Results

nNOS and eNOS expression
Western blotting analyses using the anti-nNOS antibody revealed the presence of one band at 155 KDa. In the GTN Group, the intensity of this band was significantly increased in the hypothalamus and medulla, when compared to the control group (Fig. 1). By contrast, no change in eNOS expression (135KDa) was detected in any of the cerebral areas under evaluation after GTN administration (Fig. 2).
Fig. 1 nNOS expression in homogenates of hypothalamus (a), mesencephalon (b) and medulla (c) of rats injected with glyceryl trinitrate (GTN) or vehicle (CT). The histograms illustrate the densitometric analysis representing expression levels of nNOS (155KDa), evaluated as the ratio vs β-actin (39 kDa). The latter protein was used as a housekeeping protein on the same membrane previously incubated with nNOS. nNOS expression was evaluated after 4 h of GTN or vehicle injection. In the right of each panel are illustrated representative western blots of nNOS protein. Data are expressed as mean ± SD. Mann Whitney test, *p < 0.05 vs vehicle (CT)
Fig. 2 eNOS expression in homogenates of hypothalamus (a), mesencephalon (b) and medulla (c) of rats injected with glyceryl trinitrate (GTN) or vehicle (CT). The histograms illustrate the densitometric analysis representing expression levels of eNOS (130KDa) as ratio vs β-actin (39 kDa). The latter protein was used as a housekeeping protein on the same membrane previously incubated with eNOS. In the right of each panel are illustrated representative western blots of eNOS protein. eNOS expression was evaluated after 4 h of GTN or vehicle injection. Data are expressed as mean ± SD. Mann Whitney test, *p < 0.05 vs vehicle (CT)

AMDA levels
ADMA levels were significantly increased in the hypothalamus and medulla of GTN treated rats, when compared to CT group. Conversely, we did not detect any significant differences in mesencephalon (Fig. 3). No significant difference was observed in serum ADMA concentrations between GTN and CT groups (Fig. 4).
Fig. 3 Cerebral levels of ADMA in rats injected with glyceryl trinitrate (GTN) or vehicle (CT). ADMA levels were evaluated after 4 h of GTN or vehicle injection. Data are expressed as mean ± SD, Mann Whitney test, *p <0.05 vs vehicle (CT)
Fig. 4 Serum levels of ADMA in rats injected with glyceryl trinitrate (GTN) or vehicle (CT). ADMA levels in serum were evaluated after 4 h of GTN or vehicle injection. Data are expressed as mean ± SD

DDAH-1mRNA expression
DDAH-1 mRNA expression was significantly decreased in the hypothalamus and in the medulla of rats treated with GTN when compared to CT group. No significant difference in DDAH-1 mRNA expression was found in the mesencephalon of rats treated with GTN when compared to CT group (Fig. 5).
Fig. 5 DDAH-1 mRNA expression in specific brain areas, 4 h after glyceryl trinitrate (GTN) or vehicle (CT) administration. Data are expressed as mean ± SD, Mann Whitney test, *p <0.05 vs vehicle (CT)