leupeptins and 7-nitroindazole

Nitric oxide is linked with neurodegeneration in Parkinson's disease (PD) through the involvement of both inducible (iNOS) and neuronal nitric oxide synthase (nNOS). While non-selective NOS inhibitors are neuroprotective, the role of nNOS has not been determined using selective NOS inhibitors. The present study investigated the neuroprotective effect of selective iNOS and nNOS inhibitors on MPP(+)- and MG-132-induced cell death in cell lines with differing levels of nNOS expression. Inhibition of endogenously expressed nNOS by 7-NI and ARR17477 enhanced the toxicity of MPP(+) and MG-132 in N1E-115 cells, whereas in transfected SH-SY5Y cells overexpressing nNOS, ARR17477 and 7-NI protected against MPP(+)- and MG-132-induced cell death. In contrast, inhibition of iNOS by 1400W was ineffective in preventing MPP(+) and MG-132 toxicity in these cell lines. These results suggest a dual role for NOS in dopaminergic cell viability. nNOS is protective against toxic insult when produced endogenously. When nNOS is overexpressed, it becomes neurotoxic to cells suggesting that inhibition of nNOS may be a promising strategy to prevent cell death in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amidines; Animals; Asparagine; Caspase 3; Cell Death; Cell Line, Tumor; Citrulline; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Indazoles; L-Lactate Dehydrogenase; Leupeptins; Mice; Neuroblastoma; Neurotoxins; Nitric Oxide Synthase Type I; Thiophenes; Transfection; Tritium; Tyrosine 3-Monooxygenase; Vesicular Monoamine Transport Proteins

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x(L) phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.

Topics: Animals; Apoptosis; Caspase 3; Caspase 9; Cell Survival; Cysteine Proteinase Inhibitors; Indazoles; JNK Mitogen-Activated Protein Kinases; Leupeptins; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; PC12 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Reactive Nitrogen Species; Signal Transduction