Roy S, Balasubramanian S, Wang Jinghua, Chandrashekhar Y, Charboneau R, Barke Roderick
Department of Pharmacology, University of Minnesota, Minneapolis Veterans Affairs Medical Center, Minneapois, MN 55417, USA.
Surgery. 2003 Aug;134(2):336-44. doi: 10.1067/msy.2003.247.
Vascular endothelial growth factor (VEGF) may contribute to the resolution of myocardial ischemia by stimulating collateral circulation. Morphine analgesia after myocardial ischemia is thought to increase infarct size. We hypothesize that morphine inhibits myocardial VEGF expression by inhibiting hypoxia-induced factor 1alpha (HIF-1alpha) and the signal transduction mechanisms involving Erk-1,2 MAP kinase (p42/p44), and PI3 kinase activity (phospho-Akt).
(1) In vitro: primary cultures of rat cardiac myocytes; (2) in vivo: rat coronary ligation model; (3) mRNA measurement: real-time reverse transcriptase-polymerase chain reaction; (4) protein measurements: enzyme-linked immunosorbent assay, Western immunoblot, electromobility shift assay (EMSA), and immunohistochemistry.
Using rat cardiac myocytes in vitro, we show that morphine: (1) decreases hypoxia-induced VEGF(121) and VEGF(165) mRNA expression and VEGF protein concentration through an opioid receptor mechanism; (2) decreases HIF-1alpha protein expression (immunoblot) and nuclear protein binding to the VEGF HIF-1alpha DNA response element (EMSA); and (3) inhibits phospho-Erk-1,2 MAP kinase (immunoblot) and phospho-Akt kinase activity (immunoblot). Using a rat coronary ligation model, we show that morphine treatment: (1) decreases myocardial VEGF protein expression (immunohistochemistry); (2) decreases HIF-1alpha protein expression (immunoblot); and (3) decreases phospho-Erk-1,2 and phospho-Akt expression.
(1) Morphine inhibits hypoxia-induced VEGF transcription, in part, through an HIF-1alpha-mediated mechanism and (2) morphine inhibition of hypoxia-induced HIF-1alpha may be mediated by inhibition of ERK 1,2 MAP kinase activity and PI3 kinase activity.
血管内皮生长因子（VEGF）可能通过刺激侧支循环促进心肌缺血的缓解。心肌缺血后的吗啡镇痛被认为会增加梗死面积。我们假设吗啡通过抑制缺氧诱导因子1α（HIF-1α）以及涉及细胞外调节蛋白激酶1、2（Erk-1,2 MAP激酶，p42/p44）和磷脂酰肌醇3激酶活性（磷酸化Akt）的信号转导机制来抑制心肌VEGF表达。
（1）体外：大鼠心肌细胞原代培养；（2）体内：大鼠冠状动脉结扎模型；（3）mRNA测量：实时逆转录聚合酶链反应；（4）蛋白质测量：酶联免疫吸附测定、蛋白质免疫印迹法、电泳迁移率变动分析（EMSA）和免疫组织化学。
利用体外培养的大鼠心肌细胞，我们发现吗啡：（1）通过阿片受体机制降低缺氧诱导的VEGF(121)和VEGF(165) mRNA表达以及VEGF蛋白浓度；（2）降低HIF-1α蛋白表达（免疫印迹法）以及与VEGF HIF-1α DNA反应元件结合的核蛋白（EMSA）；（3）抑制磷酸化Erk-1,2 MAP激酶（免疫印迹法）和磷酸化Akt激酶活性（免疫印迹法）。利用大鼠冠状动脉结扎模型，我们发现吗啡治疗：（1）降低心肌VEGF蛋白表达（免疫组织化学）；（2）降低HIF-1α蛋白表达（免疫印迹法）；（3）降低磷酸化Erk-1,2和磷酸化Akt表达。
（1）吗啡部分通过HIF-1α介导的机制抑制缺氧诱导的VEGF转录，（2）吗啡对缺氧诱导的HIF-1α的抑制可能由ERK 1,2 MAP激酶活性和PI3激酶活性的抑制介导。
