Yura T, Fukunaga M, Khan R, Nassar G N, Badr K F, Montero A
Renal Division, Department of Medicine, Emory University, Atlanta, Georgia, USA.
Kidney Int. 1999 Aug;56(2):471-8. doi: 10.1046/j.1523-1755.1999.00596.x.
Free-radical-generated F2-isoprostane stimulates DNA synthesis and endothelin-1 (ET-1) expression on endothelial cells. 8-Iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a member of the recently discovered family of prostanoids, the F2-isoprostanes, produced in vivo by cyclooxygenase-independent, free-radical-catalyzed lipid peroxidation. The goal of our study is to establish the effect of isoprostane on ET-1 production by endothelial cells, as well to determine the receptors responsible for these effects.
The proliferative effect of isoprostanes was measured as an increase of viable cell number and [3H]-thymidine uptake. ET-1 gene expression and protein synthesis were determined by Northern blot and radioimmunoassay, respectively. We also determined inositol 1,4,5-trisphosphate synthesis. Thromboxane A2 (TXA2) receptor antagonist SQ29,548 was used to establish the role of TXA2 receptor in isoprostane effect, as well as to determine the type of receptors involved in these effects.
Our results show that physiological concentrations of 8-iso-PGF2alpha stimulated cell proliferation, DNA synthesis, and ET-1 mRNA and protein expression in bovine aortic endothelial cells (BAECs). The proliferative effect was partially abolished by treatment with anti-endothelin antibody. 8-Iso-PGF2alpha also increased inositol 1, 4,5-trisphosphate formation in these cells. These effects were partially inhibited by SQ29,548. In competitive binding assays, two binding sites were recognized on BAECs with dissociation constants (Kd) and binding site densities at equilibrium similar to those previously described in smooth muscle cells and likely represent [3H]-8-iso-PGF2alpha binding to its own receptor (high-affinity binding site) and cross-recognition of the TXA2 receptor (low-affinity binding site).
These studies expand the potential scope of the pathophysiologic significance of F2-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.
自由基生成的F2 -异前列腺素刺激内皮细胞的DNA合成和内皮素-1（ET-1）表达。8-异前列腺素F2α（8-iso-PGF2α）是最近发现的前列腺素家族F2 -异前列腺素的成员，它在体内由不依赖环氧化酶的自由基催化脂质过氧化产生。我们研究的目的是确定异前列腺素对内皮细胞产生ET-1的影响，并确定介导这些作用的受体。
通过活细胞数量增加和[3H]-胸苷摄取来测定异前列腺素的增殖作用。分别通过Northern印迹和放射免疫测定法测定ET-1基因表达和蛋白质合成。我们还测定了肌醇1,4,5-三磷酸的合成。血栓素A2（TXA2）受体拮抗剂SQ29,548用于确定TXA2受体在异前列腺素作用中的作用，以及确定参与这些作用的受体类型。
我们的结果表明，生理浓度的8-iso-PGF2α刺激牛主动脉内皮细胞（BAECs）的细胞增殖、DNA合成以及ET-1 mRNA和蛋白质表达。用抗内皮素抗体处理可部分消除增殖作用。8-iso-PGF2α还增加了这些细胞中肌醇1,4,5-三磷酸的形成。这些作用被SQ29,548部分抑制。在竞争性结合试验中，在BAECs上识别出两个结合位点，其解离常数（Kd）和平衡时的结合位点密度与先前在平滑肌细胞中描述的相似，可能代表[3H]-8-iso-PGFα与其自身受体的结合（高亲和力结合位点）以及TXA2受体的交叉识别（低亲和力结合位点）。
这些研究扩展了氧化损伤期间释放的F2 -异前列腺素病理生理意义的潜在范围，包括内皮细胞生物学的改变。
