Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP+-induced neuronal toxicity

Nitrative stress is a key component of the pathogenic process in Parkinson's disease (PD), but the relative roles of constitutive neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) in glial cells remain unresolved. We have investigated the effects of a range of concentrations of the selective n-NOS inhibitor ARR17477, and the selective i-NOS inhibitor 1400W, on MPP+-induced cell death in foetal ventral mesencephalic (VM) dopaminergic cultures. MPP+ induced a loss of TH-positive neurones accompanied by an increase in immunoreactivity for GFAP and OX-6 as markers of astrocytes and activated microglia, respectively, and induced i-NOS immunoreactivity. Unexpectedly, MPP+ treatment did not induce 3-NT immunoreactivity in the cultures. ARR17477 and 1400W alone had no effect on the number of TH-positive cells or on the number of GFAP or OX-6 positive cells. ARR17477 did not prevent the MPP+-induced decrease in TH-positive neurones and had no effect on the increased number of GFAP- and OX-6-positive cells. By contrast, 1400W caused a concentration-dependent preservation of TH-positive neurones in the presence of MPP+. It also significantly reduced the number of OX-6-immunoreactive cells and there was a small reduction in GFAP immunoreactivity. The results suggest a major role for i-NOS-mediated nitrative stress in microglia in MPP+-induced dopaminergic cell death and this may have important implications for developing neuroprotective strategies for PD.