Effect of P2X
receptor antagonists on cell viability after rotenone and MPTP pretreatment in PC12 cells. A. and B. Expression levels of the P2X7 (A) and P2X4 (B) purinergic receptors in PC12 cells after 1 μM MPTP treatment. Quantitative SYBR Green real-time PCR was performed using specific primers. The experiments were repeated twice with similar results. The expression level of the P2X receptors was normalized to the expression level of the distinct housekeeping gene, 18S rRNA. Data are displayed as the mean ± S.E.M. Asterisks indicate significant differences from the corresponding control (*P < 0.05, Student's t-test). C and D. Concentration-dependent inhibition of cell viability by rotenone (C) and MPTP (D) in the MTT assay. Cells were treated with various concentrations of the toxins ranging from 0.01 μM to 30 μM, and the reduction of MTT into formazan was measured 20 h later. Cell viability is expressed as a percentage of the respective controls. E and F. Effects of BBG and AZ10606120 on toxicity induced by rotenone (E) and MPTP (F) measured in the MTT assay. Cells were pretreated with L-deprenyl and with the P2X7 antagonists, BBG and AZ10606120, in 10 and 100 nM concentrations for 1 h before treatment with 1 μM rotenone or 10 μM MPTP for 20 h. Data are expressed as the percentage of values of control cultures and are the means ± S.E.M. of four experiments. G. and H. Effects of BBG and AZ10606120 on toxicity induced by rotenone (G) and MPTP (H) measured in the LDH assay. Treatments of the cells were identical to the MTT assay. The released LDH is expressed as the percentage of total LDH measured after freeze/thaw lysis of cells. These data are then expressed as the percentage of values of control cultures and are the means ± S.E.M. of four experiments. * P < 0.05, ** P < 0.01, significance vs. controls using an ANOVA followed by the Dunnett test. E-H. Note that the concentration of rotenone and MPTP are indicated in μM, whereas the concentration of other drugs in nM.