Free-radical-generated F₂-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells

Background. Free-radical-generated F₂-isoprostane stimulates DNA synthesis and endothelin-1 (ET-1) expression on endothelial cells. 8-Iso- prostaglandin F(2α) (8-iso-PGF(2α)) 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. Methods. 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. Results. Our results show that physiological concentrations of 8-iso-PGF(2α) 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-PGF(2α) 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-PGF(2α) binding to its own receptor (high-affinity binding site) and cross-recognition of the TXA₂ receptor (low-affinity binding site). Conclusion. These studies expand the potential scope of the pathophysiologic significance of F₂-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.