The plasma NO levels were evaluated by NO derivatives nitrate and nitrite,

as previously described [28]. Blood samples were collected into EDTA-coated tubes and plasma was immediately separated by low-speed centrifugation (1500 × g). The concentration of nitrate in blood was determined by chemiluminescence, elicited by the reaction of NO with ozone after nitrate reduction with VCl3 saturated solution in 1 mol/L HCl, at 90 °C, using a NO analyzer (NOA™280 Sievers Instruments Inc., Boulder, CO, USA). Nitrite was determined after reduction with 1% KCl solution in glacial acetic acid to convert nitrite to NO. Basal NO in mesenteric arterioles was determined by using a fluorescent cell permeable dye for NO, 4,5 diaminofluorescein diacetate (DAF-2 DA, Alexis, USA), as previously described [10].

Once inside the cell, DAF-2 DA is hydrolyzed by cytosolic Bleomycin esterases thus releasing DAF-2. The reaction between DAF-2 and NO yields the corresponding bright green-fluorescent triazolofluoresceins (DAF-2T). The mesenteric arterioles were dissected, immersed in medium for cryosectioning and cut AZD6738 molecular weight into 10 μm thick sections (Leica CM 1850 cryostat, Leica Instruments, Germany). In order to stimulate NOS activation and provide optimal levels of substrate, slices were pre-incubated with phosphate buffer (PB) solution containing CaCl2 (0.45 μmol/L) and l-arginine (100 μmol/L) during 30 min at 37 °C. Slices were washed, incubated with PB containing DAF-2 DA (10 μmol/L) for 30 min at 37 °C and observed on a microscope (Axiovert 100 M – Carl Zeiss SMT, Germany) equipped with fluorescein filter (excitation at 488 nm and measuring emission at 515 nm). Fluorescence emitted in response to NO production was quantified

through optic densitometry (arbitrary units, a.u.) using the AxioVision 4.8. digital images analysis software (Carl Zeiss). The semi-quantitative analysis of basal NO production was determined, at least, in three slices from each animal. Significant auto-fluorescence Vitamin B12 was discarded by experiments performed in the absence of DAF-2DA. NOS activity was measured by the biochemical conversion of l-[3H] arginine to l-[3H] citrulline according to the method described by Rees et al. [33]. Mesenteric vessels were dissected, washed, homogenized in ice-cold buffer and stored at −80°C. On the day of assay, homogenates were incubated (37 °C/60 min) in a buffer containing FMN and FAD 4 μmol/L, calmodulin 10 μg/mL, Ca2+ 1.25 mmol/L, NADPH 1 mmol/L, l-arginine 120 nmol/L, l-[3H] arginine 50 nmol/L (NEN Life Science Products, USA) and BH4 10 μmol/L. For the determination of iNOS activity, experiments were performed in the absence of Ca2+. Reaction was terminated by the addition of cation-exchange resin (Dowex 50WX8-400) to remove the excess of substrate.