These studies expand the potential scope of the pathophysiologic significance of F2-isoprostanes, released during oxidant injury, to include alteration of endothelial cell biology.
The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B4 biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct antiinflammatory role for 15-LO during immune-mediated tissue injury.
In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.
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