Gauthier Kathryn M, Baewer David V, Hittner Sarah, Hillard Cecilia J, Nithipatikom Kasem, Reddy D Sudarshan, Falck J R, Campbell William B
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Am J Physiol Heart Circ Physiol. 2005 Mar;288(3):H1344-51. doi: 10.1152/ajpheart.00537.2004. Epub 2004 Nov 4.
Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC) AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K(+) channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone.
乙酰胆碱刺激内皮衍生的花生四烯酸（AA）代谢产物的释放，包括前列环素和环氧二十碳三烯酸（EETs），这些物质可使冠状动脉舒张。然而，内皮细胞（EC）中AA激活的机制仍不明确。我们提出2-花生四烯酸甘油酯（2-AG）在该途径中起重要作用。通过质谱法鉴定从牛冠状动脉内皮细胞中分离出的一种AA代谢产物为2-AG。在用脂肪酸酰胺水解酶抑制剂重氮甲基花生四烯酸酮（DAK；20微摩尔/升）预处理的内皮细胞中，乙酰甲胆碱（10微摩尔/升）刺激的2-AG释放被磷脂酶C抑制剂U-73122（10微摩尔/升）或二酰基甘油脂肪酶抑制剂RHC-80267（40微摩尔/升）阻断。在U-46619预收缩的牛冠状动脉环中，10微摩尔/升时平均100%的2-AG舒张作用被去除内皮、DAK、水解酶抑制剂甲基花生四烯酰氟磷酸酯（10微摩尔/升）、环氧合酶抑制剂吲哚美辛（10微摩尔/升）抑制，但不被CB1大麻素受体拮抗剂SR-141716（1微摩尔/升）抑制。细胞色素P-450抑制剂SKF-525a（10微摩尔/升）和14,15-环氧二十碳-5Z-烯酸EET拮抗剂（14,15-EEZE；10微摩尔/升）进一步减弱了吲哚美辛耐药的舒张作用。不可水解的2-AG类似物诺拉汀醚、2-AG酰胺和14,15-EET甘油酰胺未诱导舒张。对乙酰甲胆碱的N-硝基-L-精氨酸耐药性舒张也被U-73122、RHC-80267和DAK抑制。14,15-EET甘油酯使分离的平滑肌细胞贴壁片中大电导钾（+）通道的开放增加了12倍，并诱导平均95%的舒张。这些结果表明，乙酰甲胆碱通过激活磷脂酶C和二酰基甘油脂肪酶刺激内皮细胞产生2-AG。2-AG进一步水解为AA，AA再代谢为血管活性类二十烷酸。这些研究揭示了2-AG在内皮细胞AA释放和冠状动脉张力调节中的作用。
