Abstract
Elevated synaptic levels of dopamine may induce striatal neurodegeneration in l-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that approximately 60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-l-arginine methyl ester and genistein, wortmannin, or NF-kappaB SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-kappaB, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.
Highlights
Striatonigral neurodegeneration is implicated in the clinical expression of several human disorders and addictive states involving dopamine neuronal dysfunction, such as MSA,1 L
SK-N-MC cells (90% confluence) were incubated for 16 h with increasing concentrations of either dopamine or H2O2, and free radical production was indexed by measuring nitrite levels, a stable byproduct of nitric oxide (NO) released into the growth medium (Fig. 1A)
Similar to dopamine and H2O2, SKF R-38393 caused an increase in nitrite production that was comparable in magnitude to that obtained with H2O2 but was significantly (p Ͻ 0.01) less than that seen with dopamine (Fig. 1A)
Summary
Neuroprotective and anti-parkinsonian effects (19, 20), supporting the concept of co-participation of D1 receptors in the maintenance and/or pathogenesis of postsynaptic neurodegeneration and oxidative stress. To investigate the overall role of extracellular dopamine in degeneration of striatonigral neurons and of D1 dopamine receptors in particular, studies were conducted in both rat primary neuronal striatal cultures and in human SK-N-MC neuroblastoma cells. The latter was chosen as a postsynaptic striatal cell model system because it endogenously expresses the D1 dopamine receptor and contains the appropriate receptor-linked dopaminergic signaling machinery, a feature lacking in transfected cells (21). Dopamine caused a marked overexpression of NOS, primarily iNOS and nNOS, with concomitant increases in NO levels, oxidative stress, and cytotoxicity These results highlight a previously unknown role of D1 dopamine receptors in neurodegeneration and provide a heuristic framework in which to view the mechanisms underlying, and treatment of, striatal neurodegeneration
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