Pregnancy is a state of oxidative stress arising from increased placental mitochondrial activity and production of reactive oxygen species (ROS), mainly superoxide anion. The placenta also produces other ROS including nitric oxide, carbon monoxide, and peroxynitrite which have pronounced effects on placental function including trophoblast proliferation and differentiation and vascular reactivity. Excessive production of ROS may occur at certain windows in placental development and in pathologic pregnancies, such as those complicated by preeclampsia and/or IUGR, overpowering antioxidant defenses with deleterious outcome. In the first trimester, establishment of blood flow into the intervillous space is associated with a burst of oxidative stress. The inability to mount an effective antioxidant defense against this results in early pregnancy loss. In late gestation increased oxidative stress is seen in pregnancies complicated by diabetes, IUGR, and preeclampsia in association with increased trophoblast apoptosis and deportation and altered placental vascular reactivity. Evidence for this oxidative stress includes increased lipid peroxides and isoprostanes and decreased expression and activity of antioxidants. The interaction of nitric oxide and superoxide produces peroxynitrite, a powerful prooxidant with diverse deleterious effects including nitration of tyrosine residues on proteins thus altering function. Nitrative stress, subsequent to oxidative stress is seen in the placenta in preeclampsia and diabetes in association with altered placental function.

We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellularregulated kinase (ERK1 ⁄2 ), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK1 ⁄2 was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 M, ERK1 ⁄2 activity significantly increased. VEGF produced a time-and concentration-dependent activation of ERK1 ⁄2 . Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK1 ⁄2 activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitor N -monomethyl-Larginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK1 ⁄2 induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK1 ⁄2 activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK1 ⁄2 is necessary for VEGF-induced endothelial cell proliferation.

In kidney epithelial cells, arachidonic acid and other fatty acids are important signal transduction molecules for G protein-coupled receptors. We now demonstrate that arachidonic acid induced a time-and dosedependent activation of JNK, a member of the mitogenactivated protein kinase family, as assessed by phosphorylation of the transcription factor ATF-2. Increments in JNK activity were detectable at 5 M arachidonic acid and plateaued at 30 M. Activation was specific to arachidonic acid and linoleic acid, since other fatty acids of the n ؊ 3 and n ؊ 6 series and͞or various degrees of saturation were without effect. Specific inhibitors of cyclooxygenase-, lipoxygenase-, and cytochrome P450-dependent metabolism did not affect arachidonic acid-induced JNK activity. We further demonstrated that the free radical scavenger N-acetylcysteine blocked arachidonic acid-induced JNK activation, while H 2 O 2 , a reactive oxidative molecule, activated JNK in a dosedependent manner, providing additional support for a redox mechanism. Moreover, arachidonic acid activated NADPH oxidase (EC 1.6.-.-, EC 1.6.99.-) in a dose-dependent manner, and the potency of superoxide generation paralleled that of JNK activation by other fatty acids. We conclude that in kidney epithelial cells arachidonic acid activates JNK by means of NADPH oxidase and superoxide generation, independent of eicosanoid biosynthesis.