Free-radical-generated F-2-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells

Background. Free-radical-generated F-2-isoprostane stimulates DNA synthesis and endothelin-l (ET-1) expression on endothelial cells. S-Iso-prostagland in F-2 alpha (8-iso-PGF(2 alpha)) is a member of the recently discovered fa mily of prostanoids, the F-2-isoprostanes, produced in vivo by cyclooxygena se-independent, free-radical-catalyzed lipid peroxidation. The goal of our study is to establish the effect of isoprostane on ET-1 production by endot helial cells, as well to determine the receptors responsible for these effe cts. Methods. The proliferative effect of isoprostanes was measured as an increa se of viable cell number and [H-3]-thymidine uptake. ET-1 gene expression a nd protein synthesis were determined by Northern blot and radioimmunoassay, respectively. We also determined inositol 1,4,5-trisphosphate synthesis. T hromboxane A(2) (TXA(2)) receptor antagonist SQ29,548 was used to establish the role of TXA(2) 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(2 alpha) stimulated cell proliferation, DNA synthesis, and ET-1 mRNA and prot ein expression in bovine aortic endothelial cells (BAECs). The proliferativ e effect was partially abolished by treatment with anti-endothelin antibody . 8-Iso-PGF(2 alpha) also increased inositol 1,4,5-trisphosphate formation in these cells. These effects were partially inhibited by SQ29,548. In comp etitive binding assays, two binding sites were recognized on BAECs with dis sociation constants (Kd) and binding site densities at equilibrium similar to those previously described in smooth muscle cells and likely represent [ H-3]-8-iso-PGF(2 alpha) binding to its own receptor (high-affinity binding site) and cross-recognition of the TXA(2) receptor (low-affinity binding si te). Conclusion. These studies expand the potential scope of the pathophysiologi c significance of F-2-isoprostanes, released during oxidant injury, to incl ude alteration of endothelial cell biology.