| Recent studies have demonstrated that the D3 preferring agonist pramipexole (PPX) can prevent neurotoxicity produced by levo-dopa in mesencephalic cultures and by MPP+ in the neuroblastoma SH-SY5Y cell line. However, the neuroprotective effects of PPX were not dopamine receptor mediated in the undifferentiated SH-SY5Y cells. We characterized differentiation conditions for the SH-SY5Y neuroblastoma cell line, cells differentiated with RA and TPA exhibited the high levels of expression TH, D2R, D3R and DAT but low levels of VMAT. Characterization of the kinetics of [3H]DA uptake and [3H]MPP+ uptake to DAT in RA/TPA differentiated cells was similar to that of rat and mouse caudate-putamen synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of MPP+ (LD50 of 100 muM), and the neurotoxic effects of MPP+ were blocked with the DAT inhibitor GBR12909. The neuroprotective effect of PPX was dose-dependent (EC50 = 41 mum) and was completely antagonized by D2-like antagonists spiperone and U99194A but not by the D1 antagonist SCH23390.; To test if D3 receptor preferring agonists S32504 and pramipexole act through D2 and/or D3 receptors, we utilized the RA/TPA differentiated SH-SY5Y cell line. The cytotoxic effects of MPP+ were antagonized only by S32504 (EC50 = 2.0 muM) and pramipexole (EC 50 = 64.3 muM) at all time points. Neuroprotection afforded by EC 50 doses of S32504 and pramipexole were antagonized by D3 antagonists; S33084, U99194A, SB269652, D2/D3 antagonist raclopride, and D2 antagonist LY741626. Raclopride and L741626 provided complete anatagonism and U99194A, S33084 and SB269652 produced nearly maximal antagonism; and the neuroprotection by pramipexole and S32504 was ineffectively antagonized by SCH23390, haloperidol and spiperone. Thus, S32504 and pramipexole partly act through D3 receptor to protect against MPP+-induced toxicity in terminally differentiated SH-SY5Y neurons. |
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