Chen Zhengju, Zhang Xu, Ying Lei, Dou Dou, Li Yanhui, Bai Yun, Liu Juan, Liu Limei, Feng Han, Yu Xiaoxing, Leung Susan Wai-Sum, Vanhoutte Paul M, Gao Yuansheng
Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China;
Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China; Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China;
Am J Physiol Heart Circ Physiol. 2014 Aug 1;307(3):H328-36. doi: 10.1152/ajpheart.00132.2014. Epub 2014 Jun 6.
cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.
环磷酸鸟苷（cGMP）被认为是可溶性鸟苷酸环化酶（sGC）响应一氧化氮（NO）合成的唯一介质。然而，纯化的sGC可以合成其他几种环核苷酸，包括肌苷3',5'-环磷酸（cIMP）。本研究旨在确定cIMP在离体猪冠状动脉缺氧收缩中的作用。通过测量等长张力来检测血管反应。通过高效液相色谱串联质谱法测定环核苷酸。使用蛋白质免疫印迹分析和酶联免疫吸附测定试剂盒，通过测量肌球蛋白磷酸酶靶亚基1的磷酸化来确定Rho激酶（ROCK）活性。在有内皮的动脉中，缺氧会使cIMP水平升高，但在无内皮的动脉中则不会（除非用二乙三胺（DETA） NONOate处理）；cIMP的增加被sGC抑制剂1H-[1,2,4]恶二唑并[4,3,-a]喹喔啉-1-酮（ODQ）抑制。如果用DETA NONOate处理，缺氧（氧分压：25 - 30 mmHg）会增强有内皮和无内皮动脉的收缩；这些缺氧收缩被ODQ阻断。在无内皮的动脉中，外源性cIMP也能引起缺氧增强的收缩。在有内皮的动脉中，5'-三磷酸肌苷（cIMP的前体）进一步增强了缺氧增强的收缩。缺氧或cIMP引起的收缩增强伴随着肌球蛋白磷酸酶靶亚基1在苏氨酸（853）位点的磷酸化增加，而ROCK抑制剂Y-27632可阻止这种增加。分离动脉上清液中的ROCK活性以浓度依赖的方式被cIMP刺激。这些结果表明，sGC合成的cIMP可能是冠状动脉血管收缩缺氧增强的介质，部分原因是激活了ROCK。
