vascular relaxation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Peng. / "Ca" on title page is subscript. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 115-133). / Abstracts also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077730 |
| Date | January 2014 |
| Contributors | Zhang, Peng , active 2014 (author.), Yao, Xiaoqiang (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvi, 134 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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