Original Article
Kidney International (1999) 56, 471–478; doi:10.1046/j.1523-1755.1999.00596.x
Free-radical–generated F2-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells
Takafumi Yura1, Megumu Fukunaga2, Rizwan Khan, George N Nassar, Kamal F Badr and Angel Montero
Renal Division, Department of Medicine, Emory University and Veterans Affairs Medical Center, Atlanta, Georgia, USA
Correspondence: , Renal Section (111C), VA Medical Center, 1501 San Pedro, SE, Albuquerque, New Mexico 87108, USA.
1Current address: The First Department of Internal Medicine, Osaka-Minami National Hospital, Osaka, Japan.
2Current address: Department of Integrated Medicine, Kagawa Medical University, Kagawa, Japan.
Received 30 September 1998; Revised 11 February 1999; Accepted 8 March 1999.
Abstract
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 F2
(8-iso-PGF2) 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-PGF2 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-PGF2 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-PGF2 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.
Keywords:
oxidant injury, endothelial cell, isoprostanes, lipid peroxidation, prostanoids, vasoconstrictor
Abbreviations:
BAECs, cultured bovine aortic endothelial cells; Bmax, maximal binding capacity; ET-1, endothelin-1; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ir-ET, immunoreactive endothelin; IP3, inositol 1,4,5-trisphosphate; 8-iso-PGF2, 8-iso-prostaglandin F2; Kd, dissociation constant; PGF2, prostaglandin F2; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; TXA2, thromboxane A2
