4.4.3. Griess Assessment of nNOS Activity

We determined total nitrites (after the reduction of nitrates), as the end products of neuronal nitric oxide synthase (nNOS), with the previously employed Griess method [76,77] modified using vanadium (III) instead of cadmium for the reduction of nitrates to nitrites [78].

For the assessment of total nitrites, the sample (50 µL) was treated with vanadium (III) chloride 0.8% in HCl 1M (50 µL) and Griess modified reagent 4% (100 µL), incubated at RT for 30 min and OD measured at 540 nm. A standard curve of NaNO₂ was measured for each experiment and results are reported as µM NO₂⁻ to protein ratio.

Further, we determined the activity of nNOS incubating tissue homogenates samples (50 µL) with arginine 0.013% (10 µL), FAD (10 µL) and NADPH2 (50 µL) for 60 min · 37 °C, followed by the addition of VCl₃ 0.8% in HCl 1M (50 µL) and Griess modified reagent (100 µL), reading at 540 nm after 30 min at RT, versus a sample blank without enzyme substrate and cofactors. nNOS activity is expressed as the % increase of total nitrites = 100 × (DO_sample − DO_{sample blank})/DO_{sample blank}.