Levi-Rosenzvig Reut, Beyer Andreas M, Hockenberry Joseph, Ben-Shushan Rotem Shelly, Chuyun Dimitry, Atiya Shahar, Tamir Snait, Gutterman David D, Szuchman-Sapir Andrea
Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
Department of Medicine and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA.
Free Radic Biol Med. 2017 Feb;103:87-94. doi: 10.1016/j.freeradbiomed.2016.12.022. Epub 2016 Dec 19.
Prominent among the endothelium-derived hyperpolarizing factors (EDHFs) are the Cytochrome P450 (CYP) epoxygenase-derived arachidonic acid metabolites-the epoxyeicosatrienoic acids (EETs), that are known as vasodilators in the microcirculation. Among the EET isomers, 5,6-EET undergoes rapid lactonization in aqueous solution to the more stable 5,6-δ DHTL (5,6-dihydroxytrienoic lactone) isomer. It is unclear whether this metabolic transformation maintains its vasodilator potential and what is the mechanism of action. Thus, the aim of this study was to investigate the capacity of the lactone isomer, 5,6- δ DHTL, to induce dilation of arterioles and explore the endothelial Ca response mechanism.
In isolated human microvessels 5,6- δ DHTL induced a dose dependent vasodilation, that was inhibited by mechanical denudation of the endothelial layer. This 5,6- δ DHTL -dependent dilation was partially reduced in the presence of L-NAME (NOS inhibitor) or the NO-scavenger, cPTIO (by 19.7%, which was not statistically significantly). In human endothelial cells, 5,6- δ DHTL induced an increase in intracellular Ca([Ca]i) in a dose dependent manner. This increase in [Ca]i was similar to that induced by the 5,6-EET isomer, and significantly higher than observed by administering the hydrolytic dihydroxy isomer, 5,6-DHET. Further experiments aimed to investigate the mechanism of action revealed, that the 5,6-δ DHTL-mediated ([Ca]i elevation was reduced by IP3 and ryanodine antagonists, but not by antagonists to the TRPV4 membrane channel. Similar to their effect on the dilation response in the arteries, NO inhibitors reduced the 5,6-δ DHTL-mediated ([Ca]i elevation by 20%. Subsequent 5,6-δ DHTL -dependent K ion efflux from endothelial cells, was abolished by the inhibition of small and intermediate conductance K.
The present study shows that 5,6-δ DHTL is a potential EDHF, that dilates microvessels through a mechanism that involves endothelial dependent Ca entry, requiring endothelial hyperpolarization. These results suggest the existence of additional lactone-containing metabolites that can be derived from the PUFA metabolism and which may function as novel EDHFs.
细胞色素P450（CYP）环氧合酶衍生的花生四烯酸代谢产物——环氧二十碳三烯酸（EETs）是内皮源性超极化因子（EDHFs）中的重要成员，在微循环中作为血管舒张剂发挥作用。在EET异构体中，5,6-EET在水溶液中会迅速内酯化形成更稳定的5,6-δ DHTL（5,6-二羟基三烯酸内酯）异构体。目前尚不清楚这种代谢转变是否能维持其血管舒张潜能以及作用机制是什么。因此，本研究旨在探究内酯异构体5,6-δ DHTL诱导小动脉舒张的能力，并探索内皮细胞钙反应机制。
在分离的人体微血管中，5,6-δ DHTL可诱导剂量依赖性血管舒张，这种舒张作用可被机械性剥脱内皮细胞所抑制。在存在L-NAME（一氧化氮合酶抑制剂）或一氧化氮清除剂cPTIO的情况下，这种依赖于5,6-δ DHTL的舒张作用部分减弱（降低了19.7%，无统计学意义）。在人内皮细胞中，5,6-δ DHTL可剂量依赖性地诱导细胞内钙（[Ca]i）升高。这种[Ca]i的升高与5,6-EET异构体诱导的情况相似，且显著高于水解二羟基异构体5,6-DHET所观察到的升高水平。旨在探究作用机制的进一步实验表明，5,6-δ DHTL介导的（[Ca]i升高可被肌醇三磷酸（IP3）和兰尼碱拮抗剂降低，但不受瞬时受体电位香草酸亚型4（TRPV4）膜通道拮抗剂的影响。与它们对动脉舒张反应的作用类似，一氧化氮抑制剂可使5,6-δ DHTL介导的（[Ca]i升高降低20%。随后，内皮细胞中依赖于5,6-δ DHTL的钾离子外流在小电导和中电导钾离子通道受抑制后被消除。
本研究表明，5,6-δ DHTL是一种潜在的EDHF，它通过一种涉及内皮依赖性钙内流的机制使微血管舒张，这需要内皮细胞超极化。这些结果提示可能存在其他源自多不饱和脂肪酸（PUFA）代谢的含内酯代谢产物，它们可能作为新型EDHF发挥作用。
