Loeb A L, Izzo N J, Johnson R M, Garrison J C, Peach M J
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Am J Cardiol. 1988 Oct 5;62(11):36G-40G. doi: 10.1016/0002-9149(88)90030-6.
The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.
内皮细胞中类二十烷酸和内皮衍生舒张因子（EDRF）的释放被认为涉及一个钙依赖性步骤。我们以培养的牛主动脉内皮细胞作为模型系统，研究了激动剂诱导的内皮细胞内肌醇多磷酸和钙水平的变化以及细胞外钙对EDRF释放的影响。在一个灌流级联系统中，通过将无内皮的兔主动脉环置于培养的内皮细胞柱下方并观察其舒张情况来检测EDRF。缓激肽或蜂毒素刺激内皮细胞可诱导生物测定环产生剂量依赖性舒张。此外，缓激肽和蜂毒素刺激使负载fura-2的内皮细胞内钙浓度升高，并用3H-肌醇预标记的细胞中肌醇1,4,5-三磷酸（Ins[1,4,5]P3）增加。缓激肽刺激使Ins(1,4,5)P3、fura-2荧光短暂增加，并使EDRF短暂释放。蜂毒素刺激诱导内皮细胞柱中EDRF释放时间延长，这与fura-2信号和Ins(1,4,5)P3水平的持续升高相关。从细胞灌流液中去除钙可减弱但不能消除缓激肽诱导的EDRF释放和钙瞬变，而蜂毒素诱导的反应仅略有减弱。内皮细胞在响应Ins(1,4,5)P3时，能清楚地显示磷脂酶C的受体激活以及从亚细胞位点释放储存的钙。这些结果表明，在没有细胞外钙的情况下，EDRF释放与细胞内钙水平升高相关。然而，EDRF的持续释放确实需要通过一种未知机制使细胞外钙流入。
