In addition to ROS production, EtOH/Ach can potentially activate the nitric oxide synthase (NOS) pathway generating NO in neurons. The nNOS in neurons is usually present in its constitutive form, and therefore produces a minimal level of NO in a calcium-dependent manner, serving as an effective neurotransmitter. Although clearly detectable, the insignificant changes in 160-kDa nNOS protein content that we observed here did not reflect the significant increase in NO production (47-57%) after EtOH/Ach exposure (Figs. 6A-C), suggesting that enhanced NO level was not triggered by nNOS stimulation. Thus, we investigated the role of calcium-independent cytokine inducible NOS in neuronal cell cultures after treatment with EtOH/Ach in the presence or absence of the iNOS-specific inhibitor L-NAME. Our results demonstrated that 45% increase in low molecular weight (10 kDa) iNOS protein content after EtOH/Ach exposure correlated with the 47-57% increase in NO levels (Figs. 6D and E), and augmented NO production was effectively inhibited by L-NAME, similar to the findings in flap vessels or in rat renal endothelial membrane compartments [42,43]. We also detected two other iNOS-immunoreactive bands (molecular weights of
