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 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.


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-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).


CONCLUSIONS
These studies expand the potential scope of the pathophysiologic significance of F2-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.
Yura
T
T
Renal Division, Department of Medicine, Emory University, Atlanta, Georgia, USA.
Fukunaga
M
M
Khan
R
R
Nassar
G N
GN
Badr
K F
KF
Montero
A
A
D000818:Animals; D001011:Aorta / Q000166:cytology; D001667:Binding, Competitive / Q000502:physiology; D002417:Cattle; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D019332:Endothelin-1 / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D028441:F2-Isoprostanes; D005609:Free Radicals / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis / Q000378:metabolism; D015227:Lipid Peroxidation / Q000502:physiology; D018384:Oxidative Stress / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D017482:Receptors, Thromboxane / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014316:Tritium; D014662:Vasoconstrictor Agents / Q000378:metabolism
D019332:Endothelin-1; D028441:F2-Isoprostanes; D005609:Free Radicals; D012333:RNA, Messenger; D017482:Receptors, Thromboxane; D014662:Vasoconstrictor Agents; D014316:Tritium; C075750:8-epi-prostaglandin F2alpha; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D015237:Dinoprost