We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

We previously reported that nitric oxide (NO) is first detected in the uterus of a pregnant rat on gestational day 13.5 (GD13.5) and that NO levels peak on GD17.5. In addition, NO production in the uterus is mainly derived from the decidua and not the myometrium. The aim of the present study was to reveal the role of NO that peaked on GD17.5 of gestation in the decidua. To inhibit NO production, pregnant rats were continuously administered by an nitric oxide synthase inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME) for 48 h. In the control group, saline was infused instead of L-NAME. After treatment, the decidua were obtained from GD13.5, GD17.5 and GD21.5 rats. Apoptosis and activated caspase-3-positive cells were observed by transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry, respectively. The caspase-3 enzyme activity was also measured in the cell lysate from the decidua. The numbers of TUNEL-positive cells and activated caspase-3-positive cells each increased and the amount of caspase-3 activity also increased significantly in rats on GD17.5 than in rats in the control group, but no changes were observed in rats on GD13.5 and GD21.5. Furthermore, enzyme activity regarding the initiator caspases, caspase-8 and -9, upstream factors for caspase-3 in the caspase cascade, was measured simultaneously on GD17.5 under the same treatment. Caspase-8 and -9 enzyme activities increased significantly in the control group; an increment of caspase-8 activity was especially prominent. The present results indicate that an inhibitor of NO production caused apoptosis through typical apoptotic signals in the decidua on GD17.5, suggesting that an NO peak in the decidua is essential to cell survival and the maintenance of uterine formation.